African Journal of Biotechnology Volume 14 Number 6, 11 February, 2015 ISSN 1684-5315
ABOUT AJB
The African Journal of Biotechnology (AJB) (ISSN 1684-5315) is published weekly (one volume per year) by Academic Journals.
African Journal of Biotechnology (AJB), a new broad-based journal, is an open access journal that was founded on two key tenets: To publish the most exciting research in all areas of applied biochemistry, industrial microbiology, molecular biology, genomics and proteomics, food and agricultural technologies, and metabolic engineering. Secondly, to provide the most rapid turn-around time possible for reviewing and publishing, and to disseminate the articles freely for teaching and reference purposes. All articles published in AJB are peer- reviewed.
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George Nkem Ude, Ph.D Prof. Dr. AE Aboulata Plant Breeder & Molecular Biologist Plant Path. Res. Inst., ARC, POBox 12619, Giza, Egypt Department of Natural Sciences 30 D, El-Karama St., Alf Maskan, P.O. Box 1567, Crawford Building, Rm 003A Ain Shams, Cairo, Bowie State University Egypt 14000 Jericho Park Road Bowie, MD 20715, USA Dr. S.K Das Department of Applied Chemistry and Biotechnology, University of Fukui, Japan Editor Prof. Okoh, A. I. N. John Tonukari, Ph.D Applied and Environmental Microbiology Research Department of Biochemistry Group (AEMREG), Delta State University Department of Biochemistry and Microbiology, PMB 1 University of Fort Hare. Abraka, Nigeria P/Bag X1314 Alice 5700, South Africa
Dr. Ismail TURKOGLU Department of Biology Education, Education Faculty, Fırat University, Elazığ, Turkey
Prof T.K.Raja, PhD FRSC (UK)
Department of Biotechnology
PSG COLLEGE OF TECHNOLOGY (Autonomous)
(Affiliated to Anna University)
Coimbatore-641004, Tamilnadu,
INDIA.
Dr. George Edward Mamati Horticulture Department, Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000-00200, Nairobi, Kenya.
Dr. Gitonga Kenya Agricultural Research Institute, National Horticultural Research Center, P.O Box 220, Thika, Kenya.
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African Journal of Biotechnology
International Journal of Medicine and Medical Sciences
Table of Contents: Volume 14 Number 6, 11 February, 2015
ARTICLES
Structural, physical, functional and nutraceutical changes of freeze-dried fruit Liliana Serna-Cock, Diana Patricia Vargas-Muñoz and Alfredo Ayala Aponte
Examination of genetic diversity in common bean (Phaseolus vulgaris L.) using random amplified polymorphic DNA (RAPD) markers Asifa Bukhari, M. A. Bhat, Mushtaq Ahmad and Noorul Saleem
Feedstuffs potential of harvest by-products from two oleaginous cucurbits A. I. Touré, A. L. Loukou, K.K. Koffi, I. Mouaragadja, G. L. Bohoua, B. Mbatchi and B. I. A. Zoro
Influence of rural processing methods and postharvest storage treatments on quality characteristics of kola nut (acuminata Schott & Endl.) Eze, S. C., Ameh, G. I., Ugwuoke, K. I., Onyeke, C. C. and Ozioko, J. O.
Allelopathic potential of some biocontrol agents for the control of fungal rot of yellow yam (Dioscorea cayenensis Lam) Dania, V. O., Fadina, O. O., Ayodele, M. and Lava Kumar, P.
Growth of mycotal species on the eggs of Cyprinus carpio in limnologically and trophically different water bodies Bazyli Czeczuga, Adrianna Semeniuk- Grell and Ewa Czeczuga- Semeniuk
Exploitation of molecular genetics in microbial degradation and decolorization of industrial waste water effluent Shah M. P.
Influence of temperature on the anaerobic stabilization of organic solid residues Jorge Marcell Coelho Menezes, Valderi Duarte Leite, Aldre Jorge Morais Barros, Wilton Silva Lopes, José Tavares de Sousa and Andrezza Raphaella Costa Campos
Table of Contents: Volume 14 Number 6, 11 February, 2015
Understanding the efficacy of influent waste water on microbial community structure of activated sludge process Shah M. P.
Ethanol production using hemicellulosic hydrolyzate and sugarcane juice with yeasts that converts pentoses and hexoses Juliana Pelegrini Roviero, Priscila Vaz de Arruda, Maria das Graças de Almeida Felipe and Márcia Justino Rossini Mutton
Antitrypanosomal activity of Khaya senegalensis and Anogeissus leiocarpus stem bark on Trypanosoma brucei brucei infected rats Awobode, Henrietta O., Fagbemi, Folasade T. and Afolayan, Funmilayo I. D.
Fungal infection in freshwater fishes of Andhra Pradesh, India S. A. Mastan
Vol. 14(6), pp. 442-450, 11 February, 2015 DOI: 10.5897/AJB2014.14189 Article Number: B98D7FF50317 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Review
Structural, physical, functional and nutraceutical changes of freeze-dried fruit
Liliana Serna-Cock1*, Diana Patricia Vargas-Muñoz2 and Alfredo Ayala Aponte3
1Facultad de Ingeniería y Administración, Universidad Nacional de Colombia, Colombia. 2Centro Agropecuario de Buga, Regional Valle, SENA, Colombia. 3Facultad de Ingeniería, Universidad del Valle, Colombia.
Received 16 September, 2014; Accepted 9 February, 2015
This review examines the structural, physical, functional and nutraceutical changes of lyophilized fruits. Collapse, porosity, color, glass transition temperature, rehydration capacity, ability to retain water, volatile compounds, phenolic compounds, ascorbic acid, and beta-carotene, were defined, and the causes of changes in these parameters, during freeze-drying, were analyzed. Advantages and limitations of the freeze-drying, were shown, and strategies to reduce the costs associated with its use were proposed. It was concluded that lyophilized fruit retained to a greater proportion characteristics of fresh fruits, compared with other methods of dehydration. The effects of freeze-drying on physical and chemical properties vary in accordance with factors intrinsic to the fruit, and with extrinsic properties inherent to process. Most fruits maintain their color using freeze-drying. The porosity of freeze-dried fruit depends on the freezing speed. The glass transition temperature of dry solid would be an important optimization parameter for the freezing-drying process. The majority of phenolic acids and volatile compounds were conserved in freeze-drying. Freeze-drying increases the rehydration capacity of dried fruits, to a greater extent the hydrophilic groups which are responsible for interaction with water. However, dried fruits by freeze-drying can show structural collapse. The long processing time and energy costs are limiting the application of technology. The researchers recommend using combined to potentiate the benefits of freeze-drying and to lessen their limiting technologies.
Key words: Freeze-drying, fruit, dehydration.
INTRODUCTION
Fruits are necessary in the human diet because of their of fruit per day (Montenegro et al., 2009; Orrego et al., vitamin, mineral and antioxidant content; they are remark- 2009; Shofian et al., 2011). However, the perishability able for their exuberant flavors, colors and smells. These and seasonal availability of many fruits means that they properties ought to be preserved in agro-industrial are not always available to consumers. To combat this processing (Kirmaci et al., 2008). The World Health problem, the industry often offers processed fruits that Organization (WHO) promotes the consumption of 400 g have partially or completely lost their physical, nutraceutical
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Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Serna-Cock et al. 443
and nutritional properties (Sijtsema et al., 2012). Madiouli et al., 2012). According to Harnkarnsujarit and Dehydrated fruits are excellent alternative, making Charoenrein (2011), collapse is the viscous flow that year-round availability more feasible and extending the occurs when the viscosity diminishes beyond the glass shelf life of fruits (Marques et al., 2007). The methods of transition temperature (Tg). Loss of structure occurs when fruit dehydration include drying in hot air (Giraldo et al., the material is incapable of supporting its own weight. If 2010), refraction window (Ocoro and Ayala, 2012), osmotic the temperature of a porous product is above the glass dehydration (Ayala et al., 2010), frying (Villamizar and transition temperature, the viscosity of the solid material Giraldo, 2010), drying by microwave (Duan et al., 2010; may not be able to support the structure, causing collapse Jiang et al., 2010) and freeze-drying (Ayala et al., 2010; and contraction (de Oliveira et al., 2010). Collapse is Ceballos et al., 2012). These methods diminish water alternatively defined as a decrease in volume or an increase activity and therefore reduce the number of enzymatic, in apparent specific density (Cui et al., 2008). chemical and microbiological reactions that take place Collapse is noted by the shrinkage of the dried product. (Hincapié et al., 2010). Dehydration methods should These structures are sensitive to physical, chemical and minimize the loss of nutrient and antioxidant contents microbiological changes, which reduce the shelf life and (Santos and Silva, 2008); however, because some stability of the product (Queiroz et al., 2008). Several anti0oxidant compounds are weakly bonded to water in studies have shown that collapse slows some chemical the fruit, some nutritional content will be lost in the reactions, such as oxidation (Prado et al., 2006) as well dehydration process. Generally, fruits with the greatest as the liberation of trapped volatile compounds (Levi and initial water content lose a greater portion of soluble Karel, 1995). solids during dehydration (Ceballos et al., 2012). The primary drying temperature can be manipulated to Freeze-drying is the removal of water from a product control collapse. A high freeze-drying temperature nega- through sublimation (Rothmayr, 1975). Sublimation is the tively affects the humidity of the final product, which can conversion from ice directly to vapor without passing lead to structural loss or collapse. Collapsed products are through a liquid state. In water, sublimation occurs when tougher, are have less aroma, and have less rehydration the vapor pressure and the temperature of the ice’s capacity (Harnkarnsujarit and Charoenrein, 2011). surface are below the triple point [4.58 mm Hg (610 The structural collapse of fruit can be physically controlled, Pascal), 0°C] (Jennings, 2002). The process of freeze- by modifying the product structures before freeze-drying, drying has three essential steps: freezing, primary drying and can be chemically controlled, through the addition of and secondary drying. Approximately 90% of the water is compounds that modify the collapse temperature. Products removed from fruits in the first drying phase (Welti et al., with high sugar content, such as fruit juice, have low 2005). This article reviews the structural, physical, collapse temperatures (de Oliveira et al., 2010). Collapse nutraceutical and functional changes of freeze-dried fruit. can be decreased, by controlling variables in the process Advantages and limitations of the freeze-drying process such as the freezing speed and temperature (Ceballos et as well as strategies for improvement are discussed. al., 2012). Ayala et al. (2010) describe collapse (19.15% volume loss) during the osmotic dehydration of yellow pitahaya. Evidence of this collapse can be observed in STRUCTURAL CHANGES THAT CAN OCCUR IN THE the deformation of cell walls and diminished cell turgor. FREEZE-DRYING PROCESS Freeze-dried pitahaya, even without pre-treatment, did not collapse, losing only 2.6% of the original volume. The sought-after freeze-drying products are porous fruits Similarly, freeze-dried papaya did not present collapse that maintain their volume, can have fast and nearly (Marques et al., 2009). complete rehydration when water is added and do not Collapse is related to product porosity and rehydration. shrink (Duan et al., 2010). However, some freeze-dried When collapse is not visually obvious, collapse can be products undergo undesirable structural changes. evaluated through rehydration because structural collapse Microscopy can be used to study structural changes in is known to diminish rehydration capacity (Marques et al., freeze-dried fruits and to find a relationship to some 2009). physical properties (Yeom and Song, 2010). Table 1 Freeze-drying tropical fruits such as pineapple, guayaba, presents a variety of fruits, processing variables, physical mango and Barbados cherries does not produce signs of changes and the structural and nutraceutical effects of collapse (Marques et al., 2006). The same is true for freeze-drying. acerola pulp, which does not show visible signs of collapse (Marques et al., 2007). However, acerola and guayaba have a low rehydration capacity, which can be described Collapse as collapse. Mango, papaya and pineapple are readily rehydrated and are not thought to collapse in the freeze- Collapse and contraction are terms used to describe the drying process (Marques et al., 2009). de Oliveira et al. loss of structure. A collapsed product may show reduced (2010) describe the freeze-drying of peki, a Brazilian fruit. pore size and volumetric contraction (Khalloufi et al., 2010; The pulp was pretreated with sucrose and ethanol, which 444 Afr. J. Biotechnol.
Table 1. Process variables and physical, structural and nutraceutical changes in freeze-dried fruit.
Nutraceutical changes Fruit Processing variables Physical and structural changes Citation Fresh fruit Freeze-dried fruit Rapid freezing (-100°C). Liquid Minimal collapse. Acerola (Malpighia nitrogen. Marques et al. Low Tg (-32.1°C). Vitamin C 1021 mg/100 g Vitamin C 153.4 mg/100 g. glabra L.) Primary drying (-32.1°C). (2007) High rehydration capacity (10.1 kg/kg). Secondary drying (35°C)
Vitamin C was conserved. Firmness 0.56 Newton Rapid freezing (-80°C for 24 h). Vitamin C 50.7 mg/100 g Strawberry Color: L*57, a*24, b*26, changed to L*58 a*23 b*26. Phenolic acids and aromatic substances Yurdugül (2008) Freeze-drying time (24 h). Firmness 0.1 Newton were conserved. Anthocyanin was diminished
Freeze-dried fruit: Aw: 0.382 Freezing (-35°C). Porosity: 48.17 Yellow pitahaya Sublimation: 8 Pa vacuum Rehydratability: 1,982 Kg water/Kg mass Ayala et al. (Selenicereus pressure (2010). megalanthus) Freeze-dried fruit: Volume was preserved in sliced fruit. Drying from -35 to 35°C Aw: 0.364 Porosity: 84.52 Rehydratability: 2.614 Kg water/Kg dry mass
Sublimation: (0.0998 kPa, - Particulates smaller than 1.20 mm. Peki (Caryocar de Oliveira et al. 40°C), 72 h. Aw: 0.06 – 0.25 brasiliense Camb.) (2010). Secondary drying (35°C). Collapse.
TPC (181.71 mg/100 g) TPC (137.95 mg/100 g) Vitamin C (4.99 mg/100 g) Carambola Vitamin C (4.67 mg/100 g) Beta-carotene (30.79 Beta-carotene (25.94 mg/100 g) mg/100 g)
TPC (99.69 mg/100 g) TPC (76.57 mg/100 g) Freezing (-20°C), 24 h Vitamin C (8.36 mg/100 g) Shofian et al. Mango Vitamin C (8.34 mg/100 g) Vacuum drying (-50°C), 3 days. Beta-carotene (660.27 (2011) -Beta-carotene (487.34 mg/100 g) mg/100 g)
TPC (16.71 mg/100 g) TPC (14.97 mg/100 g) Vitamin C (2.24 mg/100 g) Melon Vitamin C (2.75 mg/100 g) Beta-carotene (508.18 Beta-carotene (523.26 mg/100 g) mg/100 g) Serna-Cock et al. 445
accelerates the freeze-drying process. This pretreatment Yellow pitahaya porosity is high (84.52%) compared to protected the structure from collapse. the porosity of fresh pitahaya (2.12%) and osmotically There are reports that freeze-dried apple and carrot dehydrated pitahaya (48.17%) (Ayala et al., 2010). also undergo collapse; however, compared with those dried Freeze-dried papaya is less porous than freeze-dried by hot air or microwave, these freeze-dried products lose guayaba and has a pore diameter less than 0.003 mm substantially less volume than their counterparts (Cui et (Hawlader et al., 2006). al., 2008).
PHYSICAL CHANGES TO FRUIT DURING THE Variation in porosity FREEZE-DRYING PROCESS
Using scanning electron microscopy (SEM), changes in a Color model system’s microstructure can be studied, and the pore size of freeze-dried fruit can be measured. Similarly, The conservation of color is considered an indication of SEM can be used to determine the size and shape of the quality in dried fruits given that non-enzymatic browning particulates in pulverized fruits (Yeom and Song, 2010). processes develop during the drying process (Ceballos et Porosity is defined as the relationship between fractional al., 2012). Freeze-dried fruits better maintain red and pore volume and the total food volume (Rahman et al., yellow colors than fruits dried using traditional methods
2005). Porosity affects the texture and quality of dry foods (Shishehgarha et al., 2002). However, kinetic color analyses and foods with moderate moisture levels. This charac- of freeze-dried fruit show some deterioration of reds and teristic is more important in freeze-dried foods than in yellows (Guiné and Barroca, 2012). microwaved or air-dried foods (Purnama et al., 2010). Strawberries that are freeze-dried at temperatures below The pores of dried products are composed of the small -50°C retain their color better than strawberries freeze- or large spaces formed by ice crystals during the freezing dried at temperatures above -50°C. This effect is due to a process. These spaces facilitate the diffusion of water higher concentration of solutes (reduction of water) and vapor during the drying process. The porosity of freeze- the effects of pH on anthocyanin (Shishehgarha et al., dried fruit depends on the freezing speed, which, when 2002). Fresh alpine strawberries maintained their color after slowed or augmented, can cause the ice to form smaller freeze-drying; color values for freeze-dried strawberries or larger pores, respectively (Voda et al., 2012). Slow were L* = 58, a* = 23, and b* = 26, while these values freezing leads to the formation of voluminous crystals and were and L* = 57, a* = 23 and b* = 26 for fresh straw- pores that facilitate dehydration and subsequent rehydra- berries (Yurdugül, 2008). tion (Jennings, 2002). Pore size has a small effect on Color variation in freeze-dried Granny Smith apples vapor flow (Pardo and Niranjan, 2006). was lower (3,537 ± 1,717) than the variation after convective Information about the characteristics of individual pores drying [5,279 ± 0.989], convective drying in a vacuum and structural characteristics of dried fruit can be used to (11,308 ± 1,729) and microwave drying (5,958 ± 0,580) design processes, determine product quality and (Valencia et al., 2011). estimate other properties such as thermic conductivity, Moßhammer et al. (2006) found that freeze-drying density, water diffusivity and characteristics related to the drastically affected the color of nopal, a change that can extraction of bioactive components (Rahman and be attributed to the formation of soluble melanoidins. Sablani, 2003; Madiouli et al., 2012). Pulverized nopal does not exhibit the same color change. Porosity in fresh and freeze-dried fruit can be Sliced carrots maintain their color throughout the measured by comparing the apparent (ρu) and true freeze-drying process. Sliced apples exhibit minor color density (ρr). Pore thickness can be measured using a variation (Cui et al., 2008). The loss of color in freeze- method proposed by Madiouli et al. (2012) that includes dried green peppers is minor compared to the color loss open and closed pores. Direct experimental methods do in air-dried peppers. However, comparing the color not permit measurement of the formation of closed pores. values L*, a* and b* of fresh peppers (37.22 -14.11 and Freeze-dried ginseng has a total porosity of 77.15 ± 22.52, respectively) and freeze-dried peppers (44.12 - 0.93%. Freeze-dried pineapple, cherry, guayaba, papaya 12.11 and 19.59, respectively), green color loss is noted, and mango all have porosity values between 84 and 93% due to the decomposition of chlorophyll (Guiné and (Marques et al., 2006). Barroca, 2012). Freeze-dried guanabana has medium-sized pores on Guiné and Barroca (2012) reported that freeze-dried the dry outer layer that are associated with rapid freezing squash had L*, a* and b* values of 77.70, 15.25 and (2.4 and 3.1°C/min), which causes the formation of 41.43, respectively, exhibiting color losses in red and smaller ice crystals. Medium-sized pores on the dry layer yellow compared to fresh squash (68.97, 18.21 and make sublimation more difficult and therefore increase 49.82). the moisture level of the freeze-dried product (Ceballos et Rapid freezing of guanabana (from 1.1 to 3.1°C/min) al., 2012). produced a more intense white value (L* = 85.10 to 88.92). 446 Afr. J. Biotechnol.
This higher color intensity can be explained by small mentioned above, porosity also influences the rehydra- pores, (an effect of rapid freezing), due to, the small tion capacity of freeze-dried fruits. The formation of large pores dispersing more light than large pores (an effect of water crystals facilitates large pore formation, which in slow freezing) (Ceballos et al., 2012). Freeze-dried turn facilitates rehydration (Jennings, 2002). papaya was more luminescent, and freeze-dried guayaba The quality of a freeze-dried product is marked by the maintained better color than the same fruits dehydrated speed and ease of reconstitution or rehydration. Rehydra- in a vacuum (Hawlader et al., 2006). tion capacity is a key characteristic of freeze-dried fruit and is dependent on the size, geometry, composition, water content and porosity of the fruit (Sanjuán et al., Glass transition 2001). Rehydratability is also affected by the method of rehydration utilized and the temperature, time and condi- The glass transition temperature (Tg) is a key physical tions of agitation (Arriola et al., 2006), as well as pre- parameter that explains the chemical and physical behavior treatment factors. Sliced yellow pitahaya, for example, of a food system. Freeze-dried foods must have stable has 24% less rehydration capacity when osmotically moisture levels and good packaging during storage. dehydrated prior to freeze-drying (Ayala et al., 2010). In Absorption of additional moisture can lead to a state of avocado, the speed and capacity of rehydration are amorphous disequilibrium, which brings with it a trans- independent of immersion temperature (Arriola et al., formation from a glass solid state to a plastic fluid state 2006). when the glass transition temperature is reached (Duan The rehydration capacities of freeze-dried carrots and et al., 2013). The glass transition temperature of dry solid apple chips (3.94 and 5.61, respectively) were similar to would be an important optimization parameter for the the values obtained by combined methods (microwave- freezing-drying process. This parameter can be used as freeze-drying) (3.95 and 5.22, respectively). Porosity was useful tool for the choice of the most appropiate materials similar in both of these foods (Cui et al., 2008). Freeze- to be freezing-dried (Ratti, 2001). dried Chilean guava has an external appearance similar Freeze-dried cubed apples were analyzed by differential to that of the fresh fruit, although the freeze-dried product scanning calorimetry (DSC). The Tg was below -30°C is gelatinous inside (Reyes et al., 2010). (Duan et al., 2013). The Tg of freeze-dried tomatoes was The rehydration capacity of freeze-dried acerola is high determined using DSC. The thermogram revealed the (10.1 kg/kg) when it is frozen with liquid nitrogen at existence of two similar but distinct Tg values, which 73.6°C/min, which does not cause cell rupture in the generated a water plasticization effect in hygroscopic samples (Marques et al., 2007). The rehydration capacity regions of the product (Tg= -50°C) (Telis and Sobral, of freeze-dried yellow pitahaya is high, reaching a 2002). Guizani et al. (2010) studied freeze-dried Deglet moisture content nearly equal to that of the fresh fruit Nour palm dates using DSC. The thermograms generated (3.393 kg water/kg m.s) (Ayala et al., 2010). showed a diminishing glass transition temperature (-13.8 to -48.7°C). However, the Tg rose with an increase of the solid content of the fruit. The Tg of the common mushroom CHANGES IN VOLATILE COMPOUND CONTENT IN (Agaricus bisporus) was as low as -77.9°C after freeze- FRUIT DURING THE PROCESS OF FREEZE-DRYING drying (Shi et al., 2012). Freeze-dried kiwi [Grosella espinosa China] had a Tg of -57.2°C for optimal freezing- Fruit aromas and flavors are transmitted by volatile concentration conditions (Wang et al., 2008). compounds, such as esters, alcohols, terpenes (derived from the metabolism of mevalonic acid), aldehydes, carbonyl compounds, acids, brass, phenols, hydrocarbons, FUNCTIONAL CHANGES DURING THE FREEZE- arylpropanoids (derived from the shikimic acid metabolic DRYING PROCESS pathway), sulfur compounds and glucosinolates (derived from the amino acid metabolic pathway), among others Rehydration capacity and water retention (Wang et al., 2007; De Torres et al., 2010). Volatile compound content changes as fruit ripens. Ripe cocona, Rehydration capacity (RC) is the ability to reabsorb water for example, has an increased concentration of esters relative to the water lost during dehydration, while water and alcohols, decreased concentration of carbonyl retention capacity (WRC) is defined as the ability to compounds and high concentrations of methyl salicylate absorb and retain water relative to mechanical force. and 훼-terpineol, as well as (Z)-3-hexenol, though in lesser These indicators are related to the structure, the tissue proportion. The concentrations of the aldehydes (Z)-3- and the capacity to retain absorbed water. Increases or hexenal, (Z)-2-hexenal and (E)-2-hexenal diminish as the decreases in these indicators can be attributed to the fruit ripens (Quijano and Pino, 2006). denaturation and/or aggregation of proteins due to heat, The volatile compounds in freeze-dried fruits are salt concentration, desorption of water and destruction of retained by entrapment in microregions of dry material. pectins in the cell membrane (Sanjuán et al., 2001). As Loss of volatile compounds is due to adsorption of water Serna-Cock et al. 447
by the dry product, which increases the permeability of (Rojano et al., 2011). The polyphenol content in fresh these dry regions and therefore, the loss of these com- Chilean guava is 1460 mg/100 g (dry mass), a value that pounds. Carbohydrate-carbohydrate bonds are replaced drops at least 40% after freeze-drying. Freezing speed by carbohydrate-water bonds. However, for the critical affects polyphenol content; rapid freezing led to a water content, Xc, microregions remain sealed and cease polyphenol content of 793 mg/100 g (dry mass), and slow to lose volatile compounds. Xc is defined as the water freezing produced 815 mg/100 g (dry mass) (Reyes et content at the transition point between the primary and al., 2010). secondary drying phases (Jennings, 2002). In watermelon, papaya, carambola, mango and melon, Wang et al. (2007) demonstrated that freeze-dried the levels of phenolic compounds decreased by 48.23, plantains maintain their ester content. Esters are the 39.73, 24.08, 23.19 and 10.41%, respectively, during the most important volatile compound in plantains, though freeze-drying process (Shofian et al., 2011). The phenolic unlike enols, which are lost, esters have little influence on compounds found in strawberries, esters, terpenoids, the aromatic profile of the plantains. Coffee that is freeze- carbonyl compounds and various alcohols and acids, do dried under conditions with slow freezing and rapid drying not change during the freeze-drying process (Yurdugül, retains aromatic compounds (Sagara et al., 2005). Grape 2008). peels in freeze-dried grapes retain terpenes, sesquiter- Reyes et al. (2011) demonstrated that freeze-drying penes norisoprenoids, aldehydes and esters, among diminished the phenolic compound content of blueberries other compounds, which ensure wine quality (De Torres from 832.9 mg/100 g to 769.2 mg/100 g (dry mass); et al., 2010). however, this loss was insignificant compared to other Analysis of apples by mass spectrometry and gas dehydration methods. Freeze-dried Chilean guava lost chromatography [GC/MS] showed that the volatile com- 33.3 and 67.5% of the original total antioxidants after pounds most common in apples-ethyl acetate, ethyl rapid and slow freezing, respectively (Reyes et al., 2010). butyrate and methyl anthranilate-were retained during the freeze-drying process (Krokida and Philippopoulos, 2006). Ascorbic acid The chalarina (white zapote), a Peruvian fruit, retained a larger proportion of volatile and thermosensitive Ascorbic acid content can decrease in high-temperature compounds, such as vitamin C, when dehydrated via treatments, with this compound degrading when oxidized freeze-drying (Castañeda et al., 2010). The majority of to dehydroascorbic acid and hydrolyzed to 2,3- phenolic acids and volatile compounds were conserved in diketogulonic acid. The latter can further polymerize to freeze-dried strawberries (Yurdugül, 2008). form other nutritionally inactive products (Chang et al., 2006). Some studies have indicated that freeze-drying allows the retention of ascorbic acid when the freeze- CHANGES IN THE NUTRACEUTICAL CONTENT OF drying is conducted at low temperatures, minimizing FREEZE-DRIED FRUIT deterioration of this water-soluble vitamin (Shofian et al., 2011). Total phenolic compounds (TPC) Chang et al. (2006) compared the ascorbic acid content of fresh, freeze-dried and hot-air dried tomatoes and Phenolic compounds are a large group of antioxidants found that freeze-drying reduces the loss (8.2%) that are present in nearly all plant-based foods. The compared to hot-air drying (56-61%). The losses of vitamin measured total phenolic compound content depends on C in the pulp of cherries, papaya, pineapple, mango and the type of fruit, ripeness, agricultural and climactic guava due to freeze-drying were 3.59, 24.2, 26.92 and factors, post-harvest storage conditions and method of 37%, respectively (Marques et al., 2006). Vitamin C loss anti-oxidant extraction (Shofian et al., 2011). was 10% in freeze-dried nopal and 50-55% in spray-dried Freeze-drying fruit can diminish the phenolic compound nopal (Moßhammer et al., 2006). content because ice crystals formed during freezing can Freeze-dried apples retain a larger proportion of vitamin cause cell wall rupture and the subsequent release of C (97.0%) than microwave-dried (89%), air dried (63.7) or oxidative and hydrolytic enzymes that degrade phenolic microwaved and freeze-dried (91.8%) apples (Cui et al., compounds (Chang et al., 2006). However, there are 2008). Chalarina (white zapote) lost 22.55% of its fresh fruits, such as tomatoes and blueberries that have ascorbic acid content when dried (29.75 mg/100 g) significantly higher levels of phenol compounds after (Castañeda et al., 2010). freeze-drying. This effect is due to the liberation of The original ascorbic acid content was preserved in phenolic compounds that had been trapped in the cell freeze-dried carambolo, mango, papaya, melon and water- wall (Reyes et al., 2011). melon. This study concluded that freeze-drying maximizes The antioxidant content of freeze-dried açaí was 255.1 ascorbic acid retention and that freezing had a minimal mg/100 g of freeze-dried fruit for cyanidin-3-glucoside degradation effect on vitamin C (Shofian et al., 2011). and 25 mg/100 g of freeze-dried fruit for ferulic acid The ascorbic acid content of freeze-dried guanabana 448 Afr. J. Biotechnol.
declined from 81 mg/100 g to 40.57 mg/100 g under rapid rehydration with water or other adequate solvents, and freezing conditions (3.1°C/min), and from 81 mg/100 g to greater porosity is directly related to a greater rehydration 53.76 mg/100 g under slow freezing conditions (1.1°C/min) capacity and is an indicator of product quality (Ayala et (Ceballos et al., 2012). al., 2010). The reduction of volume during freeze-drying The acerola is a Brazilian fruit with a high vitamin C is minimal (Shishehgarha et al., 2002). The temperatures content that varies according to maturity. The green fruit reached in the freeze-drying process are outside of the has 179.7 mg/100 g, the red-yellow fruit has 176.4 mg/100 range of temperatures that induce chemical changes in g, and the red fruit has 149 mg/100 g. The vitamin C fruit. There is minimal loss of volatile compounds at freeze- content declines significantly during the freeze-drying drying temperatures (Yurdugül, 2008). process, to values as low as 55.1 and 72.1 mg/100 g in Freeze-dried fruits have a very low moisture content, green and red fruits, respectively. In the yellow-red fruit, which allows them to be stored for long periods; freeze- vitamin C loss was 13% (Marques et al., 2007). Freeze- dried goods are highly stable products (Shishehgarha et dried blueberries lose 60.43% of their initial vitamin C al., 2002). Freeze-dried fruits can be used as antioxidants content (151.9 mg/100 g dry mass) during drying (Reyes and colorants in natural foods (Yurdugül, 2008). Freeze- et al., 2011). drying allows the production of powdered fruit that can be used as base materials in the food and pharmaceutical industries. Freeze-drying is an ideal technology for con- β-Carotene content sumers without access to fresh produce, such as astronauts (Aguilera and Stanley, 1999). β-Carotenes are compounds found in the lipid membranes Among the limitations of freeze-drying is the cost. or vacuoles of leafy green vegetables and yellow to orange Freeze-drying requires long processing times and con- fruits and vegetables. The degradation of phenolic com- sumes large amounts of energy to freeze the fruit, pounds can lead to the degradation of β-carotenes sublimate the ice, dry the product and condense the (Shofian et al., 2011). Freeze-dried carrots retain 95.4% vapor while maintaining vacuum conditions (Sadikoglu et of their initial β-carotene; this value is a larger proportion al., 2006; Benlloch et al., 2012). The vacuum pump and than that obtained through other dehydration methods, sublimation account for 26 to 45% of the total energy including microwave and hot air dehydration (Cui et al., consumed in the process (Menlik et al., 2010). Equally 2008). In the process of freeze-drying watermelon, mango, important to energy costs are general operating conditions, carambola and papaya, 43.1, 26.19, 15.75 and 8.16% of such as the chamber pressure and heating and freezing their initial β-carotene was lost, respectively (Shofian et speeds (Arriola et al., 2006). Structural collapse is a al., 2011). limiting factor for some fruits for which freeze-drying causes a wrinkled and compromised structure that cannot be reversed (Lee et al., 2006). ADVANTAGES AND LIMITATIONS OF FREEZE- A final limitation, closely linked to freeze-dried product DRYING FRUIT stability, is hygroscopicity. Freeze-dried products are highly hygroscopic, and further extension of the shelf life can be As has been demonstrated in the studies described, the gained with the addition of solutes such as Arabic gum, tricalcium phosphate and maltodextrin that can increase effects of freeze-drying on physical and chemical properties vary in accordance with factors intrinsic to the fruit and product stability and act as a barrier to water absorption extrinsic properties inherent to the process. Regardless, (Kaushik and Roos, 2007; de Oliveira et al., 2010; Fabra general advantages and limitations can be identified. et al., 2011; Benlloch et al., 2012; Ceballos et al., 2012; Among the advantages of freeze-drying are enhanced Mosquera et al., 2012). stability, rapid and easy solubility, prolonged shelf life, In an effort to reduce energy costs when maintaining a and increased availability and permanent access to fruit. high-quality product, investigators have sought to combine Freeze-drying guarantees high-quality products with technologies such as microwave (Cui et al., 2008), hot air superior sensory and nutritional properties. (Giraldo et al., 2010) and osmotic dehydration (Ayala et As with traditional dehydration methods, freeze-drying al., 2010) to eliminate a portion of the water before is not completely reversible by rehydration; however, freeze-drying (Zhang et al., 2006). Table 2 outlines the damage to the hydrophilic groups responsible for the investigations of combined dehydration technologies as a interaction with water is lower than that of other methods. cost-saving measure.
Freeze-dried products rapidly rehydrate and regain water content and organoleptic properties similar to those of the CONCLUSIONS fresh product (Jiang et al., 2010). In comparison to products obtained through other The structural, physical, functional and nutraceutical effects dehydration methods, freeze-dried fruits have a more of freeze-drying produce are dependent on intrinsic porous structure as well as superior flavor and aroma factors that are inherent to the fruit and to extrinsic retention (Ceballos et al., 2012). High porosity facilitates factors that are inherent to the process. Freeze-drying Serna-Cock et al. 449
Table 2. Combining methods to reduce freeze-drying costs.
Fruit Pre-treatment Results Citation 4.87% of the total Vitamin C was lost during osmotic dehydration. Chalarina (Casimiroa Osmotic dehydration Castañeda et al. When osmotic dehydration was combined with freeze-drying, the Vitamin C loss edulis) 63.57°Brix, 679.06 mmHg and 45.3°C. (2010) was 49.63%.
Microwave dehydration in a vacuum MWV carrots retained 94.7% of their original carotenoids Carrot Cui et al. (2008) (MWV) MWV+freeze-dried carrots retained 94.9% of their original carotenoids.
MWV apples retained 89% of their original Vitamin C Apple MWV dehydration Cui et al. (2008) MWV+freeze-dried apples retained 91.8% of their original Vitamin C.
technology offers advantages and has limitations. Arriola E, García T, Guatemala G, Nungaray J, González O, transition. Food Bioproducts Process. 91(4):534-538. Ruíz, J. (2006). The behavior of freeze-dried hass avocado Duan X, Zhang M, Mujumdar A, Wang R (2010). Trends in Freeze-drying is an ideal method for heat-sensitive during the rehydration process. Revista mexicana de microwave-assisted freeze-drying of foods. Drying Technol. fruits that require special care during processing. ingeniería química 5(1):51-56. 28(4):444-453. In many fruits, properties such as shape, dimension, Ayala A, Giraldo C, Serna L (2010). Kinetics of osmotic Fabra M, Márquez E, Castro D, Chiralt A (2011). Effect of appearance, flavor, color, texture and nutraceutical dehydration of yellow pitahaya fruit (Selenicereus maltodextrins in the water-content–water activity–glass megalanthus). Revista Interciencia 35(7):539-554. transition relationships of noni (Morinda citrifolia L.) pulp ingredients are retained after freeze-drying, adding Ayala A, Serna L, Mosquera E (2010). Freeze-drying in yellow powder. J. Food Eng. 103(1):47-51. value of approximately 120%. The technology is pitahaya (Selenicereus megalanthus). Vitae 17(2):121-27. Giraldo, A, Arévalo, A, Silva, A, Silva, P, Valdes, J, Pavlak, M, equally valuable when a high rehydration capacity Benlloch M, Moraga G, del Mar M, Martínez N (2012). (2010). Kinetic drying experimental data and mathematical is required, as is the case for powdered freeze- Combined Drying Technologies for High-Quality Kiwifruit model for cupuaçu pulp (Theobroma grandiflora) slices. dried fruit. Limitations to the application of this Powder Production. Food bioprocess Technol. 6(12):3544- Food Sci. Technol. (Campinas). 30(1):179-82. 53. Guiné R, Barroca M (2012). Effect of drying treatments on technology include the prolonged processing time, Castañeda J, Arteaga H, Siche R, Rodriguez G (2010). texture and color of vegetables (pumpkin and green high-energy costs, high product hygroscopicity Comparative study of the loss of vitamin C in chalarina pepper). Food Bioproducts Process. 90(1):58-63. and undesirable physical changes such as collapse. (Casimiroa edulis) by four methods of dehydration. Scientia Guizani N, Al-Saidi G, Rahman M, Bornaz S, Al-Alawi A Investigators have sought to lower freeze-drying Agropecuaria 1:75-80. (2010). State diagram of dates: Glass transition, freezing Ceballos A, Giraldo G, Orrego C (2012). Effect of freezing rate curve and maximal-freeze-concentration condition. J. Food costs through the use of pretreatments and the on quality parameters of freeze-dried soursop fruit pulp. J. Eng. 99(1):92-97. combination of freeze-drying with other dehydration Food Eng. 111(2):360-365. Harnkarnsujarit N, Charoenrein S (2011). Influence of technologies, such as hot air, microwave and Chang C, Lin H, Chang C, Liu Y (2006). Comparisons on the collapsed structure on stability of β-carotene in freeze-dried osmotic dehydration. antioxidant properties of fresh, freeze-dried and hot-air-dried mangoes. Food Res. Int. 44(1):3188-94. tomatoes. J. Food Eng. 77(3):478-485. Hawlader M, Perera C, Tian M, Yeo K (2006). Drying of guava
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Vol. 14(6), pp. 451-458, 11 February, 2015 DOI: 10.5897/AJB2014.14281 Article Number: FC1101C50321 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Examination of genetic diversity in common bean (Phaseolus vulgaris L.) using random amplified polymorphic DNA (RAPD) markers
Asifa Bukhari, M. A. Bhat, Mushtaq Ahmad* and Noorul Saleem
Division of Genetics and Pant Breeding Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir Shalimar campus, Srinagar -191 121, India.
Received 28 October, 2014; Accepted 2 February, 2015
To study the pattern of genetic diversity in 45 genotypes of common bean, 19 RAPD primers were used. Of 253 bands produced, 236 bands (94.22%) were polymorphic in which maximum number (20 polymorphic bands) were observed in the profiles of the primer OPB-07. Highest PIC value (0.79) was observed for the primers OPG-14 and OPE-1, whereas lowest PIC value (0.34) was recorded for the primer OPA-11 with an average PIC value of 0.54. Similarly, highest effective multiplex ratio value (0.11) was scored for the primers OPA-04 and OPG-6 and the lowest value (0.05) was recorded for the primer OPB-07. Primer OPG-14 and OPE-1 exhibited the highest marker index value (0.078) and the lowest value (0.024) was recorded for OPA-11. Pair-wise genetic similarity coefficients of genotypes varied from 0.56 to 0.92 with an average of 0.70 amongst 45 genotypes. The maximum similarity coefficient (0.92) was noticed between G-9 and G-8. Cluster analysis separated 45 genotypes into seven major clusters which were further grouped into various sub-clusters. Among these seven clusters, the maximum number of genotypes (12) were recorded in cluster I, while cluster VII formed a mono-genotypic cluster.
Key words: Genetic diversity, polymorphism, cluster analysis, random amplified polymorphic DNA markers (RAPD), common bean.
INTRODUCTION
The common bean (Phaseolus vulgaris L.) belongs to the and complex carbohydrates. Common bean is produced family Leguminosae, subfamily Papilionoideae, and tribe in a wide range of climatic conditions and is also cultivated Phaseoleae. It is considered the most important grain as a source of nitrogen in crop rotations (Hillocks et al., legume for human consumption in the world. Beans are 2006). In fact, it is a good genetic model because of its considered an excellent source of proteins and minerals, small genome and diploid in nature with 2n = 2x =22 for example iron, magnesium, manganese, and to a (Santalla et al., 2010). lesser extent, zinc, copper, calcium, and group B Analysis of genetic relationships in crop species is an vitamins. In addition, beans have high amounts of fiber important component of crop improvement programs, as
*Corresponding author. E-mail: [email protected].
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License
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Table 1. Base sequence of RAPD primers used for DNA fingerprinting.
S/N Primer name 5' sequence 3’ 01 OPA-01 5’-CAGGCCCTTC-3 02 OPA-03 5’-AGTCAGCCAC-3’ 03. OPA-04 5’-AATCGGGCTG-3 04. OPA-05 5’-AGGGGTCTTG-3’ 05 OPA-07 5’-GAAACGGGTG-3’ 06 OPA-09 5’-GGGTAACGCC-3’ 07 OPA-10 5’-GTGATCGCAG-3’ 08 OPA-11 5’-CAATCGCCGT-3’ 09 OPA–17 5’-CACCGCTTGT-3 10 OPB-07 5'-GGTGACGCAG-3' 11 OPB-10 5’-CTGCTGGGAC-3’ 12 OPC-02 5’-GTGAGGCGTC-3’ 13 OPC-08 5’-TGGACCGGTG-3’ 14 OPD-07 5’-TTGGCACGGG-3’ 15 OPG–06 5’-GTGCCTAACC-3’ 16 OPG–14 5’GGATGAGACC-3’ 17 OPE-01 5’CCCAAGGTCC-3’ 18 OPE-06 5’AAGACCCCTC-3’ 19 OPE-12 5’TTATCGCCCC-3’
it serves to provide information about genetic diversity, Technology Park, Sher-e-Kashmir University of Agricultural and is a platform for stratified sampling of breeding Sciences and Technology of Kashmir, Habak during Kharif 2010- populations. Traditionally, diversity is assessed by 2011 and 2011-2012 and molecular analysis was carried out in Molecular Laboratory, Centre for Plant Biotechnology, Sher-e- measuring variation in phenotypic traits such as flower Kashmir University of Agricultural Sciences and Technology of color, growth habit or quantitative agronomic traits like Kashmir. The experimental material comprised of forty five (45) yield potential, stress tolerance, etc., which are of direct genotypes of common bean (Phaseolus vulgaris L.) collected from interest to users. This approach has certain limitations: different pulse growing areas of Jammu and Kashmir. The field genetic information provided by morphological characters experiment was laid out in a randomized block design with three replications. The experimental materials were provided the cropping is often limited and expression of quantitative traits is geometry of 30 cm distance between the rows and 10 cm within the subjected to strong environmental influence. row. The experimental fields were well prepared and standard With the development of the polymerase chain reaction recommended package of practices were followed to raise a good (PCR) in particular, numerous molecular technologies crop. such as two random amplified polymorphic DNA markers
(RAPD) and ISSR techniques have been developed DNA extraction and PCR (Zietkiewicz et al., 1994). RAPD markers offered an opportunity to reduce the time and expense taken in Total genomic DNA from 20 gof fresh young leaf tissue, collected RFLP for genetic diversity and molecular mapping. RAPD from five random plants per accession, was extracted following the analysis can be used to characterize DNA variation cetyl trimethyl ammonium bromide (CTAB) method as described by patterns within species and among closely related taxa. Saghai-Maroof et al. (1984). Nineteen 10-mer oligonucleotides Within grain legume crops alone, RAPD markers have primers (Operon Technologies Inc., CA. USA) were used for characterization of genotypes (Table 1). In vitro amplification using been widely used for the identification of genetic relation- polymerase chain reaction (PCR) was performed in a 96 well ships a) among cultivars, b) among wild forms and c) BioRad Mode-II T-gradient thermoblock using 50 ng of genomic between wild forms and cultivars (Xu et al., 2000). DNA of each genotype in a final volume of 20 l per reaction. Keeping this in view, the present investigation was To 20 l of the amplified product, 3.33 l of 6x loading dye was undertaken with an objective to assess genetic diversity added so as to make the final concentration of the loading dye in in common bean genotypes using RAPD markers. the reaction samples to 1x. The PCR products were resolved on 1.5% super fine resolution agarose gel. The gel was prepared in 0.5x TBE buffer or 1x TAE. Ethidium bromide was added at MATERIALS AND METHODS concentration of 0.5 mg/l. The gel was run at 10 V, visualized under UV light and photographed (Figure 2a and 2b) using ultra The research was carried out at the Experimental Farm of Urban cam digital imaging (A6 rc canon camera).
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Table 2. Percentage of polymorphism and polymorphic information content (PIC) obtained by PCR amplification of DNA in common bean (Phaseolus vulgaris L.) genotypes using RAPD primers.
Total number Number of Percentage of Polymorphic Effective Marker Primers of bands polymorphic bands polymorphism information content multiplex ratio index OPA-01 15 13 86.67 0.64 0.07 0.042346 OPA-03 17 16 94.12 0.46 0.06 0.027331 OPA-04 9 9 100.00 0.56 0.11 0.062096 OPA-05 17 14 82.35 0.63 0.06 0.051176 OPA-07 17 15 88.24 0.56 0.06 0.051176 OPA-09 14 14 100.00 0.44 0.07 0.031150 OPA-10 17 16 94.12 0.74 0.06 0.051176 OPA-11 14 14 100.00 0.34 0.07 0.024453 OPA–17 18 14 77.78 0.58 0.06 0.032426 OPB-07 21 20 95.24 0.61 0.05 0.029123 OPB-10 12 11 91.67 0.44 0.08 0.036767 OPC-02 10 10 100.00 0.36 0.10 0.035773 OPC-08 11 11 100.00 0.51 0.09 0.046662 OPD-07 12 12 100.00 0.37 0.08 0.030788 OPG–06 9 9 100.00 0.49 0.11 0.054940 OPG–14 10 10 100.00 0.79 0.10 0.078588 OPE-01 10 10 100.00 0.79 0.10 0.078588 OPE-06 10 10 100.00 0.42 0.10 0.042320 OPE-12 10 8 80.00 0.53 0.10 0.053419 Total 253 236 - - - - Mean 13.32 12.42 94.22 0.54 0.08 0.045279
Clearly resolved, unambiguous bands were scored visually for their value based on Nei and Li (1979) Sorensen-Dice coefficient of presence or absence. The scores were obtained in the form of a similarity (Dij) was calculated as: matrix with ‘1’ and ‘0’, which indicate the presence and absence of bands in every accession. The binary data scored was used to 2a calculate the similarity coefficient between the accessions and Dij = construct a dendrogram. (2a + b + c)
Polymorphism information content Where ‘a’ represents matched fragments ‘b’ and ‘c’ are unmatched fragments. The similarity matrix was then generated and Polymorphism information content (PIC) or expected heterozygosity dendrogram was constructed using unweighted pair group method scores for each RAPD marker was calculated based on the formula: using arithmetic averages (UPGMA) available in NTSYS.
n 2 RESULTS AND DISCUSSION PIC = 1-Σ (Pij)
i =1 In the present investigation, a total of 253 bands were Where, Pi is the allele frequency for the jth allele in ith primer and detected (Table 2). The number of bands detected per summation extended over ‘n’ patterns (Nei, 1973). primer ranged from 9 to 21 with 13.32 per primer. The highest number of scorable bands (21) was recorded for
Cluster analysis primer OPB-07 which was closely followed by OPA-17 with 18 bands and OPA-03, OPA-05, OPA-07 and OPA- Cluster analysis based on molecular data was conducted using 10 with 17 bands each (Table 2). This value is relatively computer software Numerical Taxonomic and Multivariate Analysis high in comparison with the other studies performed System (NTSYS-PC) version 2.02e (Rohlf, 1997). Data from all the using RAPD markers. Sadeghi and Cheghamirza (2012) 19 primers were used to estimate the similarity based on the number of shared amplified bands. Similarity was estimated using reported that among 33 primers, maximum number of SIMQUAL function of NTSYS, which computes a variety of similarity clear bands was 20 bands for primer OPE02. Muthusamy coefficient for quantitative data (nominal data). Similarity matrix et al. (2008) also reported lower number of clear bands
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G-1 G-14 G-4 G-20 G-10 G-11 Cluster I G-33 G-34 G-35 G-7 G-8 G-9 G-2 G-3 G-17 G-18 G-19 Cluster II G-24 G-26 G-25 G-27 G-5 G-6 G-45 G-28 G-29 Cluster III G-30 G-31 G-32 G-12 G-13 G-15 Cluster IV G-16 G-22 G-23 G-36 G-44 G-40 Cluster V G-41 G-42 G-43 G-21 G-38 Cluster VI G-39 G-37 Cluster VI I 0.14 0.26 0.38 0.49 0.61 0.73 Coefficient
Figure 1. UPGMA dendrogram depicting genetic relationship among 45 genotypes of common bean (Phaseolus vulgaris L.) based on RAPD data using Dice similarity coefficient.
as compared to present findings. The possible reason for noticed that maximum EMR (0.11) was recorded for the this could be the utilization of primers with 60 to 70% GC markers OPA-04 and OPG-6; which were closely content in our experiments. An increase in the number of followed by OPA-4, OPG-14, OPE-1, OPE-6 and OPE-12 amplified bands was observed with increase in primer GC with EMR of 0.10 (Plate 2a and b), while the minimum content, and its reason could be the higher stability of G- (0.05) EMR was recorded for OP-07 (Table 2). Similarly, C complementation with respect to A-T pairing (Fukuoka the marker index for 19 primers was in the range of 0.024 et al., 1992). The studies of Szilagyi et al. (2011) and (OPA-11) to 0.078 (OPG-14 and OPE-1) with mean of Barelli et al. (2011) in common beans also reported less 0.045. polymorphism by RAPD markers as compared to our Pair-wise genetic similarity coefficients of genotypes study. These high values could be due to nature of RAPD varied from 0.56 to 0.92 with average of 0.70 between 45 markers. The maximum number of scorable bands 21 genotypes. The maximum similarity coefficient (0.92) was was obtained from primers (OPB-07), followed by 18 noticed between G-9, G-8, G-13 and G-12 which was bands from OPA-17. followed by G-34, G-33 (0.89), G-26 and G-24 (0.88); The polymorphic information content (PIC) was while minimum similarity coefficient was noticed between calculated for all 19 RAPD markers and are presented in G-6 and G-1 (0.56). The difference at molecular level can Table 2. The PIC values ranged from 0.34 (OPA-11) to be explained by the long span of cultivation in the areas 0.79 (OPG-14 and OPE-1) with mean of 0.54. The higher by different soil types, climatic conditions and cultivation the PIC value, the more informative the RAPD marker is. practices. Hence, the primers OPG-14 and OPE-1 were found to be Cluster analysis of fingerprints generated by means of highly informative, while OPA-11 was least informative. RAPD markers resulted in a dendrogram (Figure 1) that With regard to the effective multiplex ratio (EMR), it was grouped bean genotypes into seven clusters using 0.16
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Table 3. Composition of the clusters of common bean (Phaseolus vulgaris L.) genotypes as observed in the UPGMA dendrogram.
Cluster Number of genotypes Name of genotypes I Ia 2 G-1, G-14 Ib 2 G-4, G-1420 Ic 2 G-10, G-141 Id 3 G-33, G-34, G-35 Ie 3 G-7, G-8, G-9
II IIa 2 G-2, G-3 IIb 3 G-17, G-18, G-19 IIc 4 G-24, G-25, G-26, G-27
III IIIa 3 G-5, G-6, G-45 IIIb 3 G-28, G-29, G-30 IIIc 2 G-31, G-32
IV IVa 2 G-12, G-13 IVb 2 G-15, G-16 IVc 2 G-22, G-23
V Va 2 G-36, G-44 Vb 4 G-40, G-41, G-42, G-43
VI VIa 1 G-21 VIb 2 G-38, G-30
VII 1 G-37
similarity coefficients as a cutoff point. Among these difficulty in performing a large number of crosses in self- seven clusters, cluster I included the maximum number of pollinating species suggest the need to cross genotypes genotypes (12) and also showed maximum number of that present great genetic variability (Broughton et al., sub-clusters (5); while cluster VI was mono-genotypic. 2003). As recommended by Cruz et al. (2004) the joint Other clusters included between 3 and 9 genotypes use of clustering methods and graph dispersion has been (Table 3). The present findings are in agreement with the best suitable alternative in genetic diversity studies. those of Palomino et al. (2005) who studied genetic Although the genotypes used in this study were very diversity of common bean cultivars of the close genetically, it was possible to differentiate them by commercialCarioca group using RAPD markers. They using RAPD molecular marker technique. The results reported that the RAPD markers were efficient in have been satisfactory because even with reduced differentiating and clustering the genotypes and showed variability, different groups were always formed, showing the wide genetic variability within the group of Carioca the efficiency of this technique. Information about genetic common bean cultivars. Tiwari et al. (2005) investigated diversity is essential for breeding analysis and genetic variability in common beans collected in the germplasm conservation. In these cases, molecular Central Himalaya region using RAPD markers and markers may be used as an efficient instrument for obtained 12 groups with different similarity levels. detecting similarity/divergence and identifying accessions The close genetic relationship among common bean among common bean cultivars (Alzate-Marin et al., genotypes from Carioca commercial group and the 2003).
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OPA-01 OPA-03
OPA-10 OPA-11
Figure 2a. Images of agarose gels stained by ethidium bromide showing RAPD markers amplified in 45 common bean (Phaseolus vulgaris L.) genotypes. M = Ladder; C = Control Sr. No. 1-45 are the 45 genotypes of common bean.
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OPA-17 OPG-06
OPG-14 OPE-12
Figure 2b. Images of agarose gels stained by ethidium bromide showing RAPD markers amplified in 45 common bean (Phaseolus vulgaris L.) genotypes. M = Ladder; C = Control Sr. No. 1-45 are the 45 genotypes of common bean.
458 Afr. J. Biotechnol.
Breeding strategies need to exploit the existing variation Muthusamy S, Kanagarajan S, Ponnusamy S (2008). Efficiency of within and between wild beans and landraces. Hybri- RAPD and ISSR markers system in accessing genetic variation of rice bean (Vigna umbellata) landraces. Electr. J. Biotechnol. dization programme can be initiated between the 11(3):32-45. identified diverse genotypes in order to create variation Nei M, Li W (1979). Mathematical model for studying genetic variation in and for incorporation of the desire trait. The molecular terms of restriction endonucleases, Proceedings of the National markers, especially SCAR can be utilized for transfer of Academic Science of United States of America. 76:5256-5273. Nei M (1973). Analysis of gene diversity in subdivided populations. the desired trait in short duration. These bean germplasm Proceedings of the National Academic Science of United States of lines when crossed can help to broaden the genetic base America. 70: 3321-3323. of commercial beans to develop high yielding cultivars. Palomino EC, Mori ES, Zimback L, Tambarussi EV, Moraes CB (2005). Genetic diversity of common bean genotypes of Carioca commercial group using RAPD markers. Crop Breed. Appl. Biotechnol. 5(1):80- 85. Conflict of interests Rohlf FJ (1997). NTSYS-PC (Numerical Taxonomy and multivariate Analysis System) ver 2.02e. Applied Biostatic, New York. The authors did not declare any conflict of interest. Sadeghi A, Cheghamirza K (2012). Efficiency of RAPD and ISSR marker systems for studying genetic diversity in common bean (Phaseolus vulgaris L.) cultivars. Ann. Biol. Res. 3(7):3267-3273. Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984). REFERENCES Ribosomal DNA spacer length polymorphism in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Alzate-Marin AL, Costa MR, Sartorato A, Del Peloso MJ, Barros EG, Natl. Acad. Sci. USA. 83:1757-1761. Moreira MA (2003). Genetic variability and pedigree analysis of Santalla M, De Ron AM, De La Fuente M (2010). Integration of genome Brazilian common bean elite genotypes. Sci. Agric. 60:283-290. and phenotype scanning gives evidence of genetic structure in Barelli MAA, Poletine JP, Thomazella C, Filho PSV, Souto E, Pacheco Mesoamerican common bean (Phaseolus vulgaris L.) land races from CMNA, Neves LG, Elias JCF and Vidigal MCG (2011). Evaluation of the South-west of Europe. Theor. Appl. Genet. 120(8):1635-1651. genetic divergence among traditional accessions of common bean by Szilagyi L, Tayyar S, Ciuca M (2011). Evaluation of genetic diversity in using RAPD molecular markers. J. Food Agric. Environ. 9(2):195- common bean (Phaseolus vulgaris L.) using RAPD markers and 199. morpho-agronomic traits. Romanian Biotechnol. Lett. 16(1):98-105. Broughton WJ, Hernandez G, Blair M, Beebe S, Gepts P, Vanderleyden Tiwari M, Singh NK, Rathore M, Kumar N (2005). RAPD markers in the J (2003). Bean (Phaseolus spp.); model food legumes. Plant and analysis of genetic diversity among common bean germplasm from Soil. 252:55-128. Central Himalaya. Genet. Resour. Crop Evol. 52:315-324. Cruz CD, Regazzi AJ, Carneiro PCS (20040. Biometrical Models Xu RQ, Tomooka N, Vaughan DA, Doi K (2000). The Vigna angulars Applied to Genetic Breeding. UFV, Vicosa. complex: genetic variation and relationships revealed by RAPD Fukuoka S, Hosaka K, Kamijima O (1992). Use of random amplified analysis and their implications for in situ conservation and polymorphic DNA’s (RAPD’s) for identification of rice accessions. domestication. Genet. Resour. Crop Evol. 47:123-134. Japn. J. Genet. 67:243-252. Zietkiewicz E, Rafalski A, Labuda D (1994). Genome finger printing by Hillocks RJ, Madata CS, Chirwa R, Minja EM, Msolla S (2006). simple sequence repeat (SSR): anchored PCR amplification. Phaseolus bean improvement in Tanzania, 1959-2005. Euphytica. Genomics. 20:176-183. 150:215-231.
Vol. 14(6), pp. 459-465, 11 February, 2015 DOI: 10.5897/AJB2014.14164 Article Number: 64E58CE50327 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Feedstuffs potential of harvest by-products from two oleaginous cucurbits
A. I. Touré1,2, A. L. Loukou3, K.K. Koffi1, I. Mouaragadja2, G. L. Bohoua3, B. Mbatchi2 and B. I. A. Zoro1*
1Division of Plant production, Crop Husbandry and Breeding Unit; University Nangui Abrogoua, P. O. Box 801 Abidjan 02, Cote d’Ivoire. 2Agrobiology Unit, Sciences and Techniques University of Masuku, Franceville, P. O. Box 901, Gabon. 3Division of Food Security, University Nangui Abrogoua, P. O. Box 801 Abidjan 02, Cote d’Ivoire.
Received 8 September, 2014; Accepted 2 February, 2015
Among the generated crop residues and by-products in tropical agriculture, those of cucurbits represent great opportunities for animal nutrition. Nutritive profile of harvest by-products (dried leaves, fermented fruits, non fermented fruits and seeds shells) of two oleaginous cucurbits (Citrullus lanatus and Lagenaria siceraria) were investigated in order to explore their potential use as feedstuffs. The moisture, ash, and crude fibres contents were 4.81 to 12.87, 9.93 to 18.29, and 2.18 to 16.35%, respectively. Shells of L. siceraria seeds yielded the highest carbohydrate content (84.80 ± 2.78 %) while the highest calorific value (380.92 ± 11.40 kcal/100 g) was obtained in C. lanatus bebu. The contents of threonine (Thr), lysine (Lys) and methionine (Met) in dried leaves of C. lanatus bebu were 4.16, 6.86 and 6.89 g/100 g proteins, respectively. The content of methionine (Met) was 5.81 g/100 g proteins in fermented fruits of C. lanatus (wlêwlê). The harvest by-products analyzed in this study contained remarkably high amounts of potassium (671.78 – 4738.79 mg/100 g) and calcium (342.08 – 2963.95 mg/100 g) with highest value (4738.79 ± 230.10; 2963.95 ± 135.74 mg/100 g) for non-fermented fruits of L. siceraria and dried leaves of C. lanatus wlêwlê, respectively. The analyzed plants parts were also notable sources of magnesium, ranging from 221.45 ± 1.96 mg/100 g (non-fermented fruits of L. siceraria) to 872.10 ± 48.49 mg/100 g (dried leaves of C. lanatus wlêwlê). All these results suggest that the studied by-products could be used as valuable feedstuffs.
Key words: Harvest by-products, nutritive value, cucurbits, feedstuffs.
INTRODUCTION
Shortages of feed resources often impose major constraints derable quantities of by-products and crop residues on the development of animal production in tropical zones generated every year and tending to accumulate can be (Aregheore, 2000). In most developing countries, consi- utilized as feedstuffs (Ensminger et al., 1990). A by-product
*Corresponding author. E-mail :[email protected] . Tel: +225 07 39 02 31.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 460 Afr. J. Biotechnol.
feedstuff is a product that has value as an animal feed are rich in minerals (calcium, potassium, phosphorus, and is obtained during the harvesting or processing of a iron, magnesium and zinc), proteins (36%) and fats commodity in which human food or fibre is derived (50%) (Achu et al., 2005; Loukou et al., 2011). In addition, (Fadel, 1999). oils extracted from cucurbit seeds contain relatively high Among the generated crop residues and by-products in content of essential fatty acid, particularly linoleic acid tropical agriculture, those of cucurbits have a great and this property made them recommended for healthy economical importance (Dupriez and De Leener, 1987; human nutrition (Achu et al., 2005; Anhwange et al., Pitrat et al., 1999). Cucurbits belonging to the family 2010). Cucurbitaceae have common features: large leaves, Data from agro-ecological, cultural, genetic, and nutria- creeping or climbing stems with simple or branched tional investigations (Pitrat et al., 1999; Yaniv et al., 1999; tendrils, and fleshy fruits containing numerous seeds Besognin, 2002; Zoro Bi et al., 2006; Dane and Liu, 2007; (Ajuru and Okoli, 2013). Considering their consumption Fuller et al., 2010; Loukou et al., 2011; Sarao et al., mode, two major groups are distinguished: those with 2014) suggest that the oleaginous forms of C. lanatus edible flesh (watermelon, melon, squash, etc.) and those and L. siceraria have a potential to play crussial roles in with oleaginous seeds (bitter cucumber, African melon, the improvement of food security in tropics, particularly pumpkin, etc.) which are consumed as soup thickeners or though helping the poor for subsistence and income, cakes (Zoro Bi et al., 2003). Cucurbits cultivated for seed reducing risks of over-reliance on major crops, increasing consumption are well adapted to extremely divergent agriculture sustainability by mean of reduction in inputs, agro-ecosystems and various cropping systems and contributing to food quality (Frison et al., 2011; characterized by minimal inputs (Yaniv et al., 1999; Mayes et al., 2012). Despite these potentialities, these Besognin, 2002). cucurbits are somehow neglected and classified as minor Citrullus lanatus (Thunberg) Matsumura & Nakai, and crops. Furthermore, there is a lack of scientific data Lagenaria siceraria (Molina) Standley are among the concerning nutritive profile of some harvest by-products, most widely cultivated oilseed cucurbits in tropical zones derived from cucurbits, such as dried leaves, dried fruits, (Levi et al., 2001, 2009; Zoro Bi et al., 2006; Dane and dried fermented fruits and seed shells. The study was Liu, 2007; Sarao et al., 2014). C. lanatus is a monoecious undertaken to promote the harvest by-products from the species, yellow flowered, and creeping annual vine, oleaginous forms of C. lanatus and L. siceraria by presenting leaves deeply divided into 5-7 more or less exploring their nutritive profile for their potential use as subdivided lobes. The fruits are round or oval, uniformly feedstuffs. light green or mottled light and dark green and contain a white bitter flesh embedding about 200 seeds. Based on the morphology of seeds, two distinct cultigroups have MATERIALS AND METHODS been described (Zoro Bi et al., 2006). The first one, Plant material “wlêwlê”, is characterized by glossy seeds with a tapered proximal extremity. Within the second cultigroup, “bebu”, Fruits and leaves of two edible-seeded cucurbit species, namely seeds are heavier and have a flat ovoid shape with two cultivars of C. lanatus (wlêwlê and bebu) and a soft-shelled L. rugged and thick ends (Zoro Bi et al., 2003). L. siceraria siceraria were harvested at maturity on May 2012 from an is also a monoecious species but white-flowered. Fruit experimental farmland (15 m × 10 m) located at Anyama (longitudes 4°0352.7-4°0353.9 W; latitudes 5°2954.4-5°2954.6 N), a suburb and seeds shape and size are reported to be highly of Abidjan (Côte d’Ivoire). The collected plant parts were washed variable in the genus Lagenaria (Besognin, 2002). There with distilled water, drained at ambient temperature and subjected are two varieties in L. siceraria, which are distinguishable to processing. by the hardness of their fruit exocarp and seed tegument. The variety with hard-shelled fruit and inedible seed is Processing methods mainly cultivated for its dry fruit that is used mostly as containers, musical instruments, fishing floats, etc. The Fruits and leaves of the studied cucurbits were processed as immature and tender fruits of some forms of this variety depicted in Figure 1. The leaves were sun-dried on clean papers for are also eaten as vegetable in some communities. The 10 days with constant turning over to avert fungal growth. The fruits variety with soft-shelled fruit produces oleaginous edible containing seeds were entirely cut up using a stainless steel laboratory knife. The pieces obtained were divided into two lots. seeds. The seeds of this variety are extracted after a 7- The first lot was packaged in sterile airtight polythene bags and day fermentation period of cut fruit containing a juicy pulp allowed to ferment for 7 days in the darkness. Afterwards, the to which they are attached (Zoro Bi et al., 2003). This fermented pieces of fruits were oven-dried (Memmert, Germany) at process is expected to facilitate the seed separation from 50°C for 7 days. The seeds of fermented fruits were removed, sun- the surrounding tissues, and increase their nutritive value dried for 3 days, sorted to remove bad ones and manually shelled. and germination percentage (Achinewhu and Ryley, The second lot was immediately oven-dried at 50°C for 7 days. The dried materials (leaves, fermented fruits, non-fermented 1986). fruits and shells) obtained were ground with a laboratory crusher Many studies on physicochemical and nutritive (Culatti, France) equipped with a 10 µm mesh sieve. The powdered characteristics of cucurbit seeds have revealed that they samples obtained were stored in airtight polythene bags at 4 °C Touré et al. 461
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Figure 1. Processing diagram of fruits and leaves from C. lanatus and L. siceraria. Figure 1. Processing diagram of fruits and leaves from C. lanatus and L. siceraria.
until further analyses. Three replicates were performed for each sodium citrate 0.2 N, pH 2.3 and protein hydrolysis was performed selected by-product. with hydrochloric acid 6 N at 110°C for 24 h. After evaporation, the aliquots were re-suspended in sodium borate buffer 0.4 N, pH 9.5. The homogenized mixture was filtered through a 0.45 mm Millipore Physicochemical analysis membrane (Sartorius AG, Goettingen, UK). The extracts obtained were stored at -20°C until HPLC analysis. An aliquot of 20 µL of Proximate analysis filtered sample was injected for HPLC analysis after derivatisation Moisture, ash, proteins and lipids were determined using AOAC of the amino acids on a pre-column with orthophtaldialdehyde. The (1990) official methods. For crude fibres, 2 g of dried powdered HPLC equipment was composed of a pump (Shimadzu LC-6A sample were digested with 0.25 M sulphuric acid and 0.3 M sodium Liquid Chromatograph), an UV detector (Shimadzu SPD-6A UV hydroxide solution. The insoluble residue obtained was washed spectrophotometry detector) and an integrator (Shimadzu CR 6A with hot water and dried in an oven (Memmert, Germany) at 100°C Chromatopac). Chromatographic separation of amino acids was until constant weight. The dried residue was then incinerated, and performed with an ion exclusion column Hypersil C8 (25 cm x 4.6 weighed for the determination of crude fibres content. Carbohydrates mm x 5 µm, Interchom, France) maintained at 35°C. The mobile and calorific value were calculated using the following formulas phase was a buffer solution of phosphate/methanol (88:12, v/v) with (WHO/FAO, 1985): a flow rate of 0.6 mL/min and the detector was set at 280 nm. The standards were filtered and injected separately and each analysis Carbohydrates = 100 – (% moisture + % proteins + % lipids + % was performed in triplicate. Amino acids were identified and ash + % fibres) quantified by comparison of their retention time and peak area with those of standard solutions.
Calorific value = (% proteins x 4) + (% carbohydrates x 4) + (% lipids x 9) Mineral analysis
Polyphenols content was determined using the method reported by The mineral content was estimated by dry ashing of dried powdered Singleton et al. (1999). A dried powdered sample (1 g) was soaked sample (5 g) in a muffle furnace (Pyrolabo, France). The ash in 10 mL of methanol 70% (w/v) and centrifuged at 1000 rpm for 10 obtained was dissolved in 5 mL of HCl/HNO3 and analyzed using min. An aliquot (1 mL) of supernatant was oxidized with 1 mL of the atomic absorption spectrophotometer (AAS model, SP9). Folin–Ciocalteu’s reagent and neutralized by 1 mL of 20% (w/v) sodium carbonate. The reaction mixture was incubated for 30 min Statistical analysis at ambient temperature and absorbance was measured at 745 nm using a spectrophotometer (PG Instruments, England). The All the analyses were performed in triplicate and data were polyphenols content was obtained using a calibration curve of gallic analyzed using SPSS 11.5. Values were expressed as mean ± acid (1 mg/mL) as standard. standard deviation (SD). Treatments (species and processing
methods) were compared using two-way analysis of variance Amino-acids analysis (ANOVA2). Differences between means were evaluated by Duncan’s test. Statistical significant difference was stated at p < Free amino acids were extracted from 5 g dry matter with 20 mL 0.05. 462 Afr. J. Biotechnol.
Table 1. Physicochemical characteristics of harvest by-products from C. lanatus (wlêwlê and bebu) and L. siceraria.
Moisture Ash Fibres Polyphenols Lipids Proteins Carbohydrates Calorific value Cucurbit By-product (%) (%) (%) (mg/100 g) (%) (%) (%) (kcal/100 g) DL 11.72±0.27b 17.00±0.20bc 13.38±0.46de 428.82±3.18b 3.61±0.16bc 6.79±0.00a 62.09±4.88c 307.87±19.70bcd FF 9.42±0.13d 16.27±1.17c 16.05±0.26a 166.60±3.38h 1.50±0.09g 2.44±0.14g 66.13±5.25bc 287.78±20.52cd C. lanatus (wlêwlê) NFF 9.50±0.33d 16.93±0.30bc 14.16±0.72cd 230.76±3.97g 1.50±0.16g 3.65±0.22d 66.86±4.71bc 295.58±18.55bcd S 11.60±1.04b 9.93±0.32e 3.18±0.52f 89.32±0.92i 3.99±0.28b 4.35±0.00c 66.86±4.71bc 374.21±11.73a
DL 10.43±1.12c 16.93±8.55bc 13.05±0.48e 265.34±7.62f 1.23±0.10g 6.37±0.12b 65.00±5.28c 296.40±20.58bcd FF 9.62±0.14d 17.45±0.43ab 16.35±0.09a 361.37±6.29e 2.27±0.12f 2.93±0.00f 63.95±5.09c 287.92±20.01cd C. lanatus (bebu) NFF 8.39±0,27e 13.07±0.90d 14.47±0.55c 389.21±0.68c 3.36±0.22cd 3.11±0.08f 68.72±3.37bc 317.48±13.70b S 12.87±0.15a 10.13±0.21e 2.78±0.46fg 91.84±4.04i 5.00±0.32a 1.76±0.08i 82.21±2.80a 380.92±11.40a
DL 10.70±0.35c 17.47±0.42ab 14.32±0.06c 396.08±5.20c 2.68±0.20ef 6.75±0.26a 61.53±5.44c 297.05±20.75bcd FF 9.60±0.13c 18.29±0.31a 15.68±0.92ab 379.02±5.25d 1.58±0.17g 3.41±0.08e 64.45±4.98c 285.86±20.14d L. siceraria NFF 4.81±0.16f 10.33±0.31e 14.92±0.29bc 443.25±1.51a 1.71±0.48g 2.05±0.00h 72.78±2.96b 315.88±10.74bc S 11.70±0.05b 10.03±0.15e 2.18±0.45g 54.68±3.98j 1.89±0.68de 1.96±0.00h 84.80±2.78a 372.75±11.83a
Data are represented as means ± SD (n = 3). Means in the columns with no common superscript differ significantly (p < 0.05). DL, dried leaves; FF, fermented fruits; NFF, non-fermented fruits; S, shells.
RESULTS of C. lanatus bebu seeds) to 6.79 ± 0.00% (C. Tyrosine (Tyr), methionine (Met), lysine (Lys), and lanatus wlêwlê dried leaves). Shells of L. siceraria cystine (Cyst) were determined. The contents of Physicochemical properties seeds showed the highest carbohydrate content these amino acids differed significantly (p < 0.05) (84.80 ± 2.78%) while the highest calorific value and ranged from 0.045 to 6.89 g/100 g proteins. The proximate composition of harvest by-products (380.92 ± 11.40 kcal/100 g) was obtained with C. The contents of Thr, Lys, and Met in dried leaves (leaves, fruits and shells) from C. lanatus and L. lanatus bebu. The analysis of polyphenols of C. lanatus bebu were 4.16, 6.86, and 6.89 siceraria is presented in Table 1. The physico- revealed that non-fermented fruits of L. siceraria, g/100 g proteins, respectively. The content of Met chemical parameters generally differed signifi- dried leaves of C. lanatus wlêwlê, and dried was 5.81 g/100 g proteins in fermented fruits of C. cantly (p < 0.05) among species. Moisture of all leaves of L. siceraria were major sources with lanatus (wlêwlê). samples varied from 4.81 to 12.87%. The ash contents of 443.25 ± 1.51, 428.82 ± 3.18, and content ranged from 9.93 ± 0.32% (shells of C. 396.08 ± 5.20 mg/100 g, respectively. lanatus wlêwlê seeds) to 18.29 ± 0.31% (fermented Mineral composition fruits of L. siceraria). There was a significant variation in the fibres content of the plant parts Amino acid composition Mean values for mineral content of analyzed examined, ranging from 2.18 ± 0.45% (shells of L. samples are presented in Table 3. The harvest siceraria seeds) to 16.35 ± 0.09% (fermented The amino acids contents of leaves, fruits, and by-products analyzed in this study contained fruits of C. lanatus bebu). The fat content of the shells from C. lanatus and L. siceraria are remarkably high amounts of potassium (671.78 – remaining plant parts was in the range 1.23 - 5%. presented in Table 2. Seven major amino acids, 4738.79 mg/100 g) and calcium (342.08 – 2963.95
Proteins content ranged from 1.76 ± 0.08% (shells namely threonine (Thr), proline (Pro), serine (Ser), mg/100 g) with the highest values (4738.79 ± 230.10; Touré et al. 463
Table 2. Amino-acids composition (g/100g proteins) of harvest by-products from C. lanatus (wlêwlê and bebu) and L. siceraria.
Cucurbit By-product Thr Pro Ser Tyr Met Lys Cyst DL 3.99±0.02b 3.60±0.00b 1.27±0.00b 0.44±0.00d 6.61±0.00a 4.07±0.01b 0.90±0.01c FF 0.94±0.01d 2.84±0.01c 0.53±0.00e nd 5.81±0.02b 1.26±0.05c nd C. lanatus (wlêwlê) NFF nd nd 0.12±0.05f nd 2.28±0.05d nd nd S 0.10±0.00g 0.49±0.01e nd nd 0.96±0.01e 0.31±0.00e nd
DL 4.16±0.01a 6.01±0.05a 5.83±0.03a 1.82±0.01a 6.89±0.05a 6.86±0.00a 6.27±0.03a FF 0.51±0.01e 5.83±0.04a 0.64±0.00e 0.51±0.00c 2.47±0.01d 1.40±0.00c 0.85±0.01c C. lanatus (bebu) NFF nd nd 0.61±0.01e 0.68±0.09c 2.45±0.05d nd 0.71±0.01d S nd nd nd 0.86±0.06b nd 1.11±0.03d nd
DL 3.65±0.02b 0.87±0.03d 1.39±0.03b 0.07±0.00e 4.23±0.01c 1.33±0.05c 1.23±0.01b FF 1.98±0.01c 0.49±0.03e 1.07±0.01c nd 2.74±0.06d nd 0.76±0.01d L. siceraria NFF 0.38±0.00f 0.22±0.00f 0.045±0.00g nd 0.99±0.01e nd nd S 0.34±0.00f nd 0.80±0.00d nd nd nd nd
Data are represented as means ± SD (n = 3). Means in the columns with no common superscript differ significantly (p < 0.05). DL, dried leaves; FF, fermented fruits; NFF, non-fermented fruits; S, shells; dw, dry weight; nd, non-detected. Thr , Threonine ; Pro, proline; Ser, serine ; Tyr, tyrosine; Lys ,lysine; Met , methionine; Lys, lysine; Cyst, cystine.
Table 3. Mineral composition (mg/100g dw) of harvest by-products from C. lanatus (wlêwlê and bebu) and L. siceraria.
By- Cucurbit Na Mg P K Ca Fe Zn Cu product DL nd 872.10±48.49a 510.57±30.56ef 1017.17±54.51g 2963.95±135.7a 117.02±17.20ef nd 31.73±3.83abc FF nd 349.32±26.44g 443.90±27.32fg 1325.46±92.00f 775.87±60.17e 566.66±28.94b 14.63±12.67a nd C. lanatus (wlêwle) NFF nd 570.60±20.82e 624.52±62.61cd 1885.27±100.5e 1479.43±81.39d 583.87±86.98b nd 38.68±3.42ab S 29.82±1.79b 288.43±7.64h 769.69±23.84b 671.78±7.74h 419.47±1.49gh 352.20±17.81d nd 16.90±7.81cd
DL nd 810.30±34.03b 437.90±23.76g 988.17±18.29g 1494.67±86.80cd 373.81±13.80d nd 38.68±4.58ab FF nd 753.71±11.00c 573.46±4.10de 4159.17±40.50b 1586.13±11.16c 75.23±17.39fg 14.18±4.46a 28.93±3.03abc C. lanatus (bebu) NFF 17.19±8.67b 352.18±17.65g 359.37±33.33h 3912.56±98.80c 594.67±42.85f 25.90±7.65g nd 21.77±5.58cd S 254.85±7.21a 420.30±32.52f 434.30±8.47g 794.64±19.83h 499.90±4.41fg 686.14±10.02a nd 23.32±6.96 bcd
DL nd 673.96±51.60d 524.09±20.96e 1059.82±15.89g 2372.03±58.19b 357.28±6.24d nd 16.30±16.20cde FF 23.52±21.33b 412.36±30.20f 1158.09±103.04a 4738.79±230.10a 593.53±77.78f 129.02±3.30e 10.68±1.90a 41.48±11.37a L. siceraria NFF 24.47±9.48b 221.45±1.96i 652.10±3.63c 2740.10±61.37d 342.08±11.93h 25.85±1.40g 11.55±6.46a 11.89±1.79de S 31.46±18.39b 391.39±25.35f 546.51±42.05e 1269.73±20.60f 395.74±13.00gh 451.80±30.02c 7.20±0.35ab 17.74±6.85cd
Data are represented as means ± SD (n = 3). Means in the columns with no common superscript differ significantly (p < 0.05). DL, dried leaves; FF, fermented fruits; NFF, non- fermented fruits; S, shells; dw, dry weight. nd, non-detected. Na, sodium; Mg, magnesium; P, potassium; K, potassium; Ca, calcium; Fe, iron; Zn, zinc; Cu, cupper. 464 Afr. J. Biotechnol.
2963.95 ± 135.74 mg/100 g) from non-fermented fruits of amino acids which cannot be synthesized by animals and L. siceraria and dried leaves of C. lanatus wlêwlê, need to be supplied by feedstuffs (Mendonca and respectively. The analyzed plant parts were also notable Jensen, 1989). The content of dried leaves of C. lanatus sources of magnesium, ranging from 221.45 ± 1.96 bebu in these essential amino acids was higher than mg/100 g (non-fermented fruits of L. siceraria) to 872.10 those of standard protein (WHO/FAO, 1985): 2.80, 2.20, ± 48.49 mg/100 g (dried leaves of C. lanatus wlêwlê). and 4.20 g/100 g protein, respectively. Therefore, the The phosphorus content varied from 359.37 ± 33.33 consumption of dried leaves of C. lanatus bebu by mg/100 g (non-fermented fruits of C. lanatus bebu) to animals could increase their adequate growth and 1158.09 ± 103.04 mg/100 g (fermented fruits of L. productivity (NRC, 1994). siceraria). The samples contained substantial quantities The macro mineral (Ca, P, Mg, K) concentrations of the of iron that varied from 25.85 ± 1.40 mg/100 g (non- analyzed by-products were lower than the maximum fermented fruits of L. siceraria) to 686.14 ± 10.02 mg/100 tolerable levels (10-40 g/kg) for poultry (McDowell, 1992). g (shells of C. lanatus bebu seeds). In addition, the contents of these macro minerals could meet the requirements (200-750 mg/100g) of poly- and monogastric animals such as beef cattles, sheep and DISCUSSION pigs (NRC, 2001). The copper (Cu) content of the shells from seeds of C. lanatus and L. siceraria was lower than The relatively lowest values of moisture obtained in this the maximum tolerable level (25-30 mg/100 g) for pigs study indicated that the analyzed plant parts were in and poultry (McDowell, 1992). These plants could storage condition avoiding microbial spoilage (Fennema therefore be exploited as non-toxic feedstuffs for these and Tannenbaum, 1996). The contents of all samples in monogastric animals. Indeed, copper is known to affect ash suggested that they might be good sources of entero-hepatic function in small ruminants, especially minerals for feedstuffs (NRC, 1994). The relatively high sheep and goats by decreasing the ability of liver to contents in fibres observed referred to structural carbo- metabolize this trace mineral (Church and Pond, 1988). hydrates that are composed of cellulose, hemicelluloses (pentosans, hexosans) and indigestible materials as lignin ( ottg ter, ). With respect to their contents in Conclusion crude fibres, dried leaves, fermented and non-fermented fruits of the selected cucurbits could be used as feeds for The data obtained in this study showed that the harvest ruminants which need fibres in their daily ration for stable by-products from C. lanatus and L. siceraria contained and healthy digestion ( ottg ter, 008). The dietary appreciable amount of ash, fibres, carbohydrate, essential carbohydrate contents of seed shells of C. lanatus bebu amino acids and mineral elements. Consequently, the plant and L. siceraria were in the range of that of cereals (80- parts analyzed could satisfy the nutrient requirements of 90%), which constitute important sources of energy for animals and should be used as valuable feedstuffs. poultry (Moran Jr, 1985). However, it is worth noting that However, it is necessary to consider other aspects such the energy content of by-product feedstuffs is usually as the in vivo bioavailability of these nutrients after determined by the level of residual fat and percentage of various feed formulations. fibres in the meal (Aherne and Kennelly, 1982; Schingoethe, 1991). Polyphenols are the main dietary antioxidants and Conflict of interests possess higher in vitro antioxidant capacity than vitamins and carotenoids (Gardner et al., 2000). Plant phenolics The authors did not declare any conflict of interest. include phenolic acids, coumarins, flavonoids, stilbenes, hydrolysable and condensed tannins, lignans, and lignins (Naczk and Shahidi, 2004). Tannins present in many ACKNOWLEDGEMENTS feedstuffs do not only affect the feed quality but can also be toxic to animals because they are often considered as The French University Agency (AUF, Yaoundé, Cameroon) anti-nutritional compounds (Lowry et al., 1996). With provided the fellowship for the first author. Bench fees regards to their low contents in polyphenols, seed shells were supported by Integrated Program of Human of C. lanatus and L. siceraria could be more exploited as Resources and Development (PIDRH, Libreville, Gabon) feedstuffs for monogastrics. and chemicals were financed by the Research Academy Dietary requirements for protein in feedstuffs are linked of Higher Education- Committee of Cooperation to the amino acids contents. 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Vol. 14(6), pp. 466-473, 11 February, 2015 DOI: 10.5897/AJB2014.13850 Article Number: 519864250333 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Influence of rural processing methods and postharvest storage treatments on quality characteristics of kola nut (acuminata Schott & Endl.)
Eze, S. C.1*, Ameh, G. I.2, Ugwuoke, K. I.1, Onyeke, C. C.3 and Ozioko, J. O.2
1Department of Crop Science, University of Nigeria, Nsukka, Enugu State, Nigeria. 2Department of Applied Biology and Biotechnology, Enugu State University of Science and Technology, Enugu State, Nigeria. 3Department of Plant Science and Biotechnology, University of Nigeria, Nsukka, Enugu State, Nigeria.
Received 10 April, 2014; Accepted 15 January, 2015
Quality changes of kola nut (Cola acuminata) as affected by processing methods and short-term storage environments were investigated. The experiment was conducted at the Teaching and Research Laboratory of the Department of Crop Science, University of Nigeria Nsukka, Nigeria. The treatments comprised of: three different colour plastic buckets – red, green, white and three inner linings - Newbouldia laevis leaves, Spondias mombin leaves and Black polyethylene sheets giving a 3 × 3 × 3 treatment combinations. The N. laevis, S. mombin and Black polyethylene sheets were laid inside the buckets as beddings for the kola nuts. About 0.45 kg of kola nuts (coated and uncoated) numbering 20 nuts were put in each storage container. Sensory quality of the nuts after storage was determined with quantitative descriptive analysis in expert panel, using six quality attributes. Physical parameters measured were; weight loss, pest incidence and sprouting incidence. Weight loss was significantly lower in white plastic storage container for coated kola nut while green plastic container reduced weight loss for uncoated. Black polyethylene sheet as inner-ling of the storage container significantly (P≤ 0.05) reduced weight loss and pest incidence in coated kola nut compared to uncoated. N. laevis and S. mombin significantly reduced weight loss in kola nuts. It is evident in this study that coated kola nut genotype were kept better than the uncoated kola nut.
Key words: Processing methods, kola nut, storage treatments, quality characteristics.
INTRODUCTION
Kola nut (sterculiaceae) is mostly produced in Africa and There are three species of kola which are mostly grown cultivated to a large degree in West Africa. Annual in Nigeria. These are Cola nitida, Cola acuminata and production from these countries alone is in excess of Cola verticulata. C. nitida, which is referred to as” true 250,000 tons, while the world production is about kola of commerce” has featured in the internal trade of 300,000 tones (American Horticultural Society, 2002). West Africa for a number of centuries. Kola nuts are
*Corresponding author. E-mail: [email protected]
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common sight in Nigeria markets, cities and villages. They The matured pods of kola nut (C. acuminata) were obtained from are often sold by street vendors, at motor parks and train Afor Opi main market in Enugu state, Nigeria. Fresh leaves of N. depots. laevis and S. mombin were collected and identified at the Department of Plant Science and Biotechnology, University of Many Nigerians consume kola nuts regularly, even daily, Nigeria, Nsukka. Uniformly sized plastic buckets with lids and black for its medicinal and stimulating value. The kola nut also polyethylene sheets were purchased from Nsukka main market. has social and traditional significance as it also has Two openings of 3 × 4 cm were made on each bucket to let out industrial usage in pharmaceuticals for the production of excess heat from the container during the storage. The height of soft drinks, wines and confectionaries (Ogutuga, 2001). the bucket was 45 cm and the openings were made directly opposite each other 35 cm from the base of the buckets. The Kola is offered as gifts in several ceremonies such as buckets were red, green and white in colour. The fresh leaves of N. betrothal ceremony, wedding ceremony, naming ceremony, laevis and S. mombin and the black polyethylene sheets were used burial ceremony, taking of a new chieftaincy title and as beddings for the kola nuts inside the buckets/storage containers. therefore regarded as an entertainment item which is used to demonstrate hospitality to guest in homes and Processing and storage social functions especially in Southeastern Nigeria (Russel,
2000). The kola nut pod husk, a by- product from The pods of C. acuminata were carefully and longitudinally cut open processing the nut, is widely used for feeding animals with razor blade. The nuts were removed and those nuts with slight because of its high nutritive quality. According to razor cut, disease or pest symptoms were discarded. The clean Babatunde and Hamzat (2005) broilers fed with kola nut nuts were soaked in clean water in a basin and allowed to stay on a pod husk meal diet exhibit outstanding growth laboratory bench for 24 h and the nuts were then pressed out of the performance. creamy or white coat or testa that covered the nut and washed in sterilized water. The nuts were then spread in a wide perforated It has been observed that Kola nut producing farmers wooden tray in order to drain off water. They were air dried for 48 h. are faced with the problem of pest and diseases, which Defective /infested nuts were picked out during this curing process adversely affect their rate of storability for export that usually involved considerable sweating that reduced the (Daramola, 2000). In most cases the farmers are forced moisture content of the nuts. The nuts were separated into two to sell their produce at very low prices immediately after genotypes and termed coated and uncoated. The coated were those nuts which skins are covered with thin brown inner testa while harvest to avoid total loss of the whole harvested nuts. uncoated had no inner testa. The important role C. acuminata plays in social cultural, economic and religious lives of people in southeastern Nigeria call for critical study and discovery of what must Experimental design and treatment application be done to keep the seed in good order until the next season of harvest. Traditionally, the use of Newbouldia The storage experiment was a factorial laid out in completely laevis (leaves) called “Ejuruosisi” in Igbo language and randomized design (CRD) with three replications. The treatments comprised of: three different colour plastic buckets – red, green, Spondias mombin (leaves) called “Echikara” also in Igbo white and three inner linings: N. laevis leaves, S. mombin leaves language have been used to preserve and keep Kola nut and Black polyethylene sheets giving a 3 × 3 × 3 treatment safe by the local people. The scientific basis for using combinations. The N. laevis, S. mombin and Black polyethylene these plants in preservation of Kola nut has not been sheets were laid in the buckets as beddings for the kola nuts. Each established and because of the high premium value bucket was labeled using a tag identifying the treatment combinations. The kola nuts were weighed and carefully put in the attached to C. acuminata, therefore, the need to storage containers according to the design. Each experimental unit investigate the preservation method that will keep the contained twenty (20) kola nuts (coated and/or uncoated) weighing Kola nut available all year round. about 0.45 kg. The storage containers were opened at intervals of 2 Therefore, the objective of this study was to evaluate weeks for physical measurements while sensory analysis was the effects of green, red and white coloured plastic conducted at the end of the storage period. containers inner-lined with two plant leaves, S. mombin, N. laevis and black polyethylene sheet on the quality Physical measurements indices of kola nut genotypes. The sprouts were periodically removed with a pin to slow down the emergence rate and prevent the splitting of the nuts. The duration MATERIALS AND METHODS of dormancy was determined by calculating the number of days from the start of storage to the time 50% of the nuts showed visible The experiment was conducted at the Teaching and Research sign of sprout. Percentage weight loss was determined by the Laboratory of the Department of Crop Science, University of difference between the initial weight and successive weight loss Nigeria, Nsukka. The area is located by latitude 6 52‟ N and divided by the initial weight, and multiplied by 100. These physical longitude 7 23‟ E, altitude 400 m above sea level. The mean measurements were calculated thus: Dormancy (days) = Number of annual rainfall ranges from 1600 to 2000 mm. The temperature is days from start of storage to the time 50% of nuts showed visible uniformly high throughout the year but the annual mean maximum sign of sprout. temperature does not exceed 35C. The soil is sandy loam and has been classified as Typic Kandpaleustult or Dystric Nitosol, Number of nuts with symptoms earlier scored belonging to Nkpologu series (Nwadialor, 1989) and the vegetation Pest/disease symptoms (%) = x 100 has been described as derived savannah. Original number of nuts stored 468 Afr. J. Biotechnol.
Table 1. Definitions of sensory attributes used in the qualitative descriptive analysis.
Attribute Definition Anchoring points Skin blemish Impression of black spots on the skin None – very high Nut firmness Impression of loose or tight lobes Loose – very tight Lobe blemish Visual observation of spots or insect burrows None – very high Lobe hardness Degree of force needed to chew lobe Soft – very hard Nut skin colour Visual evaluation of nut colour Light-dark brown Taste Basic taste Slightly sweet - bitter Texture Mouth-feel of homogeneous texture Very fine - coarse Overall quality Score for general sensory impression Low – high quality
Each attribute was assessed on 0 to 4 points hedonic scale where 0 is the lowest score and 4 the highest.
Table 2. Main effects of colour plastic storage container on the percentage weight loss, pest incidence and seed germination of coated and uncoated kola nut genotypes after 16 weeks in storage.
Coated kola nut Uncoated Kola nut Container Pest incidence Seed germination colour Weight Pest Seed Weight loss (%) incidence (%) germination (%) loss (%) (%) (%) Green 23.2 16.1 55.6 15.7 16.4 22.3 Red 23.0 20.3 56.0 17.6 16.5 33.1 White 22.1 20.4 48.5 20.1 25.0 23.2
LSD 0.05 0.02 0.04 .69 0.09 0.17 0.43
Statistical Analysis Software, SAS procedure (SAS, 1999), was used. Least significant Difference (LSD) test was used to determine which mean differed significantly at the P ≤ 0.50 probability level. The degree/intensity of each quality attribute score was determined Visual quality assessment was performed using a four point in a polar plot using Excel chart wizard. hedonic scale: where 1 = dull appearance, 2 = slightly bright, 3 = bright and 4 = very bright RESULTS
Sensory analysis The colour of plastic storage container did not affect Sensory evaluation of the nuts after storage was performed in a weight loss in coated kola nut but significantly (P≤0.05) closed door room to avoid distraction from outside. The nine influenced pest infestation and seed germination. The member panel of judges was selected from the University rate of development and emergence of the adult weevil communities based on their experiences in the use and application greatly reduced in red plastic container (Table 2). White of kola nut in various functions. For the evaluation proper, the quantitative descriptive analysis (QDA) was applied. At the first part and green plastic container significantly (P≤ 0.05) increased of QDA procedure, „brainstorming‟ sessions were run to select weight loss and rate of seed germination in both coated attributes for the stored kola nuts. It was a group action and an and uncoated kola nut compared with red plastic bucket interactive session among the judges. The set of 7 quality attributes container. Genotype significantly (P≤ 0.05) reduced weight and overall sensory quality were selected (Table 1). The second loss, pest infestation and seed germination in coated kola part was individual assessment by each of the judges. The samples compared to uncoated. of the kola nuts genotypes (coated and uncoated) were placed on to uniform sized milky coloured saucers and served to the judges in Black polyethylene sheet as inner-ling of the storage a manner kola nut is served in occasions and gatherings. Each container significantly (P≤ 0.05) reduced weight loss and assessor was given randomized samples. The sitting arrangement pest infestation in coated kola nut (Table 3). Similarly, of the assessors was such that there were no communications fresh leaves of N. leavis and S. mombin significantly between or among the assessors. The assessment protocols were reduced weight loss and pest incidence in both coated performed according to Eze et al. (2012) method. and uncoated kola nuts compared to polyethylene sheet.
The interaction of inner-lining and container colour was
Statistical data analysis significant (P≤ 0.05) where S. mombin leaves were inner- lining material in green plastic bucket for both coated and For the variance analysis, the general liner model (GLM) of the uncoated kola nut genotypes (Table 4). On the average, Eze et al. 469
Table 3. Main effects of inner-lining of the storage container on percentage weight loss, pest incidence and seed germination of coated and uncoated kola nut genotype after 16 weeks in storage.
Coated kola nut Uncoated kola nut Inner-lining Weight Pest Seed Weight Pest incidence Seed germination material loss (%) incidence (%) germination (%) loss (%) (%) (%) Polyethylene sheet 15.8 21.4 55.6 14.7 18.4 24.3 Spondias mombin 21.3 19.4 56.7 16.6 17.2 31.1 Newbodias leavis 21.7 16.2 46.5 19.1 23.2 22.2
LSD 0.05 0.18 0.25 0.53 0.10 0.19 0.55
Table 4. Effect of container colour and inner-lining material interaction on visual quality (Physical appearance) of coated and uncoated kola nut genotype sixteen weeks after storage.
Coated kola nut Uncoated kola nut Container Inner-lining material Inner-lining material colour Polyethelene Polyethelene S. mombin N. leavis S. mombin N. leavis Mean sheet sheet Green 4.0 3.6 2.3 3.0 2.6 2.2 2.95 Red 3.2 2.5 2.2 2.4 2.1 1.8 2.37 White 2.3 2.4 2.1 2.6 1.9 2.0 2.22 Mean 3.1 2.8 2.2 3.0 2.1 2.0 2.25
LSD 0.05 for comparing any 2 container colour means = 0.14, LSD 0.05 for comparing any 2 inner-lining material means = 0.19, LSD 0.05 for comparing any 2 container colour x lining material means = 0.25
S. mombin consistently maintained the best physical nut genotypes (Figure 2). There were lower blemishes on appearance across all the variable container colours for coated kola nut genotype whereas ratings for taste, skin both coated and uncoated kola nuts followed by N. leavis colour and firmness were higher than the uncoated kola leaves. Similarly, colour of the plastic container was nuts. The overall quality was also rated highest among important in keeping quality of kola nuts. Kola nut stored the attributes in coated kola nut. Similarly, application of in green plastic container significantly (P≤ 0.05) showed S. mombin leaves as inner-lining for storage of kola nut better physical appearance than what were obtained in improved the sensory quality of kola nut (Figure 3). Nut the other container colours. firmness, skin colour, taste and texture of coated kola nut Results of the experiment showed that the short term rated higher that the uncoated. The overall quality rating storage in those conditions significantly (P≤ 0.05) affected for coated kola was also higher than for the uncoated. some of the quality attributes of kola nuts. Generally, The coated Kola nut genotype were stored better than changes in quality characteristics appeared mostly in the the uncoated with respect to physical appearance after uncoated kola nut genotypes compared with the coated. 16 weeks of storage (Plate i). Using fresh leaves of S. Laying black polyethylene sheet inside plastic bucket mombin and N. leavis as inner-lining in the storage appeared to have increased blemishes on both lobe and containers protected the kola nuts better than whole nut firmness, and also reduced taste, texture and polyethylene sheets (Plates ii). The kola nuts stored in overall quality attributes of the stored coated kola nuts black polyethylene lined plastic buckets showed genotype after sixteen weeks of storage (Figure 1). After symptoms of heat borne after 16 weeks of storage as storage, uncoated kola nut genotype had low rating taste, shown in Plate iii. (slightly bitter), some spots on the skin and loose lobes under this storage condition. The overall quality of coated kola nut genotype stored under this condition was DISCUSSION generally higher than the uncoated by rating in all the attributes. Kola is yet to be granted a full export status by the Using N. laevis leaves as inner-lining or beddings Federal Government of Nigeria, unlike cocoa, coffee, inside plastic bucket irrespective of the colour of the cashew, palm kernel and others, which enjoy favourable bucket for storage affected the sensory qualities of Kola market prices in the international markets probably due to
470 Afr. J. Biotechnol.
Figure 1. Sensory quality profile of kola nut stored in black polyethylene lined plastic bucket.
Figure 2. Sensory quality profile of kola nut stored in Newbouldia laevis leaves lined plastic bucket.
Figure 3. Sensory quality profile of kola nut stored in Spondias mombin leaves lined plastic bucket.
Eze et al. 471
Plate i. Physical appearance of coated kola nut irrespective of the type of storage container six weeks after storage (original). dfasdfa
Plate ii. Physical appearance of uncoated kolanut stored with fresh Newbouldia laevis leaves six weeks after storage (original)
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Plate iii. Physical appearance of uncoated kolanut stored in polyethylene sheet sixteen weeks after storage (original). 472 Afr. J. Biotechnol.
non-availability of kola nut all the year round. This is weight loss, and development of postharvest diseases probably because Kola nut in the storage are attacked by are the storage temperature and humidity. It was evident storage pests which are usually controlled by application in this study that colour of storage container played a of synthetic pesticides by rural processors. It is evident in significant role in the postharvest behavior of kola nut in this study that cheap, harmless and local materials are storage. This was probably due to differences in light good alternatives to synthetic pesticides in kola intensity transmitted by the different colours of the preservation. Except for industrial uses of kola nut, it is storage containers. Variability in the intensity of light consumed raw and the application of synthetic pesticides transmitted could also account for heat buildup in the for preservation would always pose health risks on the different storage containers. There are empirical facts consumers and environment (Alexandratos, 2000). that different colours reflect different wave lengths of light In this study, application of N. laevis and green plastic with consequent effects on physiological behaviuor of storage container effectively reduced losses of kola nut in plant. White plastic container in this study enhanced storage. This result suggests that storage environment activities of kola weevils probably because of refraction of with or without external storage treatment has important infra red light wave lengths. In contrast to our finding, role in the activities of kola weevil, a notorious pest of Brown et al. (1989) reported a reduction of fungi virulence kola nuts in southeastern Nigeria. This approach can be using black polyethylene mulch. The heat borne quite practical and preferable over other methods such as observed on the skins of kola nut stored in black fumigation which lose effectiveness after some time, polyethylene lined bucket could be attributed to retained leaving the stored commodity vulnerable to re-infestation heat in the container. Similarly, application of coloured (Singh, 2000). The kola weevils are said to be field to plastic mulches in vegetable crops production systems store pest as their infestation is usually initiated in the have resulted in both plant and pest responses. field and persists in storage (Daramola, and Ivbijaro, 1999). The use of white plastic storage container appeared to Conclusions have attracted more weevils and also increased seed germination. The high incidence of weevil pest in kola nut Post-harvest losses arising from the rural processing and inside white plastic storage container could be related to storage method of kola nut is a major problem limiting its refraction of infrared light through the transparent plastic availability at affordable prices all the year round in container. However, the frequency of shoot emergence in Nigeria. green plastic container treated with S. mombin could be Evidence from this study showed that modified due to the high water content of the fresh leaves in the atmosphere storage of kola nut using botanical leaves, presence of air thereby creating necessary conditions for significantly enhanced the postharvest and culinary seed germination and shoot emergence. Weight loss qualities of the nuts when compared with the significantly reduced in black polyethylene sheet polyethylene (which represent the modern storage especially with the coated kola nut genotype. Similar system). Nut weight loss and rate of skin changes were studies carried out by Korie, (2000) on the effect of higher in polyethylene sheet sample than in those nuts packaging materials on C. nitida, confirmed the keeping stored inside S. mombin or N. leavis. After sixteen weeks ability of polyethylene sheet over other materials. Korie of storage, nuts stored in green plastic bucket were better (2000) noted that kola nuts stored in polyethylene sheet in physical appearance and culinary quality than those rated first, followed closely by those stored in botanicals stored in red or white buckets. Coated kola nut genotype in terms of firmness, freshness and crunchiness during keeps better than the uncoated kola nut. It is therefore, chewing. Result for this experiment rated N. laevis first, recommended that kola nut processors should use fresh followed by storage with black polyethylene sheet and S. leaves of botanicals in long term storage of kola nut while mombin. polyethylene may be used for short term storage. Plant Organoleptic assessment of kola nut after 16 weeks of breeders are also advised to develop more of coated kola storage revealed that uncoated kola nuts stored in green nut genotype on the basis of its good post-harvest bucket inner-lined with black polyethylene sheet had low characteristics compared with the uncoated. rating taste (slightly bitter), high blemishes and dark spots on the skin. This poor physical appearance and change in taste could be related to heat buildup inside black Conflict of interests polyethylene sheet which probably affected the sensory quality particularly visual appearance. Wills et al. (1998) The authors did not declare any conflict of interest. in their study of fruit storage life noted that storage environment was probably the most important factor affecting the development of their post-harvest quality. REFERENCES
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331-336. Korie CN (2000). Effect of different packaging Materials on the Shelf- American Horticultural Society (2002). The American Horticultural life of Kola (Cola nitida) B. Agric Tech. Crop Production Project Society Encyclopedia of Flowers. D.K., Publishing, New York. Report, Fedral University of Technology Owerri. 62p. Babatunde B, Hamzat RA (2005). Effects of Feeding Graded levels of Nwadialor BE (1989). Soil- landscape relationships in Udi Nsukka kola nut Husk meal on the Performance of Cokrels. Niger. J. Anim. Plateau, Nigeria. Catena 16:111-120. Prod. 32(10):61-66 Ogutuga DBA (2001). Chemical composition and Potential commercial Baiyeri KP (2001). Effects of ricehusk, sawdust and their admixture and uses of Kola nuts. Cola nitida (vent) Schott and Endl. Ghana J. Agric. polyethylene as storage media on the green life span and culinary Sci. 8:121-125 qualities of mature plantain (Musa sp. AAB) fruits. AgroScience Russel TA (2000). The Kola of Nigeria and the Cameroon. Tropical 2(2):19-25. Agriculture, Trinidad. 32:210-240. Brown JE, Stevens C, Osborn MC, Bryce HM (1989). Black plastic SAS Institute (1999). SAS STAT user‟s Guide, release 6.12. SAS mulch and spunbounded polyester row cover as method of southern Institute, Inc., Cary, NC, USA. blight control in bell pepper. Plant Dis. 73:931-9 Singh RP (2000). Neem for the Management of Stored grain Insect, in Daramola AM, Ivbijaro MF (1999). The Distribution and Ecology of Kola developing countries. World Conf. Bangalore, India. 2000. Sourvenir. Weevil in Nigeria. Niger. J. Plant Prot. 1(1):5-9 pp. 69-80. Daramola AM (2000). The Bionomic of Kola Weevils, Sophyhinus spp. Wills R, McGlasson WB, Graham D, Joyce D (1998). Postharvest: An (Coleoptera spp., Curculionidae).PhD. Thesis University of Ibadan introduction to the physiology and handling of fruits, vegetables and Nigeria. 3:325-329. ornamentals. CAB International, London. Eze SC, Baiyeri KP, Agbo CU, Ameh GI, Osahele S (2012). Sensory evaluation response as a selection tool in African eggplant (Solanum aethiopicum) production and breeding. Afr. J. Agric. Res. 7(37): 5247-5251.
Vol. 14(6), pp. 474-481, 11 February, 2015 DOI: 10.5897/AJB2014.13757 Article Number: 05D1CC650338 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Allelopathic potential of some biocontrol agents for the control of fungal rot of yellow yam (Dioscorea cayenensis Lam)
Dania, V. O.1,2*, Fadina, O. O.2, Ayodele, M.1 and Lava Kumar, P. 1
1International Institute of Tropical Agriculture (IITA), Ibadan, Oyo State, Nigeria. 2Department of Crop Protection and Environmental Biology, University of Ibadan, Oyo State, Nigeria.
Received 25 February, 2014; Accepted 2 February, 2015
The adverse effect of synthetic pesticides on human health and the natural ecosystem necessitate the need to explore natural mechanisms of disease control in plants. This study evaluated the allelopathic potential of five biocontrol agents: Trichoderma longibrachiatum, Trichoderma asperellum, Bacillus subtilis, Bacillus cereus and Pseudomonas fluorescens in the control of six fungal pathogens associated with tuber rot of Dioscorea cayenensis. Rotten tuber samples were randomly collected across three agro-ecological zones (AEZs): humid rainforest (HF), derived savanna (DS), and southern Guinea savanna (SGS) in Nigeria. Biocontrol agents were isolated from the yam rhizosphere using the serial dilution method; the agar pairing method was used for the in vitro trials. The destructive sampling method was used to evaluate rot control by the antagonists in vivo. Aspergillus niger had the highest incidence of 64.71% across the HF, 52.08% across the DS, and 41.98% across the SGS. B. subtilis had the highest inhibitory zone of 16.7 ± 0.05% when paired with A. niger, 15.4 ± 0.01% with Lasiodiplodia theobromae, 14.0 ± 0.33% with Penicillium oxalicum, 7.1 ± 0.14% when paired with Rhizoctonia solani; 17.1 ± 0.11% with Sclerotium rolfsii, and 10.3 ± 0.94% with Fusarium oxysporum. All biocontrol agents significantly (P = 0.05) reduced rot development of the test pathogens relative to the control in the in vivo experiment. The establishment of a distinct zone of inhibition, especially by the bacterial antagonists attests to the fact that they produced allelochemical substances. Therefore, further research is recommended to evaluate the biochemical composition of these microbial metabolites, their level of toxicity, and fate in the environment.
Key words: Allelopathic potential, biocontrol agents, tubers, allelochemicals.
INTRODUCTION
Yellow yam (Dioscorea cayenensis) is an important food being the first producer (Osunde, 2008). Babaleye (2003) crop, especially in the yam zone of West Africa which reported that yam contributes more than 200 dietary accounts for 90% of world yam production with Nigeria calories per capital daily for more than 150 million people
*Corresponding author. E-mail: [email protected]. Tel: 08034353072.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Dania et al. 475
substance, an allelochemical or antibiotic, into a medium or an environment which acts as a growth inhibitor to another organism. Biological control agents (BCA) are known to produce allelochemical substancces (He et al., 2006). Most Trichoderma strains produce volatile and non-volatile toxic metabolites such as harzianic acid, alamethacin, tricholin, viridian, and peptailbols which inhibit the growth of plant pathogens (Vey et al., 2001). Similarly, Bacillus subtilis has been reported to produce allelochemicals such as bacillomycin, surfactin (Tsuge et al., 1995), bacitracin, subtenolin, and subtilin (Manjula et al., 2005). Although several authors have researched extensively on the ability of these BCA to produce secondary metabolites as previously stated, most of these experiments often do not go beyond the in vitro trials. This, therefore, leaves an information gap on the
Plate 1. Dioscorea cayenensis tubers attacked by fungal rot applicability of the results in vivo. Hence the objective of disease under natural infection. this study was to determine the potential of the allelochemicals produced by some BCA for the control of fungal pathogens causing rot of D. cayenensis tubers in storage. in West Africa and serves as an important source of income to the people. Postharvest deterioration of yams has been attributed to insects, nematodes, respiration of MATERIALS AND METHODS the dormant tuber, sprouting, and microbial attack Collection of yam samples (Ikotun, 1989; Okigbo, 2005). Microbial rot accounts for the highest losses in yam tubers during storage, with Rot-infected tubers of D. cayenensis were collected from 14 Local fungi being responsible for greater loss than any other Government Areas (LGA) across three agro-ecological zones single cause (Amusa et al., 2003). The major fungal (AEZs) in Nigeria: humid rainforest, derived savanna/forest pathogens associated with storage rot of yams include transition, and southern Guinea savanna (plate 1). Three tubers showing symptoms of rot were randomly collected from each of five Lasiodiplodia theobromae, Penicillium oxalicum, farmers’ barn in each LGA across the AEZ. A total of 210 rotted Aspergillus niger, Rhizoctonia solani, Sclerotium rolfsii, tuber samples were screened for infecting fungi. Healthy and Fusarium oxysporum (Green et al., 1995; Dania, susceptible tubers of genotype TDc 98-172 obtained from the 2012). International Institute of Tropical Agriculture (IITA) yam barn, The use of pesticides in crop protection has been the Ibadan were used for in vivo trials. target of considerable criticism (Manjula et al., 2005). Though chemicals have played a significant role in Isolation of test pathogens maximizing productivity, extensive use of broad spectrum compounds has resulted in harmful and undesirable Infected tubers were washed with sterile distilled water (SDW) and effects on the environment (Adesiyan, 1986). In addition, cut open to expose the fresh necrotic lesion. Tissues measuring 2 × 2 mm were cut from areas in advanced necrosis, surface-sterilized the appearance of fungicide-resistant fungal pathogens with 10% sodium hypochlorite for 1 min, and rinsed 5 times in SDW. and new exigencies of laws concerning food quality and Then, the samples were plated on potato dextrose agar (PDA), environmental conservation complete the challenge for incubated at 28 ± 2°C for 2-3 days and examined for fungal growth. alternative disease management strategies in crop The cultures were further purified and pure fungal isolates viewed protection research (Dayan et al., 1999). Peasant farmers under a compound microscope and identified using standard constantly have rot problems to contend with annually in procedures (Barnett and Hunter, 1998). storage of cultivated yams. The drawbacks of chemical control now leave them with natural control options of Distribution of fungal isolates among yam species protecting their harvested yam from spoilage and avoid yield losses. This was determined in order to ascertain the occurrence of the Therefore, allelopathy has become a viable alternative fungal pathogens across the three AEZ. This was evaluated by the in natural crop protection (Francisco et al., 2003). It expression of the number of times each organism was isolated from each species per AEZ as a percentage of the total different entails biochemical processes in which secondary meta- organisms from all the AEZ. This was calculated thus: bolites from plants and microorganisms are involved, p affecting growth and development of biological systems Incidence of isolate = /Q × 100/1 (Ries et al, 1994; Francisco et al., 2003). Allelopathy in microorganisms involves the release of a chemical Where Q= Total number of micro fungi isolated per yam species in 476 Afr. J. Biotechnol.
the locations and P= number of times of occurrence of the against the pathogen isolates on healthy tubers of a susceptible individual isolate per yam species in the locations. IITA genotype TDc 98-172. Healthy yam tubers measuring 300 mm long and about 50 mm wide were used in this experiment. The yam tubers were washed in running tap water to remove any adhering Quantification of fungal spores soil particles and later rinsed in SDW. A 3-mm cork borer was used to bore holes 1 cm deep and 10 cm apart at the proximal, middle, Spore suspension of each fungal isolate was prepared by adding and distal regions on each tuber. Three replicates were inoculated 10 mL of SDW to 7-day-old culture plates to obtain 10 mL spore for each pair of organisms. A spore concentration of 106 spores/mL volume. The resultant solution was scooped with a spreader and and 108 cfu/mL was used as the inoculum concentration for each then filtered through cheese cloth. Tween 80 (0.5 ml/L) was added fungus and bacterium respectively. Each antagonist and test to the fungal culture in Petri dishes to enhance removal of their pathogen was inoculated separately. Each test fungus was spores. Fructification and spore count were determined using an inoculated singly to serve as control. All treatments and control hemacytometer. Determination of spore concentration was done by were incubated at IITA yam barn for 6 months. dispensing 0.1 mL of inoculum on the hemacytometer. Spores in the four corner squares and at the center were counted and multiplied by 104 to determine the total spore count. Final spore Measurement of zone of inhibition count and inoculum concentration were determined using the method of Berkane et al. (2002). Bacterial inoculum concentration After incubation of the agar plates for 7 days in vitro, the zone of was determined by serial dilution using the method of Okechukwu inhibition was measured using a ruler. The zone of inhibition was et al. (2000). determined by measuring the diameter of the outer circle (area of clear edge) minus the diameter of the inner circle (growth of producer strain). Three triplicates for each plate were maintained. Isolation of biocontrol agents Inhibition zones were measured in millimetres and converted to percentage.
Biocontrol agents used in this study: Trichoderma longibrachiatum,
T. asperellum, B. subtilis, B. cereus, and Pseudomonas fluorescens Data analysis were isolated from yam rhizosphere at IITA research plots using the method of Okigbo (2005). One gram of the soil sample was All numerical data were statistically analysed using generalized dissolved in 10 mL of SDW in a McCartney bottle. The solution was linear model (GLM) of SAS. Means were separated using Least left for 1 h and shaken at 20 min intervals to acclimatize the Significant Difference Test (LSD) and standard error was microorganisms at ambient temperature. Serial dilution was done to determined where applicable at 5% level of significance. obtain 106 spores/mL for fungal antagonists and 108 cfu/mL for bacterial antagonists. Thereafter, 1 mL of each dilution was dispensed into separate 9-cm Petri dishes. PDA and nutrient agar RESULTS AND DISCUSSION maintained at 45°C were poured over the suspension for fungal and bacterial antagonists, respectively. However, P. fluorescens was cultured on Kings B medium which is selective and encourages the Distribution of fungal isolates among yam species production of fluorescent pigments. A litre of the medium consisted of proteose peptone 20 g, K2HPO4 1.5 g, MgSO4.7H2O 1.5 g, The distribution of the six pathogenic fungal isolates glycerol 10ml, agar 15 g and distilled water 1000 ml. Inoculated evaluated in this study is shown in Table 1. Aspergillus Petri dishes were incubated at 28 ± 2°C for 24-48 h before being was the most predominant pathogen across the three observed for the growth of the BCA. AEZs. This was followed by L. theobromae, while S. Similarly, isolation of B. subtilis followed a definite protocol rolfsii had the least incidence. The humid rainforest had requiring the addition of supplements to enhance its growth while discouraging other bacteria. A loopful of soil obtained from decayed the highest overall incidence of fungal isolates, followed organic matter was suspended in 1 ml of sterile SDW in a test tube. by the derived savanna and southern Guinea savanna. It was mixed well and heated in a water bath at 80°C for 10 min. This could be attributed to high relative humidity Thereafter, an inoculating loop was used to streak a sample of the associated with the humid rainforest zone. This result heat treated soil on to peptone –yeast- dextrose plates (PYD) and agrees with the findings of Ikotun (1989) who reported incubated at 28 ± 2°C for 24-48 h. One litre of PYD was prepared that relative humidity and temperature are important by adding peptone 2 g, yeast extract 5 g, dextrose 15 g and agar 15 g and sterilized at1.05 kg/cm2 (121°C) for 15 min. Colonies were determinants of disease incidence. Rot-causing examined under a compound microscope and those with clear organisms can also be influenced by soil pH, especially mottled appearance and bearing endospores were again streaked by virtue of the fact that they are soil-borne saprophytes on to fresh plates for purification. These were further subjected to (Coyne, 1999). There was significant difference (P=0.05) biochemical tests following standard protocols for identification. in the abundance of A. niger and L. theobromae across the three AEZ. Similarly, the occurrence of S. rolfsii and
Screening of BCA for allelochemicals F. oxysporum differed significantly ((P=0.05) across the humid rainforest and derived savanna AEZ. The dual culture method was used to test for in vitro antagonism. P. fluorescens and B. subtilis completely inhibited Fully grown culture of each BCA was inoculated using a 3-mm cork further growth of L. theobromae, F. oxysporum and A. borer at four equidistant points, while the test pathogen was niger at four days after inoculation (Plates 2 to 4). A zone inoculated at the center of the plate in three replicates. The culture of inhibition was established, especially between the plates were incubated at 28 ± 2°C for 4 days. Radial mycelial growth was measured on a daily basis. The in vivo trial was bacterial antagonists and the test pathogens in vitro conducted in a randomized complete block design with three (Table 2). This inhibitory zone could be attributed to the replicates. Prospective BCA in the in vitro experiment were tested fact that the BCA produced allelochemical substances Dania et al. 477
Table 1. Distribution (%) of six pathogenic fungal isolates on D. cayenensis tuber rot across three AEZ in Nigeria.
Fungi Humid rainforest Derived savanna Southern Guinea savanna A. niger 64.71a 52.08a 41.98a L. theobromae 45.90b 29.44b 10.11b R. solani 18.22c 10.25d 4.42c P. oxalicum 5.81d 18.09c 9.2b S. rolfsii 4.74d 2.98e 0.0d F. oxysporum 18.92c 12.66d 10.4b
Means with the same letter along the column are not significantly different (P=0.05) using the least significant difference (LSD)
Treatment A Treatment B (Control)
Plate 2. Inhibiting effect of P. fluorescens on L. theobromae
Treatment A Treatment B (Control)
Plate 3. Inhibiting effect of B. subtilis on L. theobromae
(antibiotics) in vitro. The use of B. subtilis and P. subtilin (Manjula et al., 2005). These antibiotics cause fluorescens as BCA has been reported (Ries et al., 1994; lysis of mycelia of target organisms, which is an indication Okigbo, 2005; Manjula et al., 2005). B. subtilis has been that lytic enzymes inhibit the growth of pathogens. There reported to produce antibiotics such as bacillomycin, was no zone of inhibition between the Trichoderma sp. surfactin (Tsuge et al., 1995), bacitracin, subtenolin, and and the test pathogens (plates 5, 6 and 8), except 478 Afr. J. Biotechnol.
Treatment A Treatment B (Control)
Plate 4. Inhibiting effect of B. subtilis on A. niger
Table 2. In vitro screening of biological control agents (BCA) for inhibitory zone/allelopathy at 3 days after inoculation (DAI).
BCA zone of inhibition (%) Pathogen B. subtilis P. fluorescens B. cereus TL TA Control A. niger 16.7 ± 0.05 7.4 ± 0.07 10.1 ± 1.2 0.0 7.4±1.08 76 ± 0.09 L. theobromae 15.4 ± 0.01 4.4 ± 0.02 7.8 ± 0.31 0.0 0.0 81 ± 1.44 R. solani 7.1 ± 0.14 2.9 ± 0.7 3.7 ± 0.22 0.0 0.0 77 ± 0.15 P. oxalicum 14.0 ± 0.33 10.2 ± 1.24 9.8 ± 0.04 0.0 0.0 54 ± 1.07 S. rolfsii 17.1 ± 0.11 9.9 ± 2.13 16.7 ± 1.52 0.0 0.0 45 ± 0.22 F. oxysporum 10.3 ± 0.94 7.0 ± 0.01 6.6 ± 0.03 0.0 0.0 37 ± 0.93
TL = Trichoderma longibrachiatum; TA = Trichoderma asperellum.
Treatment A Treatment B (Control)
Plate 5. Inhibiting effect of T. asperellum on Fusarium oxysporum.
the inhibitory zone established between T. asperellum inhibition between the Trichoderma sp. and the test and A. niger (Plate 7). Although there was no zone of pathogens, antibiosis was also involved in their control Dania et al. 479
Treatment A Treatment B (Control)
Plate 6. Inhibiting effect of T. longibrachiatum on Fusarium oxysporum.
Treatment A Treatment B (Control)
Plate 7. Inhibiting effect of T. asperellum on Aspergillus niger.
Treatment A Treatment B (Control)
Plate 8. Inhibiting effect of T. longibrachiatum on A. niger. 480 Afr. J. Biotechnol.
Table 3. In vivo screening (%) of biological control agents (BCA) for tuber rot control at 6 months of storage.
Pathogen BCA L. theobromae R. solani A. niger P. oxalicum S. rolfsii F. oxysporum T. longibrachiatum 9.4a 10.0a 7.2a 8.4a 9.1a 5.8a B. subtilis 5.5b 9.4a 10.6b 8.5a 7.4a 6.2a P. fluorescens 23.3c 19.5b 23.9c 18.7b 24.2b 17.7b T. asperelum 8.2a 7.1c 8.7a 7.7a 8.8a 5.1a B. cereus 4.2a 15.5c 8.4a 15.4b 17.1c 22.2c Control 56.3d 40.2d 54.0d 40.1c 59.9d 46.7d
Means with the same letter along the column are not significantly different (P=0.05) using the least significant difference (LSD), .
mechanism through the processes of competition and (iv) Evaluation of the toxicity of the allelochemicals so mycoparasitism (Manjula et al., 2005). This was exemplified produced. in the inhibitory zone established between T. asperellum and A. niger. However, antibiosis in Trichoderma species Provision of solutions to the above questions will lead is mostly achieved through direct contact with tissues of to starting with the right selection material and extraction pathogens and do not often show inhibitory zone. This method as well as the development of a standard result is in consonance with Vey et al. (2001) who protocol for bioactivity evaluation that will compare reported that Trichoderma strains produce volatile toxic between different substances and measure effects over a metabolites such as harzianic acid, allamethacin, and wide range of target. Allelopathy research transcends tricholin. The BCA significantly reduced rot in healthy isolation, identification, and bioactivity evaluation of a tubers inoculated with the test pathogens (Table 3). natural product as an allelochemical model. Degradation Trichoderma longibrachiatum and T. asperellum did not and stability of allelochemicals in the environment should differ significantly (P > 0.05) in the control of all the also be given priority. Therefore, knowledge of the pathogens except for R. solani. interaction between the allelochemicals produced by BCA Interestingly, there was no inhibitory zone between T. and their ultimate degradation in the environment is a key longibrachiatum and A. niger, suggesting that the control to conservation of a robust ecosystem. mechanism may have been mycoparasitism (Plate 8). Several authors have reported the use of BCA in the control of postharvest plant diseases (Lewis and Conflict of interests Papavizas, 1991; Howell, 2003; Manjula et al., 2005). Although the BCA exhibited strong potential for use as The authors did not declare any conflict of interest. biopesticide, there is the need to evaluate their safety in the preservation of ware yam for consumption as well as the environmental impact assessment. ACKNOWLEDGEMENT
The author is grateful to IITA, Ibadan, Nigeria, for providing Further research on microbial allelopathy the requisite facilities to conduct this research.
The inhibition of plant pathogens by antibiotics or REFERENCES chemicals produced by BCA is an allelopathic process and its knowledge is needed to guarantee high yield of Adesiyan SO (1986). Pesticide toxicity in the environment. Real or crops in any ecosystem. Research on this area will offer speculative. Paper presented at the National Seminar on Pesticide Legislation in Nigeria. 12pp. a basis for plant growth control and crop improvement. Amusa NA, Adegbite AA, Mohammed S, Baiyewu RA (2003). Yam Following the allelopathic potential of BCA used in this diseases and its management in Nigeria. Afri. J. Biotechnol. 12:497- study, further research should be focused on the 502. following areas: Babaleye T (2003). Raising the status of yam, a major food crop in West Africa. ANB-BIA Supplement Issue/Edition. No. 463- 01/10/2003. (i) Exploration of biochemical processes responsible for Barnett HL, Hunter BB (1998). Illustrated Genera of Imperfect Fungi. 4th their bioactivity. Edition. Macmillan Inc. New York, 218pp. (ii) Determination of their mode of action. Berkane A, Cruenca-Estrella M, Gomez-Lopez A, Petrikkou E, Mellado E (2002). Comparative evaluation of two different methods of (iii) Environmental impact assessment of their fate in the inoculum preparation for antifungal susceptibility testing of ecosystem. filamentous fungi. Antimicrob. Chem. 150(5):719-722. Dania et al. 481
Okechukwu RU, Ekpo EJA, Florini DA (2000). Yield depression in Coyne MS (1999). Soil Microbiology. An exploratory approach. Delmer cowpea cultivars infected with Xanthomonas camprestis pv vignicola Publishers, Albay, New York, USA. in sudan savanna of Nigeria. J. Trop. Agric. Ext. 3(2):98-101. Dania VO (2012. Evaluation of some plant extracts and microbial Okigbo RN (2005). Biological control of post-harvest fungal rot of yam agents for the control fungal rot disease of yam (Dioscorea sp.) PhD (Dioscorea spp.). Mycopathologia 159: 307-314. dissertation, University of Ibadan. 219pp. Osunde ZD (2008). Minimizing post-harvest losses in yam (Dioscorea Dayan F, Romagni J, Tellez M, Rimando A, Duke S (1999). Managing spp.). J. Int. Union Food Sci. Technol. 1:1-11. weeds with natural products. Pesticide Outlook 17:185-188. Ries A, Siverela N, Miichereff SJ, Pereira GFA, Mariano RLR (1994). Francisco M, Macias DM, Alberto OB, Rosa MV, Ana MS, Caferrino C, Bacillus subtilis as a potential biocontrol agent of northern leaf blight Jose MG (2003). Allelopathy as new strategy for sustainable of corn. Rev. Microb. 25:255-260. ecosystems development. Biol. Sci. Space 17(1):18-23. Tsuge K, Ano H, Shoda M (1995). Characterization of Bacillus subtilis Green KR, Sangoyomi, TE, Amusa NA (1995). The importance of co-producer of lipopeptides surfactin and plipastatin. J. Gen. Appl. Rhizoctonia solani as a pathogen of yam (Dioscorea sp.). In: Microb. 41(6):541-545. Proceedings of the 6th Symposium of the International Society of Root Vey A, Hoagland RE, Butt TM (2001). Toxic metabolites of fungal and Tuber Crops Africa Branch, pp.412-418. biocontrol agents. In: But, TM, Jackson C and Morgan, N. (Eds). He L, Chen W, Liu Y (2006). Production and partial characterization of Fungi as biocontrol agents: Problems and potential. Wallingford: bacteriocin-like peptides by Bacillus linchenformis. ZJU 12. Microb. CABI Publishing. pp.311-346 Res. 23:135-141. Howell CR (2003). Mechanisms employed by Trichoderma species in the biological control of plant disease: the history and evolution of current concepts. Plant Dis. 87:4-10. Ikotun T (1989). Disease of yam tubers. Int. J. Trops. Plant Dis. 7:1-21. Lewis JA, Papavizas GC (1991). Potential for biological control of post- harvest plant diseases. Plant Dis. 58:374-378. Manjula K, Mwangi M, Bandyopadhyay R (2005). Potential of some bacteria and fungi as biocon trol agents of cassava and yam under laboratory and green house conditions. Afr. Crop Sci. Proc. 7:1395- 1400.
Vol. 14(6), pp. 482-488, 11 February, 2015 DOI: 10.5897/AJB2014.14308 Article Number: 3DD902E50346 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Growth of mycotal species on the eggs of Cyprinus carpio in limnologically and trophically different water bodies
Bazyli Czeczuga*, Adrianna Semeniuk- Grell and Ewa Czeczuga- Semeniuk
Department of General Biology, Medical University, Mickiewicza 2c, 15-222 Bialystok, Poland.
Received 9 November, 2014; Accepted 2 February, 2015
The study investigated the growth of hydromycoflora on the eggs of common carp (Cyprinus carpio L.) in five water bodies of different eutrophication levels. Thirty-three (33) species of mycotal organisms, including 28 species belonging to Peronosporomycota, 2 to anamorphic fungi and 1 each to Ascomycota, Blastocladiomycota and Zygomycota were identified on the eggs. The water from River Biala and Pond Fosa, which are more biogenic, had the largest number of mycotal species (20 and 19, respectively) on the eggs of common carp. The smallest number of those species was found on the eggs of common carp in water from Spring Cypisek, Pond Komosa and River Suprasl, which are low in biogenes (12, 11 and 11, respectively). Achlya diffusa, Aphanomyces laevis, Saprolegnia ferax, Saprolegnia parasitica and Pythium ultimum belong to the species that were most frequently found on C. carpio eggs. The following rare mycotal species were also found: Allomyces arbuscula, Aphanomyces frigidophilus, Candida albicans, Fusarium aquaeductum, F. culmorum and Zoopage phanera. Amino acid, carbohydrate and urease tests were used. Of the C. carpio eggs that were investigated, 12.7% were found to be infected with mycotal species.
Key words: Cyprinus carpio, common carp, eggs, mycotal species, infections, hydrochemistry.
INTRODUCTION
The occurrence of the carp in the inland waters of Europe breeding of this species in France dates back to 1313. was first recorded in the works of Aristotle (284-322 Carp was known to have been held in ponds in Bohemia- B.C.). This was later confirmed by Plinius (23-79 A.D.) Moravia in medieval times. In 1547, the Moravian Bishop (Wolny, 1974). The origins of the domestication of carp of Olomun-Dubravius wrote the first European manual probably took place in the estuary of the Danube River about carp farming in ponds. In terms of Germanic lands, (Dacia) during the 5th and 6th centuries B.C. Monk Albert there were descriptions in 1551 by Gessner, and for Magnus (1230-1310) reported on carp cultures in ponds France in 1555 by Belon then in 1558 by Rondelet. in Bavaria, Germany, and the first mention of the These manuals contributed greatly to the dissemination
*Corresponding author. E-mail : [email protected].
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Czeczuga et al. 483
of information about the culturing of this species. In Asia MATERIALS AND METHODS (China and Japan), the carp was domesticated much The eggs of C. carpio Linnaeus, 1758 (syn. Cyprinus regius earlier for ornamental purposes (Cyprinus carpio koi). Lesniewski, 1837; Cyprinus hungaricus Heckel, Taczanowski, Carp is now grown on all continents except antarctic. 1877; C. carpio hungaricus Heckel, Wałecki, 1889) (English name: C. carpio is distributed across Europe and Asia, in the common carp, carp) were investigated. Black, Caspian and Aral sea basins, and has been The water for these experiments was collected from five different introduced to waters throughout the world. However, wild water bodies located in the northeastern region of Poland: Spring stocks only occur naturally in rivers draining to the Black, Cypisek (53°07´N, 23°10´E): located in the south part of the Knyszynska Forest; width of 0.41 metres (m); depth of 0.17 m; Caspian and Aral seas (Kottelat and Freyhof, 2007; ITIS, discharge of 0.6 dm3/s; the limnokrenic type: River Biala (53°15´N, 2010). A rheophilic wild population in the Danube River is 22°20´E): length of 9.8 km; left-bank tributary of the River Suprasl, assumed to be the origin of the European species, but flowing through the city of Bialystok; River Suprasl (51°10´N, this population is now under threat (Kottelat, 1997; ITIS, 21°10´E): length of 106.6 km, right-bank tributary of the middle part 2010). of the River Narew, flowing through the Knyszynska Forest; Pond Adults inhabit warm, deep, slow-moving and standing Fosa (53°18´N, 23°15´E): in the Palace Park (Bialystok); an area of 2.5 hectares (ha); depth of 1.75 m; a breeding area for wild ducks; waters, such as lowland rivers and large, well-vegetated a culture of crucian carp for anglers; Pond Komosa (52°08´N, lakes. They are hardy and tolerate a wide variety of 21°12´E): an area of 12.1 ha, depth of 2.25 m; surrounded by conditions, but generally favour large water bodies with coniferous trees of the dense Knyszynska Forest slow-moving or standing water and soft bottom sediments Nineteen (19) parameters of those water samples were (ITIS, 2010). Both adults and juveniles feed on a variety measured (Table 1) according to generally accepted methods (APHA, 2005). Water samples for the analysis and for the of benthic organisms and plant material (Bryliński, 2000; experiments were collected from each reservoir at depth of 15-30 ITIS, 2010). cm at a distance of 1.5 m from the bank (except spring). In the Today, with overharvest of wild fisheries, the intensity laboratory, the samples were filtered through gauze and then of aquafarming is increasing, especially in developing poured into 1000 ml vessels. countries (Food and Agricultural Organization of the Eggs were collected (after fertilization) at the end of April from United Nations (FAO), 2012). This applies mainly to the hatchery at the Knyszyn Farm. Water samples from specific water bodies (800 ml each) were freshwater aquaculture and shrimp production, and to a placed into 1000 ml vessels, and 30 eggs were transferred to each lesser extent mariculture. In freshwater aquaculture, vessel in accordance with the general principles of culture farming of common carp species (the earliest domesticated (Watanabe, 2000). Before transfer, eggs were washed with distilled carp species in Europe) is of great economic importance. water to remove fungal spores attached to their surface. All vessels The growth of fungal species on the specimen eggs of were enclosed in Petri scales with the bed turned upside down to the common carp was first described in 1885 by prevent possible airborne contamination with fungal spores. The vessels were stored at temperature of 17± 0.5°C, with access to Walentowicz, in Kaniow, Austria. Walentowicz referred to daylight that resembled natural conditions and following the the presence of Achlya nowicki Raciborski (doubtful taxa) recommended instructions (Seymour and Fuller, 1987). The pH of and Saprolegnia monoica. Observations and experiments the water was analysed separately for every vessel (Peterson and on the Saprolegnia ferax that infected fish, including Bridge, 1994). The water analyses and experiments were carried common carp specimens, have been described by out in three parallel repetitions. Eggs were taken from each vessel, and the eggs that were covered with fungal mycelia were observed Clinton (1894). Graff (1928) has also reported on the every 3-to-4 days under a light-microscope. The presence of presence of Saprolegnia parasitica on specimens of morphological structures such as zoospores, antheridia and common carp in ponds in Western Montana, U.S.A., and oogonia that belong to aquatic fungi was recorded. Fungal species Chinapelli (1933) has looked at the experimental infection were identified using the keys of Johnson et al. (2005), Pystina of the Saprolegnia species in carp specimens in water (1998) and Petrini and Petrini (2013). The systematics of bodies of Italy. straminipiles species according to Dick (2001) were used in this experiment. The experiments were carried out for one month, and Hörter (1960) investigated the lethal mycosis of carp the results were then tested for significance using ANOVA and skin that was caused by Fusarium culmorum. Srivastava evaluated by the Scheffe test (Winer, 1997). and Srivastava (1976) have also mentioned the infection Amino acid, carbohydrate and urease tests were performed on of common carp eggs on the Indian subcontinent, while the Achlya, Aphanomyces, Leptolegnia, Pythium and Saprolegnia Czeczuga and Muszynska (1999) have described the genera, based on Yuasa and Hatai (1996). For the carbohydrate straminipilous and fungal species occurring on carp eggs utilization test, yeast nitrogen base agar was the medium used for the cultures of the fungal isolates. Glucose yeast (GY) agar was in middle and east Europe. In addition, Froelich and used for the urease test. The basal medium used in the amino acid Engelhardt (1996) have investigated the effects of assimilation test was the same as that used for the carbohydrate different antifungal agents on the successful hatching koi assimilation test. Bromo thymol blue and phenol red that was added carp eggs, and Padmakumar et al. (1985) and to the yeast nitrogen-based broth and GY broth, respectively, were Khodabandeh and Abtahi (2006) have looked at the used as indicators. These methods are described in detail in our previous paper (Czeczuga et al., 2011). effects of such agents on the development of common carp eggs. In this context, we decided to investigate the growth of mycotal species on the eggs of common carp in RESULTS water from limnologically and trophically different water bodies. Thirty three (33) mycotal species were found to be 484 Afr. J. Biotechnol.
Table 1. Chemical and physical properties of water in particular water bodies (in mg l-1) (April, 2012).
Spring River Pond Property Cypisek Supraśl Biała Fosa Komosa Temperature (C) 14.5 16.8 15.2 18.0 14.9 pH 7.2 7.1 7.3 7.1 7.2 DO 16.4 8.2 18.6 6.4 14.8
BOD5 2.6 10.8 4.2 12.8 5.1 COD (Oxidability) 9.9 16.8 11.2 20.2 9.0
CO2 37.4 26.9 29.4 22.4 18.6 -1 Alkalinity (CaCO3 mval l ) 4.6 4.3 3.6 5.8 3.8
N-NH3 0.254 0.662 0.315 0.864 0.196
N-NO2 0.015 0.128 0.037 0.114 0.018
N-NO3 0.245 0.470 0.317 0.552 0.072
P-PO4 0.920 1.820 1.02 3.598 0.706
Sulphates (SO4) 36.8 73.2 50.4 85.1 40.6 Chlorides (Cl) 40.5 66.4 50.2 79.3 35.4 Total hardness (in Ca) 110.9 98.2 93.6 24.2 76.6 Total hardness (in Mg) 21.8 17.4 17.2 20.6 18.4 Fe 0.38 0.92 0.47 1.06 0.26 Dry residue 174.2 434.0 412.0 429.0 220.0 Dissolved solids 150.0 324.0 384.0 370.0 198.0 Suspended solids 24.2 110.0 28.0 59.0 22.0
growing on the eggs of the Cyprinus carpio, including 29 water bodies examined, had the greatest number of belonging to the Peronosporomycete (Oomycetes) class mycotal species on them. The eggs of the common carp and 1 to the Zygomycete, 1 to the Endomycete and 2 to in the water from River Suprasl, Pond Komosa and the Hyphomycete classes (Table 2). Analysis of water Spring Cypisek, which had the least abundance of samples from 5 different water bodies in Northeastern biogenes, had the smallest number of mycotal species. Poland found the greatest number of mycotal species on We also observed this kind of phenomenon while the eggs of common carp from the waters of River Biala studying the growth of fungi on the eggs of certain and Pond Fosa (20 and 19, respectively) and the cyprinid species (Czeczuga and Muszynska, 1999) and smallest number of mycotal species from the waters of coregonid species (Czeczuga and Muszynska, 1998). Spring Cypisek, Pond Komosa and River Suprasl (12, 11 However, a reverse phenomenon has been observed in and 11, respectively). Species such as A. diffusa, A. Switzerland where more fungi were found on the pike laevis, S. ferax, S. parasitica and P. ultimum were found (Esox lucius L.) and perch (Perca fluviatilis L.) species in to be growing on the carp eggs in the water from all five oligotrophic lakes than on those in eutrophic lakes (Meng, water bodies. It is worth making a special note about the 1980). It is important to emphasize that in acipenserid discovery of A. arbuscula, A. frigidophilus, C. albicans, F. fish species, with the greatest amount of fungi were found aquaeductum, F. culmorum and Zoopage phanera. Table to be growing on the eggs of eutrophic waters (Czeczuga 3 shows the assimilation tests for amino acids, et al., 1995). There has been an extensive search for carbohydrates and urease for species from the Achlya, effective measures to prevent or limit mycotic infections Aphanomyces, Leptolegnia, Pythium and Saprolegnia of eggs that are in hatcheries, as well as of fish fry and genera. adult specimens in aquacultures. According to Marking et al. (1994), iodiphors and formalin are being commonly used as antifungal agents in fish cultures. DISCUSSION Mycotal species, such as A. diffusa, A. klebsiana, A. orion, Aphanomyces stellatus, A. laevis, P. ultimum and The most eutrophic water came from River Biala and S. parasitica, were frequently found on eggs in the water Pond Fosa, while the water from Spring Cypisek, River from all five water bodies. These species all belong to the Suprasl and Pond Komosa contained the lowest group of opportunistic pathogens that are sapro and amounts of biogenic compounds. necrotrophic (Bruno and Wood, 1999). The eggs of the common carp living in the water from Butler (1911) was the first to describe Allomyces River Biala and Pond Fosa, the most eutrophic of the arboscula, a species found in the water and soil on Czeczuga et al. 485
Table 2. Fungi and straminipiles from the reservoirs analysed.
Taxa Spring Cypisek River Biala River Suprasl Pond Fosa Pond Komosa Fungi Ascomycota Saccharomycetales 1. Candida albicans (Robin) Berrk. x
Blastocladiomycota Blastocladiales 2. Allomyces arbuscula Butler x x x
Zygomycota Zoopagales 3. Zoopage phanera Dreschsler x x
Straminipila Peronosporomycota Leptomitales 4.Leptomitus lacteus (Roth) Agar x x x
Pythiales 5. Pythium pulchrum Minden x x x x 6. P. ultimum Trow x x
Saprolegniales 7. Achlya diffusa J. V. Harv. et J. W. Johnson x x x 8. A. dubia Coker x 9. A. klebsiana Pieters x x x 10. A. megasperma Humphrey x x 11. A. orion Coker et Couch x x x 12. A. prolifera Nees x x 13. A. proliferoides Coker x x 14. Aphanomyces frigidophilus Kitauch. et Hatai x x 15. A. irregularis W. W. Scott x x x x 16. A. laevis de Bary x x x 17. A. parasiticus Coker x x 18. A. stellatus de Bary x 19. Dictyuchus anomalus Nagai x x x 20. D. monosporus Leitgeb x x 21. monilifera (de Bary) Kauf x x 22. Leptolegnia caudata de Bary x 23. Protoachlya polyandra (Lindst.) Apinis x x 24. Saprolegnia anisospora de Bary x x 25. S. australis Elliot x x 26. S. ferax (Gruith) Thurnet x x x x x 27. S. litoralis Coker x 28. S. parasitica Coker x x 29. S. shikotsuensis Hatai et al. x x 30. S. terrestris Cookson et Seymour x 31. Scololegnia asterophora (de Bary) M. W. Dick x x 486 Afr. J. Biotechnol.
Table 2. Contd
Anamorphic fungi Hyphomycetes Moniliales 32. Fusarium aquaeductum (Radlk et Rabh) Saccardo x 33. F. culmorum (W. G. Smith) Saccardo x Total species (the different letters indicate significant difference, p≤0.05) 12a 20b 11a 19b 11a
Table 3. Amino acids, carbohydrate and urease assimilation by straminipiles isolated from Cyprinus carpio.
Species of genus Amino acids Carbohydrate Urease Achlya Asp, Glu, Arg, Ala Fru, Glu, Man, Raf, Suc, Mal, Lac, Mel, Cel, Tre, Sta, Dex, Rha, Gly - Aphanomyces Glu, Ala, Cys Glu, Sta - Leptolegnia Asp, Glu, Ala Fru, Glu, Man, Mal, Mel, Cel, Tre, Sta, Dex, Gly + Pythium Ala, His Fru, Glu, Man, Gal, Raf, Suc, Mal, Lac, Mel, Cel, Tre, Sta, Dex, Rha, Gly, Sal + Saprolegnia Asp, Glu, Arg, Ala, His Fru, Glu, Man, Mal, Cel, Tre, Sta, Dex, Gly +
Amino acids = Ala, alanine; Arg, arginine; Asp, aspargine; Cys, cysteine; Glu, glutamine; His, histidine; Carbohydrate = Fru, froctose; Gal, galactose; Glu, glucose; Man, mannose; Mal, maltose; Mel, melibiose; Cel, cellobiose; Dex, dextrin; Gly, glycerol; Lac, lactose; Rha, rhamnose; Raf, raffinose; Sta, starch; Suc, sucrose; Tre, trehalose.
substrates of plant and animal origin. Our previous study on the eggs in water from Pond Komosa and also in the discussed our findings of Allomyces arbuscula growing bodies of certain invertebrate species (Alton, 1985) and on the eggs of cyprinid species such as Chondrostoma on the coregonidae eggs incubated in hatcheries (Czeczuga nasus, Gobio albipinatus and Scardinus erythrophthalmus and Woronowicz, 1993). F. culmorum, which developed (Czeczuga and Muszynska, 1999). A. arbuscula has also on the eggs of Cyprinus carpio in water from Spring been isolated from the eggs of C. carpio on a fish farm in Cypisek, is known to cause lethal mycosis of carp skin Thailand (Chukanhom and Hatai, 2004). Other species (Hörter, 1960). F. culmorum also afflicts the eggs of from the Allomyces genus, such as A. anomalus (Sati, coregonids (Czeczuga and Muszynska, 1998). According 1983; Khulbe et al., 1995; Czeczuga and Muszynska, to Marchenko (1988), Fusarium avenaceum var. herbarium 1999) and A. macrogynus (Czeczuga and Muszynska, has been found to cause mycosis of the swim bladders of 1999), have also been reported on Cyprinidae eggs, salmonids in areas in the Far East. Some Fusarium including carp eggs. species have also been isolated from Clarias species on A. frigidophilus was first described in Poland and in the African continent (Easa et al., 1984; Refai et al., Europe by Czeczuga et al. (2004) in salmonid eggs. 2010). In our study, the Zoopage phanera species, which Kiziewicz et al. (2013) then reported the presence of A. is rarely found in fishes, was found on eggs in water from frigidophilus in water from Polish springs. Pond Fosa and Pond Komosa. These predacious fungi In the present study, C. albicans was found on the eggs attack terricolous amoebae (Drechsler, 1935), and we of common carp in water from River Biala. This fungus found them in organic debris in the coastal area of bog- had previously been observed on the eggs of Coregonus springs and in mesotrophic-type lakes. They also occur albula in the hatchery in Wegorzewo, Poland and on the on the eggs of rainbow trout (Czeczuga and Muszyńska, fry of eel montee Anguilla anguilla, as well as on the eggs 1999). The information about the amino acid, carbo- of the rainbow trout (Czeczuga and Woronowicz, 1993). It hydrate and urease assimilation by these species that should be mentioned that Hatai and Egusa (1975) occur on the eggs of the common carp did not differ from isolated Candida sake cells from the gastro-tympanites of data obtained from fungal species that developed on the amago salmon. Findings of Fusarium aquaeductum and eggs of other fish species in the waters of Japan (Yuasa F. culmorum on common carp eggs is also worth noting and Hatai, 1996). Some species of Achlya, Aphanomyces, because these belong to an abundant genus comprising Pythium and Saprolegnia genera assimilated some other saprophitic and phytopathogenic species and the amino acids and carbohydrates on the eggs of salmonid facultative parasites of animals, mainly invertebrates species from Oncorhynchus genus (Kitancharoen and (Booth, 1971). In our study, Fusarium aquaeductum grew Hatai, 1998; Czeczuga et al., 2011). Perhaps it is Czeczuga et al. 487
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Vol. 14(6), pp. 489-499, 11 February, 2015 DOI: 10.5897/AJB2014.14311 Article Number: 8E5FCEB50352 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Exploitation of molecular genetics in microbial degradation and decolorization of industrial waste water effluent
Shah M. P.
Industrial Waste Water Research Laboratory, Division of Applied and Environmental Microbiology Environmental Technology Limited Gujarat, India.
Received 11 November, 2014; Accepted 9 February, 2015
Activated sludge samples were used to analyse microbial community structures from the anoxic and aerobic zones of a laboratory-scale modified Ludzack-Ettinger system. Fluorescent in situ hybridization and denaturing gradient gel electrophoresis were applied for analysis. With the help of DNA specific fluorochrome DAPI, approximately 75 to 80% of total cells were detected, hybridised with a specific eubacterial probe for the anoxic and aerobic zones. Results corroborate the dominance of the alpha and gamma subclasses of the Proteobacteria in the anoxic zone whilst the aerobic zone was dominated with the beta subclass of the Proteobacteria. Genetic diversity of the microbial community present in each of the anoxic and aerobic zones was employed by the DGGE technique. Results were obtained from the application of fluorescent in situ hybridisation (FISH) and PCR-DGGE yields a more precise understanding of the microbial community structure and genetic diversity present in domestic wastewater of a laboratory scale treatment process. Nitrogen mass balances indicated an upset in the nitrogen levels for wastewater batches two and seven. The carbon mass balance fell in the range of 92.4 and 105.9% and the nitrogen mass balance fell in the range of 98.4 and 160.0%.
Key words: Fluorescent in situ hybridisation (FISH), denaturing gradient gel electrophoresis (DGGE), COD and nitrogen mass balances.
INTRODUCTION
The growth of the world population, the development of suspended solids and other constituents. These biodegra- various industries and the use of fertilizers and pesticides dable organic compounds are degraded by bacteria in an in modern agriculture has overloaded not only the water aerated reactor and the biomass is allowed to settle and resources but also the atmosphere and the soil with pollu- concentrate in a clarifier (Muyima et al., 1997). The tants (Shah et al., 2013). Treatment of activated sludge system is either a continuous or semi-continuous aerobic within the modified Ludzack-Ettinger (MLE) system involves method for wastewater treatment involving carbon oxidation the removal of biodegradable organics, unsettleable and nitrification. This process has been developed for the
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Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 490 Afr. J. Biotechnol.
removal of carbon, nitrogen and phosphate and it is well Table 1. Process parameters recorded at the time of known that prokaryotic micro-organisms catalyses these sampling. main biological processes in wastewater treatments (Juretschko et al., 2002); nutrients and oxygen present Parameters Value the microbial population in the wastewater achieves pH of the digester 7.1 optimal growth and respiration (Muyima et al., 1997). The Total Solid g/L 22 dynamics and diversity of the microbial populations in Organic loading rate applied kgCOD/m3 day 4.5 activated sludge have been analysed by culture-dependent Influent COD mg/L 41,300 methods, however many members of the natural bacterial VFA mg/L 517 communities are still unculturable (Wagner et al., 1994). Acetic acid mg/L 275 Hence, microscopic identification based on morphological COD removal efficiency % 93 characteristics was researched and developed in a Methane % v/v 56 culture-independent manner by direct rRNA sequence 3 Biogas rate m /day 1200 retrieval, where nucleic acid probes which are comple- mentary to the rRNA are used as tools to monitor popula- tion dynamics amongst bacteria (Amann, 1995). FISH makes use of rRNA targeted probes which are frequently between microbial communities (Kaewpipat and Grady, applied in order to quantify the composition of microbial 2002). In this study, a combination of molecular techniques, communities present. This procedure is based on the FISH and PCR-DGGE was used to monitor the microbial composition and examine the microbial community comparative analysis of macromolecules, mostly ribosomal population shifts within a steady state laboratory-scale RNA molecules and fluorescent derivatives of such probes. These probes have been applied successfully for in situ parent anoxic and aerobic activated sludge system. enumeration of defined groups of micro-organisms present in activated sludge (Manz et al., 1994). MATERIALS AND METHODS Fluorescently monolabelled, rRNA targeted oligonu- cleotide probes detect individual cells, allowing whole-cell Sampling and bioreactor set-up hybridisation with rRNA targeted probes to be a suitable Activated sludge was collected from a 500 m3 closed digester tank tool inferring phylogenetic evolution hence the cell (CDT) for the anaerobic treatment. The CDT was operated under morphology of an uncultured microbe and its abundance mesophilic condition (32 to 39°C) for 120 days. The system was can be determined in situ (Wagner and Amann, 1997). equipped with a closed digester tank, settling tank, pumps and flow meters for biogas and the effluent. There were three sampling ports Cell numbers of the bacteria can be obtained by enume- at the top, in the middle and at the bottom of the CDT. The sludge ration under an epi-fluorescent microscope. Enumeration sample was obtained from middle sampling port. The process procedures involve the use of a semi-automated digital parameters recorded at the time of sampling are shown in Table 1. image analysis tools in order to quantify the fluorescently labelled bacteria in samples (Daims et al., 2001). The Analyses molecular technique used for analysing the structure and providing a profile of the microbial population present in The laboratory-scale modified Ludzack-Ettinger process was fed wastewaters is the denaturing gradient gel electrophoresis with 30 ℓ of mainly domestic wastewater on a 10 day sludge age (DGGE) (Muyzer et al., 1993). DGGE that is performed process. After reaching steady state, the MLE system was run for duration of 18 batches. The wastewater was diluted with tap water on 16S rRNA genes has been used to produce a genetic to give an influent feed of approximately 500 COD mg /L from the fingerprint of mixed microbial communities (Kaewpipat and original 800 COD mg /L. The process was maintained by wasting Grady, 2002). The numbers of bands that are obtained 1.5 L/day of the mixed liquor from the aerobic reactor. Samples from DGGE profiles provide an estimate of the different from the influent, anoxic, aerobic and effluent were analysed daily microbial species present. The intensity of each band for the following: chemical oxygen demand (COD), total Kjeldahl provides a reflection of the relative abundance of each nitrogen (TKN), mixed liquor suspended solids (MLSS), volatile suspended solids (VSS) and nitrate analysis (nitrite tests were not species (Nasu, 2000) because the primers used to amplify performed because the nitrite concentration was 2% less than a fragment of the 16S rRNA produces a quantitative relation- nitrates). Microbial community analyses were performed on ship between the gene copy number and the PCR-DGGE samples obtained from the aerobic and anoxic reactors. The band intensity (Kaewpipat and Grady, 2002). However, oxygen utilisation rate (OUR) was monitored within the mixed liquor many factors can prevent the formation of the number with an online dissolved oxygen (DO) controller (hi-tech micro- and intensity of the bands in the DGGE gel, therefore system) according to Randall et al. (1991). The pH was kept constant at 7.5 and the temperature was kept constant at 20°C. representing the exact number and abundance of species in a microbial community can be difficult however DGGE is a sensitive and a rapid technique that detects most Total cell counts singe-base variations when a G-C clamp is added to one Membrane filtration was performed according to Hicks et al. (1992). of the primers in the PCR process. This provides a profile Fixed samples were sonicated and diluted (dilution factor of 200) of changes that occur within a microbial community or with phosphate-buffered saline (PBS) and 1% nonidet. Nucleopore Shah 491
filters with a pore size of 0.2 μm were pretreated with 0.3% Sudan The recombinant plasmids were extracted using the Qiagen black and placed on top of a 0.45 μm backing filter. The samples plasmid DNA extraction kit (Qiagen, Germany) according to the were stained with DAPI (4,6-diamidino-2-phenylindole) (Sigma, manufacturer's instructions. Approximately, 27 nucleotides were Deisenhofen, Germany) at an end concentration of 1.25 μg/mℓ. The sequenced using an ABI 3730 XL DNA Sequencer. Chromato- Zeiss Axiolab microscope (50 W high-pressure mercury bulb and grams were edited using Chromas software (Technelysium, Zeiss filter set 01) fitted for epifluorescence microscopy was used Australia), while CHECK_CHIMERA software (Maidak et al., 1997) with the Zeiss image analysis software in order to quantify the fluo- was used to scan for potential chimeric sequences. The sequences rescing cells. were compared to known 16S rRNA sequences in the GenBank™ database, using the basic logical alignment search tool (BLAST) to locate nearly exact matches in the GenBank database. DNA Hybridization and pretreatment sequences were aligned using the program CLUSTAL W (Thompson et al., 1994) and further edited manually. Phylogenetic The fixed and sonicated samples were immobilised onto pretreated analyses were performed using the neighbor-joining (NJ) method slides and dehydrated with 60, 80% and absolute ethanol to using the MEGA ver. 3.1 (Kumar et al., 2004). prepare for whole-cell hybridisation. Samples were hybridised with appropriate hybridisation buffers and probed in situ with oligonu- cleotide probes listed in Table 1 according to Amann (1995). The PCR-based DGGE fingerprinting of methanogens probes were either labelled with tetramethylrhodamine-5- isothiocyanate or 5(6)-carboxyfluorescein-N-hydroxy sccinimide- For each RFLP group, one representative clone was selected for ester. The probe-conferred fluorescence was detected with a Zeiss further study. DGGE-PCR reactions were performed on sludge Axiolab microscope (50 W high-pressure mercury bulb and Zeiss DNA, as well as on the extracted plasmids from representative filter sets 09 and 15). Dual staining of samples with DAPI and clones. The aim was to obtain the DGGE profiles for the archaeal fluorescent rRNA probes was performed according to Hicks et al. and methanogenic species in the POME anaerobic bioreactor. (1992). Therefore, universal DGGE methanonenic and archaeal primers were used in the amplification reactions. The universal archaeal
primers PARCH340f and PARCH519r based on the E. coli 16S DNA extraction and PCR amplification rRNA gene sequence and methanogenic primers 0357F and 0691R DNA was extracted from activated sludge following the method were used to amplify 179 and 334 bp fragments (Watanabe et al., of Bourrain et al. (1999) and 16S rDNA was amplified using 2004), respectively. The GC clamp, 5‟-CGC CCG GGG CGC GCC the Wright and Pimm (2003) methanogenic specific primers Met86F CCG GGC GGG GCG GGG GCA CGG GGG-3‟ as described (5‟-GCTCAGTAACACGTGG-3‟) and Met1340R (5‟- by Chan et al. (2001), was included on the 5 end of the forward CGGTGTGTGCAAG GAG-3‟). PCR reactions (25 µL) were set up primer PARCH340f and 0357F to enable the separation of the in 0.2 mL thin-walled Eppendorf® tubes, containing 100 ng sludge fragments using DGGE. PCR reactions for both primer sets were DNA, 10 X Taq DNA polymerase buffer, 0.5 µL of 10 mM dNTP mix performed in 50 µl reaction volumes containing 0.5 µl of Taq DNA (Fermentas, Maryland, USA), 0.5 µl of 10 mM dNTPs, 2.5 µl of 25 polymerase (5 U/µl) (Fermentas, Maryland, USA), 5 µl of 10 x PCR reaction buffer, 0.5 µl of each of the primers (10 mM), 5 µl dNTPs mM MgCl2, 0.5 µl of each methanogen primer and 0.2 µl of 5 U AmpliTaq DNA polymerase (Fermentas, Maryland, USA). DNA (10 mM), 4 µl of MgCl2 (Fermentas, Maryland, USA) and 1 µl of the templates from three sludge samples obtained from the bottom and extracted DNA or plasmid. The PCR amplification conditions for the middle of the bioreactor, and from the recycling tank were amplified primer sets were as follows: initial denaturation was performed at separately for cloning. Samples were amplified using a Perkin 94°C for 3 min; followed by denaturation at 94°C for 1 min; Elmer Gene Amp system 9600. Each PCR cycle consisted of 94°C annealing at 60°C for archaeal primers and 58°C for methanogenic for 40 s, 54°C for 50 s, and 72°C for 90 s. On the 35th cycle, the primers for 1 min; and chain elongation at 72°C for 2 min. On the elongation step was increased to 10 min at 72°C. 35th cycles, the final elongation step was performed at 72°C for 8 min. The amplified products were checked on 1% agarose gels and visualized under UV light. Cloning 16S rDNA
PCR products were purified from a 1% agarose gel using a gel DGGE extraction kit according to the manufacturer‟s instructions (Qiagen, Germany). The recovered PCR fragment was cloned into pTZ57R DGGE was used to separate the 179 and 334 bp PCR products. vector (Fermentas, Maryland, USA), and transformed into Escherichia The initial parameters for the denaturing gradient were optimized coli TOP10 competent cells (Invitrogen, Carlsbad, CA). White empirically. The optimized gradient extended from 40 to 60% and colonies were randomly selected from plates and recombinant 50 to 60% of denaturant consisting of 7 M urea and 40% formamide plasmids identified by size selection on a 1% agarose gel prior to for methanogens and archaeal PCR products, respectively; this restriction fragment length polymorphism (RFLP) analysis. was established in 8% acrylamide gels. Electrophoresis was performed at 100 V for 14 h at a constant temperature of 60°C. The DNA was stained with cyber green and visualized under UV light. RFLP and sequences analysis
Cloned PCR products were digested separately with HaeIII Band selection, DNA purification and sequencing (Fermentas, Maryland, USA) in 20 µl reaction volumes containing 10 µl of PCR product, 2 µl x 10 reaction buffer and 10 units of All DGGE bands from the methanogenic profile were punched out restriction endonuclease in sterile ultrapure water. Digested PCR with a sterile pasture pipette and used as a template in a re- products were incubated for 1 h at 37°C and digested fragments amplification using the methanogens` primers 0357F and 0691R were separated using a 10% polyacrylamide gel (w/v). The resulting (Watanabe et al., 2004). The resultant PCR products were purified bands were visualized by silver-staining and each clone was using the Mag Extractor-PCR and Gel Clean up-kit. (Toyobo, assigned to an RFLP group based upon the banding patterns. Two Japan) according to the manufacturer‟s instructions and sequenced groups (groups I and II) were assigned. For each RFLP type, the using an ABI 3730 XL DNA Sequencer at the DNA Sequencing 16S rRNA insert of a few representative clones were sequenced. Facility at Kyushu Institute of Technology, Japan. The sequences 492 Afr. J. Biotechnol.
Table 2. Steady state results for the MLE anoxic and aerobic activated sludge system
COD TKN Nitrate OUR Mixed Liquor WW (in ppm) (in ppm) (in ppm) (in ppm) (in ppm) Batches INF EFF INF EFF Anoxic Aerobic Effluent VSS COD TKN 502.6 46 38.8 5.3 0.6 3.9 7.9 30.6 2352.4 3720 234.6 1 (48.8) (10.1) (6.3) (2.1) (1.0) (1.9) (1.5) (0.4) (153.1) (553.2) (20.7) 506 46.8 41.9 3.5 1.2 6.9 10.0 26.7 2341.6 3652.8 223.7 9 (16.1) (7.8) (3.4) (0.9) (1.0) (1.6) (1.3) (1.9) (251.9) (426.3) (21.9) 449.2 39.1 42.1 4.4 0.9 7.2 8.9 30.7 1914.5 2903.7 201.3 18 (40.0) (8.2) (5.4) (0.8) (0.5) (2.3) (2.5) (0.7) (239.2) (372.1) (49.3)
obtained were compared to 16S rRNA gene sequences in the aerobic zone had a higher level of concentration with an National Center for Biotechnology Information (NCBI) database average of 7 N mg/L present, indicating an increase in using the BLAST. nitrification; however, the highest concentration of nitrates
was present in the effluent with an average of 11.3 N FISH mg/L. These results suggest that a high denitrification potential of the MLE process is prominent; however, To directly analyze methanogens populations in an anaerobic complete denitrification is not possible due to the absence digester sludge sample, the probe MSMX860, complementary to of secondary reactors, which is clearly shown in the the 16S rRNA of some methanogens including Methanosarcina effluent results. The mixed liquor was analysed by perfor- spp., Methanococcoides spp., Methanolobus spp., Methanohalophilus sp. and Methanosaeta spp. was used (Crocetti ming volatile suspended solids (VSS), chemical oxygen et al., 2006). To target the sludge bacteria, the 16S rRNA probe demand (COD) and total Kjeldahl nitrogen (TKN). VSS EUB338 for the domain bacteria was used (Amann et al., 1990). analyses showed a high amount of biomass present in the mixed liquor, which accounted for the increased levels of chemical oxygen demand and total Kjeldahl Fixation and permeabilization of the cells nitrogen concentrations (results for only 3 wastewater Cells were fixed and hybridized using the protocol reported batches are shown). from Amann (1995) with some modification (Sakai et al., 2004). The sludge sample was fixed in 3% paraformaldehyde/phosphate buffer saline (PBS) (PBS; 130 mM NaCl, 10 mM sodium phosphate buffer, Screening the 16S rRNA clone library by RFLP pH 7.0) for 1 to 3 h at 4°C, pelleted by centrifugation (3500 x g, 15 min, 4°C) and then stored in a 1 : 1 mixture of ethanol and PBS. 16S rRNA was PCR amplified from the DNA extract of Fixed cell suspensions were diluted using an ethanol/PBS mixture sludge taken from an anaerobic bioreactor. From 237 clones (8 µl) and were spotted on coated glass slides, dried at 46°C for 30 screened (84 from the bottom sludge library, 75 from the min and immersed for 3 min each in 50, 80, and 99% (v/v) ethanol. Cell smears were covered with 20 µl of lysozyme (50 mg/mL PBS; middle sludge library and 78 from the recycling sludge), 37°C, 30 min). Enzymatic digestion was terminated by thoroughly all had a DNA insert of the correct size (1260 bp). Two rinsing the slides with distilled water followed by air-drying at room different HaeIII RFLP-patterns were observed from the temperature (25 ± 2°C). clone libraries and assigned as groups I and II (Figure 1). Representative examples from RFLP group I and II (SamaliEB and Samali15) are shown in Figure 1. RFLP RESULTS AND DISCUSSION group I contained 97.1% of clones and 2.9% fell in group II. Modified Ludzack-Ettinger system
The reliability of the experimental data obtained from the Phylogenetic analysis of representatives from each steady state analyses which included COD, TKN, OUR, RFLP group nitrates and mixed liquor analyses is represented in Table 2. The chemical oxygen demand concentration Partial DNA sequences were obtained from representative calculated for the MLE process was 90.9%, thus amplicons for each RFLP group. indicating that a high amount of oxygen (as shown with Approximately 27 clones were sequenced (20 from the OUR concentrations) was required for the proliferating group I and 7 from group II), and were used to search the bacteria and their biological reactions. The nitrate concen- GenBank nucleotide database with the BLAST search trations of the anoxic, aerobic and effluent zones are as tool. One representative for each RFLP group was follows: the anoxic zone supported high rates of denitrify- included in the phylogenetic tree. The sequence data set cation with an average of 1.5 N mg/L present whilst the contained 20 sequences, including Methanopyrus kandleri Shah 493
Figure 1. Calculated (a) and observed (b) HaeIII RFLP patterns that occurred among 237 cloned archaeal 16S rRNA fragments from sludge taken from bottom and middle of the bioreactor and the recycling tank. Fragment sizes were inferred from DNA sequences of representative clones. M: 100 kb molecular mass marker (Fermentas, Hanover, Maryland, USA); RFLP patterns were labeled „I‟; Samali EB, II; Samali15‟ and M. concilii (M59146).
that was used as the outgroup (Figure 2). the identification of the band as M. concilii. Band EBM2 Matches obtained for each clone sequenced had a showed a 99% sequence similarity to the uncultured similarity of ≥ 99% with those contained in the GenBank Methanosaeta sp. clone I2 that had been previously found nucleotide database. All RFLP groups I and II clones in environmental samples taken from anaerobic biofilms. sequenced were Methanosaeta concilii. Although, the Band EBM4 showed a 97% sequence similarity to RFLP group II clones had a unique HaeIII restriction Methanosarcina sp. 48. The methanogenic DGGE pattern profile, partial sequencing of the clones revealed a 99% showed that the band which belonged to Methanosaetaceae identity with M. Concilii (M59146), a sequence shared by appeared to be denser and sharper than the one of all of the samples in RFLP group I. This distribution of the Methanosarcina. The double bands observed using same species sequence in two different RFLP groups archaeal primers; PARCH340f and PARCH519r (Figure illustrates the limitation of relying on partial DNA 3b) was a result of degeneracy of the primers used sequences as well as RFLP only for representing the (Piceno et al., 1999). The archeal PCR products using diversity of samples contained in a library. clones SamaliEB and Samali15 as DNA template did not align on the DGGE gel with the band related to SamaliEB lying lower which was due to a single nucleotide difference PCR-based DGGE analysis between the two amplicons (Figure 4). As shown in the Figure 4, one base pair G/C in SamaliEB is replaced with A total of five methanogenic bands (EBM1, EBM2, EBM3, A/T in Samali15. As a matter of fact, DNA fragments EBM4 and EBM5) (Figure 3a) were excised from the richer in G/C are more stable and remain double-stranded methanogenic DGGE fingerprints, re-amplified, purified until it reached higher denaturant concentrations (lower and sequenced. Yielded sequences (334 bp) were levels of the gel). Double-stranded DNA fragments analyzed by CHECK_CHIMERA software (Maidak et al., migrate better in the acrylamide gel, while denatured 1997) to scan for potential chimeric sequences and three DNA molecules become effectively larger and stop in the were found chimeric. The remaining two (EBM2 and gel. In this study, a DNA fragment of higher G/C content EBM4) were analyzed using the BLAST program. The stoped at a lower level of gel and by providing a narrow RFLP groups‟ representatives (SamaliEB and Samali15) range of optimized gradient (50 to 60%), a subtle were in line with EBM2. Band EBM4 was found to be difference between the DNA fragments known as single Methanosarcina. Although, band EBM2 correlated with nucleotide polymorphism (SNP) was detected. This the reference clones SamaliEB and Samali15 (M. confirmed the RFLP results which found clone SamaliEB concilii), it was also excised and sequenced to confirm to be a different strain of M. concilii in comparison with 494 Afr. J. Biotechnol.
Figure 2. A phylogenetic tree, showing how clones SamaliEB and Samali15 are related to other methanogens.
Figure 3. PCR-based DGGE fingerprints of the methanogens-denaturants gradient from 40 to 60% (a), and archaea denaturants gradient from 50 to 60% (b) present in the CDT treating POME using methanogenc and archaeal primers. Lane 1 (a): Methanogens present in the anaerobic bioreactor; Lane 1 (b) Archaea present in the anaerobic bioreactor; Lane 2 and 3: Methanosaeta concilii (SamaliEB and Samali15). Shah 495
Figure 4. Alignment of the Methanosaeta concilii clone SamaliEB (EU580025) and Methanosaeta concilii clone Samali15 (EU580026) amplified by archaeal primers PARCH340f and PARCH519r using CLC Free Workbench version 4.1.2 (CLC Bio A/S, Denmark).
the other clones including clone Samali15 and the gene bank database. In combination with the DGGE fingerprint using Methanogens primers, just two of the archaeal bands.
Analysis of sludge sample by FISH
Under optimal hybridization conditions, methanogens and bacteria were specifically visualized and detected using the corresponding probes. Figures 5 and 6 show represen- tative micrographs of the fluorescent methanogenic and bacterial cells in the sludge sample double-stained with rhodamine-EUB338 and FITC-MSMX860. Solid and complex materials in digested sludge showed strong self fluorescence that pictures taken by a chilled CCD color camera with a modified RGB color balance helped distin- guish the fluorescence of FITC-probes from the self fluorescence of the refuse materials. The self- fluorescence of refuse materials was yellowish in the Figure 5. FISH staining of the sludge sample analyzed by fermented sample. The presence of members of the confocal laser microscopy of FISH cells (a, b and c) and light microscopy (d). (a) Methanosaeta concilii; (b) bacteria; (c) Methanosaetaceae and Methanosarcinaceae in sludge simultaneously hybridized with rhodamine-labeled bacterial- was also in accordance with DGGE results but, the domain probe (EUB338) (red) and FITC-labeled methanogens presence of Methanosarcinaceae was not detected by probe (MSMX860) (green) showing the consortium between PCR-cloning. All aceticlastic methanogens belong to the methanogens and bacteria; (d) Light microscopy. 496 Afr. J. Biotechnol.
assumed to form the initial nuclei followed by floc formation as well as described in Spaghetti Theory (Wiegant, 1987). Therefore, Methanosaetaceae plays an important role in floc formation in CDT and consequently higher performance of the bioreactor in terms of COD removal and methane production. However, this contrasts with the findings of Sallis and Uyanik (2003) indicating that associations between mixed bacterial populations and inert material formed the so-called nuclei at the initial stage of granule formation and the predo- minance of Methanosaetaceae occurred only when the granules had developed. This methanogen is one of the main species responsible for the conversion of acetate to methane. Species within this family use acetate as their sole energy source, which is metabolized into methane and carbon dioxide. These organisms can be found in anaerobic sediments and anaerobic sludge bioreactors Figure 6. FISH staining of the sludge samples analyzed by (Garrity and Holt, 2001). In order to maximize the efficiency confocal laser microscopy of fluorescent in situ -hybridized of the bioreactor in terms running operation; for example, cells (a, c and d) and light microscopy (b). A fluorescent in situ 3 -hybridized floc of Methanosaeta concilii (a); light micrograph of organic loading rate (OLR) (kg COD/m /day), pH and a floc of Methanosaeta concilii (b); filaments of Methanosaeta alkalinity is of great importance. In this study, high COD concilii (c); simultaneously hybridized with rhodamine-labeled removal 93% was observed which was higher than the bacterial-domain probe (EUB338) (red) and FITC-labeled findings of Choorit and Wisarnwan (2007) at mesophilic methanogens probe (MSMX860) (green) showing conditions. This was achieved due to a higher retention Methanosarcina (d). time (10 days) in comparison with seven days used in
their study. However, the methane production of 1.35 L/L[reactor]/day was lower which could be attributed to a order Methanosarcinales comprising two families, different bioreactor design (continuous stirred tank reactor Methanosarcinaceae and Methanosaetaceae. Among six (CSTR)) which enables higher application of OLR. genera belonging to Methanosarcinaceae, the genus Hence, higher methane production could be achieved. Methanosarcina can only utilize acetate as a substrate. All members of the family Methanosaetaceae are aceticlastic (Garrity and Holt, 2001). The Quantification of methanogens by FISH Methanosaetaceae and Methanosarcinaceae families differ in their physiology, biokinetics, and growth The results of FISH counting showed that the total environment with respect to acetate concentrations. The number of bacteria in the sludge sample was 1.4 x 105 family Methanosaetaceae has a high affinity for acetate cells/ml sludge. The major group in the treated sludge accompanied with a relatively low growth rate, while the sample was the Methanosaeta sp. with a count of 2 x 108 family Methanosarcinaceae has a much lower substrate cells/ml sludge. In contrast, the number of affinity but with a higher growth rate, that describes why Methanosarcina sp. was almost 1000 times lower and the POME anaerobic digester used in the present study was equal to that of bacteria. In other words, for each one was dominated by Methanosaetaceae as the acetate thousand cells of Methanosaeta sp., there was just one concentration in the sludge digester favourable to them single cell of Methanosarcina sp. present in the sludge. and acetate is the only substrate that supports the growth These results indicate that, the closed fermentation of of Methanosaetaceae (Garrity and Holt, 2001). The POME led to the domination of Methanosaeta sp. in presence of members of the Methanosaetaceae in particular M. concilii emphasizing on the importance of anaerobic bioreactors has been widely reported (Chan et the substrate. The VFAs concentration of 517 mg/L al., 2001; Batstone et al., 2004; Hulshoff Pol et al., 2004). showed high activity of methanogens which was reflected It is also generally known that the presence of by high methane production (56% v/v methane of 1200 Methanosaeta species leads to an improved granulation m3 biogas/day) and satisfactory COD removal of 93%. process and it results in a more stable bioreactor The high number of methanogenic archaea (2 x 108 performance (Hulshoff Pol et al., 2004). The bioreactor cells/ml sludge) in comparison with the bacteria cells (1.4 used in this study was designed for continuous or x 105 cells/ml sludge) might be attributed to the stage of intermittent mixing to maximize the contact between sampling at which the highest methane production was POME and the microbes. This differs from the upflow recorded. The finding also showed that quantitative FISH anaerobic sludge blanket (UASB) design in which using group and species specific rRNA-targeted probes granules are formed. However, Methanosaetaceae is could be applied to the direct analysis of semi-solid POME Shah 497
Figure 7. Light micrographs of POME sludge at a magnification of: (a) 10x; (b) 40x; (c) 100x. Gram staining test showed that the filamentaous-like Methanosaeta sp. were dominant species in the CDT sludge.
sludge to provide a good estimate of the dominant species of was not monitored. Methanosaeta sp. was normally methanogens which plays an important role in optimizing determined to be at the core of the granules formation in running conditions of the digester for achieving the UASB system (Hulshoff Pol et al., 2004). A demand to be highest COD removal and methane production. The strictly anaerobic and consume solely acetate makes PCR-cloning, DGGE analysis and species-specific FISH them to be located at the core and other archaea-like confirmed that M. concilii was the dominant species Methanosarcina sp., fermentative and facultative bacteria during the fermentation of POME. are placed on the outer granules layer to prevent oxygen contamination (Savant et al., 2002). Some other microbes that coexisted in the digester are shown in Figure 8c. SEM However, Methanosaeta-like microorganisms outnum- bered short rods and cocci. The presence of Methanosaeta sp. in the studied industrial activated sludge was corroborated by its morphology using light microscopy (Figure 7). It has been Concluding remarks reported that Methanosaeta sp. can form filaments of 10 to 300 cells (Garrity and Holt, 2001). The scanning The results indicate that filamentous acetate utilizing electron microscopy provided a better resolution of methanogens detected in anaerobic bioreactor treating Methanosaeta sp. than that obtained by conventional POME belong to the genus Methanosaeta based on the light microscopy. Filamentous like Methanosaeta sp. was cell-morphology, and the phenotypic and phylogenetic observed and found to be the dominant species in the characteristics described above. The data obtained CDT system (Figure 8a and 8b). It is clearly seen that the suggest that M. concilii is the most abundant methanogen microorganisms bend around the organic matters and in POME anaerobic digestion and that it plays an thus play an important roles in flocs/aggregate formation important role in methane production from acetate and during POME treatment. Nevertheless, the actual size of the optimum condition for its growth should be considered the flocs or aggregates formed during the CDT process when an attempt is made to treat POME anaerobically. 498 Afr. J. Biotechnol.
Figure 8. Scanning electron micrographs of sludge showing microorganisms in the flocs of POME sludge.
It also revealed the presence of possibly new strain of Juretschko S, Loy A, Lehner A, Wagner M (2002). The microbial Methanosaeta in the bioreactor for treating POME. community composition of nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analysed by the full cycle RNA approach. Syst. Appl. Microbiol. 25: 84-99. Kaewpipat K, Grady CPL (2002). Microbial population dynamics in Conflict of interests laboratory-scale activated sludge reactors. Water Sci. Technol. 46 (1):19-27. Kumar S, Tamura K, Nei M (2004). MEGA 3: integrated software for The author did not declare any conflict of interest. molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5(2):150-163. Maidak BL, Olsen GJ, Larsen N, Overbeek R, Mccaugheu MJ, Woese CR (1997). The RDP (ribosomal database project). Nucleic Acids REFERENCES Res. 25(1):109-110. Manz W, Wagner M, Amann R, Schleifer KH (1994). in situ Amann RI (1995). In situ identification of microorganisms by whole cell characterization of the microbial consortia active in two wastewater hybridisation with rrna-targeted nucleic acid probes. Molecular treatment plants. Water Res. 28 (8):1715-1723. Microbial Ecology Manual pp. 331-345. Muyima N, Momba M, Cloete T (1997). Biological methods for the Amann RI, Krumholz L, Stahl DA (1990). Fluorescent-oligonucleotide treatment of wastewaters. In: cloete te and muyima nyo (eds.) probing of whole cells for determinative, phylogenetic, and Biological methods for the treatment of wastewaters. International environmental studies in microbiology. J. Bacteriol. 172 (2):762-770. Association on Water Quality. pp. 1- 19. Batstone DJ, Keller J, Blackall LL (2004). The influence of substrate Muyzer G, Waal EC, Uitterlinden AG (1993). Profiling of complex kinetics on the microbial community structure in granular anaerobic microbial populations by denaturing gradient gel electro phoresis biomass. Water Res. 38(6):1390-1404. analysis of polynerase chain reaction-amplified genes coding for 16s Bourrain Muriel, Achouak Wafa, Urbain Vincent, Heulin Thierry (1999). rRNA. Am. Soc. Microbiol. 59 (3):695-700. DNA extraction from activated sludges. Curr. Microbiol. 38(6):315- Nasu M, Yamaguchi N, Tani K (2000). Microbial community structure 319. and their activity in aquatic environment. Proc. Symp. Establishment Chan OC, Liu WT, Fang HH (2001). Study of microbial community of and evaluation of advanced water treatment technology systems brewery-treating granular sludge by denaturing gradient gel using functions of complex microbial community. The international electrophoresis of 16S rRNA gene. Water Sci. Technol. 43(1):77-82. symposium. Tokyo. Crocetti G, Murto M, Bjornsson L (2006). An update and optimization of Piceno YM, Noble PA, Lovell CR (1999). Spatial and temporal oligonucleotide probes targeting methanogenic Archaea for use in assessment of diazotroph assemblage composition in vegetated salt fluorescence in situ hybridization (FISH). J. Microbiol. Methods marsh sediments using denaturing gradient gel electrophoresis 65(1):194-201. analysis. Microb. Ecol. 38(2):157-167. Daims H, Ramsing NB, Schleifer KH, Wagner M (2001). Cultivation- Randall EW, Wilkinson A, Ekama GA (1991). An instrument for the independent, semiautomatic determination of absolute bacterial cell direct determination of oxygen utilisation rate. Water Salination 17 numbers in environmental samples by fluorescence in situ (1): 11-18. hybridization. Appl. Environ. Microbiol. 67 (12): 5810-5818. Sakai K, Mori M, Fujii A, Iwami Y, Cukeatirote E, Shirai Y (2004). Garrity GM, Holt JG (2001). Phylum AII. Euryarchaeota phy. nov. In: Fluorescent in situ hybridization analysis of open lactic acid Boone DR, Castenholz RW. eds. Bergey‟s Manual of Systematic fermentation of kitchen refuse using rRNA targeted oligonucleotide Bacteriology. 2nd Ed. New York, Springer. pp. 211-294. ISBN 0-387- probes. J. Biosci. Bioeng. 98(1):48-56. 98771-1. Sallis PJ, Uanik S (2003). Granule development in a split-feed Hicks RE, Amann RI, Stahl DA (1992). Dual staining of natural anaerobic baffled reactor. Bioresour. Technol. 89(3):255-265. bacterioplankton with 4, 6-diamidino-2-phenylindole and fluorescent Savant DV, Shouche YS, Prakash S, Ranade DR (2002). oligonucleotide probes targeting kingdom level 16s rrna sequences. Methanobrevibacter acididurans sp. nov., a novel methanogen from a Appl. Environ. Microbiol. 58:2153-2163. sour anaerobic digester. Int. J. Syst. Evol. Microbiol. 52(4):1081- Hulshoff Pol LW, De Castro Lopes SI, Lettinga G, Lens PNL (2004). 1087. Anaerobic sludge granulation. Water Res. 38(6):1376-1389. Shah MP, Patel KA, Nair SS, Darji AM (2013). An innovative approach Shah 499
to biodegradation of textile dye (remazol black) by bacillus spp. Int. J. Watanabe T, Asakawa S, Nakamura A, Nagaoka K, Kimura M (2004). Environ. Bioremediat. Biodegrad. 1:43-48. DGGE method for analyzing 16S rDNA of methanogenic archaeal Thompson JD, Higgins DG, Gibson TJ (1994). CLUSTAL W: improving community in paddy field soil. FEMS Microbiol. Lett. 232(2): 631-637. the sensitivity of progressive multiple sequence alignment through Wiegant WM (1987). The “spaghetti theory” on anaerobic sludge sequence weighting, position-specific gap penalties and weight formation, or the inevitability of granulation. In: Lettinga G, Zehnder matrix choice. Nucleic Acids Res. 22(22):4673-4680. AJB, Grotenhuis JTC, Hulshoff Pol LW. eds. Granular Anaerobic Wagner M, Amann R (1997). Molecular techniques for determin¬ing Sludge Microbiology and Technology: Proceedings of the GASMAT microbial community structures in activated sludge. In: cloete te and Workshop. Lunteren, The Netherlands. pp. 146-152. muyima nyo (eds.) Molecular techniques for determining microbial Wright ADG, Pimm C (2003). Improved strategy for presumptive community structures in activated sludge. International Association identification of methanogens using 16S riboprinting. J. Microbiol. on Water Quality. pp. 61-70. Methods 55(2):337-349. Wagner M, Amann R, Lemmer H, Manz W, Schleifer KH (1994). Probing activated sludge with fluorescent labeled r-rna targeted oligonucleotides. Water Sci. Technol. 29 (7):15-23.
Vol. 14(6), pp. 500-508, 11 February, 2015 DOI: 10.5897/AJB2014.14008 Article Number: B2A431750359 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Influence of temperature on the anaerobic stabilization of organic solid residues
Jorge Marcell Coelho Menezes1, Valderi Duarte Leite1, Aldre Jorge Morais Barros2, Wilton Silva Lopes1,*, José Tavares de Sousa1 and Andrezza Raphaella Costa Campos1
1Departamento de Engenharia Sanitaria e Ambiental, Centro de Ciencias e Tecnologia, Universidade Estadual da Paraiba, 58429-600, Campina Grande, PB, Brazil. 2Unidade Academica de Tecnologia do Desenvolvimento, Centro de Desenvolvimento Sustentavel do Semiarido, Universidade Federal de Campina Grande, 58540-000, Campina Grande, PB, Brazil.
Received 28 June, 2014; Accepted 2 February, 2015
This study was aimed at determining the effect of temperature on the stabilization of organic solid waste conjugated with sewage sludge in anaerobic batch reactors (ABR), and to estimate the efficiency of the process in producing biogas and methane. The substrate contained 36.2 g L-1 of total solids comprising residues from fruits and vegetables enriched with anaerobic sludge from sanitary sewage (4:1, w/w). The reactors were of 1.15 L capacity and were operated at 25.5, 40 and 50°C (ABR1, ABR2 and ABR3, respectively) for 160 days. The efficiencies of transformation of total volatile solids in ABR1, ABR2 and ABR3 were, respectively, 43.2, 34.2 and 32%, and the transformation of chemical oxygen demand showed a similar tendency with efficiencies of 39.5, 33.6 and 16.6%, respectively. The mean volumes of biogas accumulated by ABR1, ABR2 and ABR3 were 28.85, 21.24 and 20.54 L, respectively, while the respective mean volumes of methane were 9.04, 7.11 and 1.11 L. The results demonstrate that the activity of methane-producing microorganisms was inhibited at higher process temperatures. It is concluded that anaerobic digestion at ambient temperature represents an economical and environmentally viable strategy for the disposal of municipal solid wastes.
Key words: Plant residues, anaerobic digestion, biofuel, methane.
INTRODUCTION
Bordering on 260, 000 tons of municipal solid waste 50% (w/w) of MSW comprises organic matter (mainly (MSW) are collected each day in Brazil and of this, 50.8% food, kitchen and green waste) that is readily biodegra- ends up in open dumping grounds, 22.5% in controlled dable and fermentable (Gossett et al., 1982). landfills and 27.7% in sanitary landfills (Instituto Brasileiro This material represents an important resource that de Geografia e Estatística, 2008). It is reported that some could be converted through anaerobic digestion into
*Corresponding author. E-mail: [email protected]. Tel: +55 83 88038416. Fax: +55 83 3315 3311.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License
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methane for use in the national energy matrix, or by development of mesophilic microorganisms, given aerobic processes into solid fertilizer for use in agriculture temperature of 21.5°C approaches the level set for (Leite et al., 2009). However, the utilization of the mesophilic microorganisms and the temperature level biodegradable organic fraction of MSW, along with other 50°C, as is favorable for the development of thermophilic recyclable fractions (such as paper, cardboard, plastics, microorganisms. Therefore, the temperature range varied glass, and ferrous and non-ferrous metals), requires from 21.5 to 50°C and expressed theoretically in a implementation of integrated and sustainable waste medium largely to the development of mesophilic micro- management policies at least in the most heavily organisms, but their possibilities at room temperature populated Brazilian cities (Maciel et al., 2011). prevailed in the presence of microorganisms and Anaerobic digestion is considered to be an efficient and psychrophilic temperature of 50°C in the presence of environmentally friendly strategy for the disposal and thermophilic microorganisms. conversion of biological waste into renewable energy in The temperature is a parameter of fundamental impor- the form of biogas (Iglesias et al., 2000; Fdez-Guelfo et tance to the process of anaerobic digestion of organic al., 2011). Moreover, anaerobic digestion of urban solid waste. There are numerous works about influence garbage is economically feasible since the biogas of temperature on the anaerobic digestion process, but produced has the potential to replace fossil fuels, with regard to the process of digesting anaerobic organic including natural gas, in heat generation and as a solid wastes with high solids concentration, there are still transportation fuel (Curry and Pillay, 2012). However, one obvious gaps to be investigated; therefore, it was anchored of the main issues impeding the wider application of in the existence of these gaps that aimed to study the anaerobic digestion is the time required for the three different temperature levels. biostabilization of solid degradable wastes. The stability According to De La Rubia et al. (2005), the thermophilic of the anaerobic process is crucial in maintaining the microorganisms are more sensitive than mesophilic balance between the various microbial populations that microorganisms for environmental changes. In terms of are responsible for the conversion of complex organic solid waste digestion, the yield and rate of biogas compounds into simple substances; such as methane, production depends mainly on operation temperature; but carbon dioxide, nitrogen, ammonia, hydrogen sulfide and other factors such as setting the reactor and the chemical other products of low molecular weight (Lay et al., 1998; composition of the waste also influence it (Bouallagui et Raposo et al., 2011). By virtue of the greater proportion al., 2003). of cellulose and lignocellulosic polymers present in solid Kim et al. (2006) indicated in their work that the degradable materials compared with liquid wastes, the temperature can influence the anaerobic biostabilization time required to attain stabilization during anaerobic of organic waste, because it has direct influence on the digestion of the former is much longer (Schievano et al., growth rate of microorganisms, substrate utilization rate 2010). and biogas production rate. Low temperature may result According to Zhang et al. (2006), temperature plays a in possible depletion of cellular energy or complete lysis, key role in anaerobic processes and exerts a significant while high temperature may contribute to reducing the effect on conversion and process stability, kinetic para- biogas production, due the production of volatile gases, meters, the quality of the effluent and consequently, on such as ammonia, which limits the activity of methano- the amount of methane produced. In the short term, genic microorganism (Fezzani et al., 2010). waste decomposition generally increases with increasing In general temperatures between 35 and 37°C are temperatures up to a limiting value (Hartz et al., 1982). considered suitable for methane production and thermo- However, since the microbial population involved in philic temperatures may cause reduction in biogas anaerobic degradation may include psychrophilic, production rate until there is complete adaptation and mesophilic and thermophilic organisms (El-Mashad et al., normal growth of thermophilic microorganisms. 2004), with respective optimal growth temperatures of < Studies conducted by Ward et al. (2008) shown that 25, 25 - 40 and > 45°C, alterations in temperature growing optimal temperatures for some methanogenic regimes will over the longer term influence the balance of microorganism were in the range 37to 45°C, with predo- the microbial population. minance for mesophilic Methanobacterium. Temperatures The optimal temperature ranges for various types of in the range 37 to 40°C for Methanobrevibacter and microorganisms that provide biostabilization process of temperatures in the range 35 to 40°C showed the presence organic matter present in liquid or solid wastes are of Methanolobus, Methanococcus, Methanoculleus and psychrophilic (12 to 18°C), mesophilic (25 to 40°C) and Methanospirillum. Temperatures in the range of 30 to thermophilic (55 at 60°C). In this work, three different 40°C were predominant in Methanoplanus and levels of temperatures: room temperature (21.5°C), 40 Methanocorpusculum. Between 55 and 50°C predo- and 50°C were studied. Considering the temperature minance was of Methanohalobium and Methanosarcina. range established by Metcalf and Eddy (2003), the three Thus, this study is aimed at: (i) determining the effect of temperature levels adopted are conducive to the temperature on the stabilization of organic solid waste
502 Afr. J. Biotechnol.
Table 1. Physical and chemical characteristics of solid vegetable waste used in the preparation of the substrate.
Total volatile Total organic Total Kjeldahl Chemical Total alkalinity Volatile fatty acids Humidity Total solids Total fixed solids N - ammonium Waste solids (TVS; carbon (TOC; nitrogen (TKN; oxygen demand pH (TA; g (VFA; g (%) (TS; g.L-1) (TFS; g.L-1) (g.L-1) g.L-1) g.L-1) g.L-1) (COD; g.L-1) CaCO3.L-1)* CH3COOH.L-1)* Chard 97.79 22.1 12.0 10.1 6.7 1.26 0.56 7.7 7.97 3.8 2.18 Potato 85.82 141.8 128.3 14.8 71.3 1.12 0.28 140.4 3.71 0 4.15 Banana 87.72 122.8 97.5 25.3 54.2 1.4 0.14 67.0 3.81 0 13.53 Eggplant 94.84 51.6 44.7 6.9 24.8 1.82 0.42 15.8 7.55 4.4 2.18 Carrot 87.78 122.2 76.9 45.3 42.7 1.82 0.28 111.1 4.26 0.8 6.76 Chayote 96.02 39.8 35.6 4.2 19.8 0.84 0.28 22.1 5.5 1.8 2.62 Coriander 92.24 77.6 39.1 38.5 21.7 1.96 0.84 26.2 8.49 15.6 3.93 Kale 93.28 67.2 51.1 16.1 28.4 1.26 0.70 11.3 8.01 4.4 1.53 Pumpkin 93.35 66.5 53.1 13.4 29.5 1.26 0.28 57.8 5.61 6 7.2 Watermelon 95.28 47.2 31.7 15.5 17.6 1.12 0.14 40.7 4.26 0.2 2.84 Melon 94.74 52.6 38.8 13.8 21.5 2.24 0.70 50.5 4.31 1.8 12.22 Papaya 93.87 61.3 51.9 9.4 28.8 2.10 0.14 44.5 3.67 0 11.78 Cucumber 97.59 24.1 16.0 8.1 8.9 1.12 0.28 23.5 5.39 3.2 3.27 Bell pepper 95.88 41.2 32.7 8.5 18.1 1.68 0.56 29.1 5.58 3.8 4.80 Gabagge 96.35 36.5 26.9 9.6 14.9 2.38 1.40 14.5 7.67 6 3.49 Tomato 97.21 27.9 17.2 10.7 9.6 1.26 0.84 24 5.58 4.2 5.45
conjugated with sewage sludge in anaerobic and vegetables, provided by the Empresa Paraibana de Grande, and the pH was increased from 4.3 to 6.0 or 6.5 batch reactors (ABR), and (ii) estimating the Abastecimento Agrícola (Campina Grande) and was used with sodium carbonate. efficiency of the process in producing biogas and as inoculum for all reactors anaerobic sludge from sanitary Tables 1 and 2 presents data on chemical sewage collected from an up-flow anaerobic sludge blanket characterization of solid vegetable waste and anaerobic methane. (UASB) reactor located at the Experimental Station for the sludge used in the preparation of the substrate, Biological Treatment of Sewage (Campina Grande, PB, respectively. In Table 3 the chemical characterization of Brazil). The UASB reactor was monitored at 25°C. The the raw substrate are presented. MATERIALS AND METHODS substrate was prepared by crushing the plant residues in a The domestic wastewater produced by the population of Trapp (Jaraguá do Sul, SC, Brazil) model TR 200 grinder the city of Campina Grande has characteristics moderate -1 -1 The experiments was conducted at the Estação and passing through a 6 mm mesh sieve until a viscous with COD around 600 mgO2.L , BOD5 300 mgO2.L , pH Experimental de Tratamento Biológico de Esgoto homogeneous mixture was obtained. Subsequently, 8 kg of 7.5 and TN 60 mgN.L-1. The vegetable solid waste used in (EXTRABES) located in the Universidade Estadual da sludge was added to 32 kg of the plant residue mixture, the the preparation of the substrate were acidic characteristics Paraíba, Campina Grande, PB, Brazil (latitude 7°13’11’’S; concentration of total solids (TS) was adjusted to 36.2 g.L-1 and the final pH was around 4.3, after the addition of longitude 35°52’31’’W; altitude 550 m). The substrate used by addition of domestic wastewater collected from the anaerobic sludge the pH incresced to 5, valour below the as feedstock in the ABRs comprises of residues from fruits eastern outfall pipe of the municipal sewer of Campina recommended process of biological waste treatment.
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Table 2. Physical and chemical characteristics of anaerobic sludge used in the preparation of the substrate.
Parameter Raw substrate Humidity (%) 93.61 Total solids (TS; g.L-1) 63.9 Total volatile solids (TVS; g.L-1) 29.62 Total fixed solids (TFS; g.L-1) 34.29 Total organic carbon (TOC; g.L-1) 16.46 Chemical oxygen demand (COD; g.L-1) 48.48 pH 8.03 -1 Total alkalinity (TA; g CaCO3.L ) 7.05 -1 Volatile fatty acids (VFA; g CH3COOH.L ) 3.00 VFA/TA ratio 0.43 Total Kjeldahl nitrogen (TKN; g.L-1) 0.70 -1 N - ammonium (g.L ) 0.14 P - orthophosphate (g.L-1) 0.61 Specific mass (g.L-1) 1029.1 C/N ratio 23.5 C/P ratio 26.9
Then, it was corrected to 6.0 by addition of sodium carbonate. Where, Py is the atmospheric pressure (atm), ρ is the specific mass Each batch reactor (Figure 1) had a capacity of 1.15 L, of which of the manometric fluid (g/cm3); g the acceleration due to gravity, h 87% was employed for substrate storage and 13% was for biogas is the height of the manometric column (cm), V is the volume of the storage (headspace). Reactors were maintained at 25.5, 40 or 50°C head space (cm3), R is the ideal gas constant (cm.atm.. mol-1. K-1), (ABR1, ABR2 and ABR3, respectively) with constant monitoring of T is the temperature treatment (K), and Xfraction is the proportion of temperature, and each experiment was performed in triplicate to the component (methane) in the biogas. give a total of nine reactors. The raw substrate and the temperature The biogas produced was analyzed qualitatively using a gas stabilized substrates discharged from the reactors after digestion chromatograph coupled to a 250 mA thermal conductivity detector were characterized chemically. The parameters used in the analysis and equipped with a stainless steel column (3 m) packed with of raw and stabilized substrates were: Total solids (TS), total Porapak matrix (mesh size Q80-100). The carrier gas was helium volatile solids (TVS) and total fixed solids(TFS) (g.L-1); total organic supplied at a flow rate of 30 L min-1, and the vaporizer, column and carbon (TOC; g.L-1); Total chemical oxygen demand (COD; g.L-1); detector were maintained at 75, 75 and 100°C, respectively. Biogas pH; Total alkalinity (TA; g.L-1); Volatile fatty acids (VFA; g.L-1); total samples were collected from the headspace by introducing a -1 -1 Kjeldahl nitrogen (TKN; g.L ); N - ammonium (g.L ); P- needle connected to a 0.5 mL syringe (equipped with a safety orthophosphate (g.L-1). The analyses followed the methods device to avoid gas loss) into the septum located on top of the described by the American Public Health Association (2005). The reactor. The volume of methane was determined from the volume of biogas produced was measured daily and once a week was biogas produced and the methane content of the biogas obtained in characterized by gas chromatography using thermic conductivity the chromatographic characterization. detector. The volumes of biogas and methane accumulated in the ABRs at The processing period was 160 days during which quantitative the end of the experimental period were submitted to analysis of and qualitative analysis of biogas was performed on a daily and variance (ANOVA) and Tukey test in order to determine if the weekly basis, respectively. The amount of biogas produced per day observed differences were statistically significant (ρ < 0.05; 95% was quantified with the aid of a U-type manometer containing confidence interval). hydrated ethanol 46° INPM (Instituto de Pesos e Medidas; equivalent to 54% ethanol by volume) as the manometric fluid. The pressure exerted by the biogas accumulated in the headspace (P ; x RESULTS AND DISCUSSION atm), the number of moles of biogas produced (nbiogas: mol), the 3 volume of biogas produced (Vbiogas; cm ), and the volume fraction of 3 the component (methane) of the biogas (Vfraction; cm ) were Characterization of the wastes estimated according to Equations 1 to 3:
The pH of the raw substrate used in the batch reactors Px = Py +(r ´ g´h) (1) was 6.52, a value that was within the pH range recom- mended for anaerobic stabilization processes. A slight nbiogas = (Px ´V) / (R´T) (2) reduction in the pH was observed in the stabilized substrates discharged from ABR1 and ABR2 after 160 days of digestion (Table 3). The total alkalinity (TA) of the Vbiogas = (nbiogas ´ R´T) / (P) (3) -1 raw substrate was equivalent to 8.95 g.L of CaCO3, of
504 Afr. J. Biotechnol.
Table 3. Physical and chemical characteristics of the raw substrate and stabilized substrates discharged from the anaerobic batch reactors (ABR) operating at different temperatures.
Raw Stabilized substrates Parameter substrate ABR1(25.5°C) ABR2(40°C) ABR3(50°C) Humidity (%) 96.38 97.41 97.22 96.89 Total solids (TS; g.L-1) 36.20 25.90 27.80 31.10 Total volatile solids (TVS; g.L-1) 21.29 12.13 14.05 14.48 Total fixed solids (TFS; g.L-1) 14.94 13.60 13.75 16.64 Total organic carbon (TOC; g.L-1) 11.83 6.74 7.81 8.04 Chemical oxygen demand (COD; g.L-1) 34.26 20.72 22.74 28.64 pH 6.52 6.27 6.03 6.48 -1 Total alkalinity (TA; g CaCO3.L ) 8.95 5.56 5.89 6.40 -1 Volatile fatty acids (VFA; g CH3COOH.L ) 8.45 4.49 4.99 5.36 VFA/TA ratio 0.94 0.81 0.85 0.84 Total Kjeldahl nitrogen (TKN; g.L-1) 0.63 0.45 0.47 0.56 -1 N - ammonium (g.L ) 0.17 0.34 0.36 0.35 P - orthophosphate (g.L-1) 0.1661 0.1698 0.1666 0.1680 Specific mass (g.L-1) 1009.6 - - - C/N ratio 18.78 14.98 16.62 14.36 C/P ratio 71.22 39.69 46.88 47.86
Figure 1. Schematic representation of the anaerobic batch reactors.
Menezes et al. 505
which a fraction of approximately 34% corresponded to C/N ratio in anaerobic digestion of organic waste was bicarbonate alkalinity. The levels of TA in the stabilized 20/1 to 30/1. substrates varied between 5.56 and 6.40, with the consumption of alkalinity being higher in ABR1 (43%) in comparison with ABR2 and ABR3 (37.7 and 28%, respec- Biogas production tively). However, because of the mechanisms inherent in the anaerobic biostabilization of solid organic solid waste The accumulation of biogas as a function of process time containing predominantly plant residues, the fractions of in ABRs operating at different temperatures is presented bicarbonate alkalinity in the stabilized structure ranged in Figure 2 and the accumulation of methane as a between 25.6 and 29%, and such small variations explain function of its processing time in ABRs operating at the insignificant differences in the pH values of the different temperatures is presented in Figure 3, while the substrates. volumes of biogas and methane accumulated after 160 The value of volatile fatty acids (VFA) decreased days of anaerobic digestion are shown in Table 4. progressively from ABR1 to ABR3. The concentration of Application of ANOVA and Tukey test revealed that the VFA present in the raw substrate reduced by 46.4, 41.6 volumes of biogas produced in the three reactors were and 35.7% in the substrates stabilized at 25.5, 40 or not significantly different. The accumulative biogas 50°C, respectively (Table 3). It is noteworthy that the ratio volumes in three reactors were quantitatively similar. of VFA to TA in the raw substrate was 0.94, a value that The volumes of methane accumulated after 160 days is well above the theoretical recommended level for of anaerobic digestion are shown in Figure 3. While the anaerobic processes. In the biostabilized substrates, this volumes of methane produced in ABR1 and ABR2, did ratio was decreased to values that ranged from 0.81 not differ significantly one from another; both were (ABR1) to 0.84 (ABR3), thereby indicating the more significantly higher (ρ < 0.05) than that generated in efficient performance of anaerobic digestion at 25.5°C. ABR3. According to Deublin and Steinhauser (2008), Along with temperature, other factors may have methane-producing microorganisms are more active at influenced the performance of the process, and these temperatures in the mesophilic range, and this explains include alterations in the particle size, heterogeneity and why the volume of methane formed in ABR3 was much stationary mass of the substrate as well as lower in comparison with ABR1 and ABR2. Considering acclimatization of the microorganisms. that the three reactors were fed with the same substrate The raw substrate fed to each reactor contained 36.2 (similar particle size and concentration of TS), and g.L-1 of total solids (TS), of which 21.29 g.L-1 (~ 59%) received comparable organic loads, it is possible to state represented total volatile solids (TVS) and 34.26 g.L-1 (~ that the volume of biogas produced was negatively 95%) corresponded to chemical oxygen demand (COD). affected by a processing temperature of 50°C, probably The efficiencies of transformation of TVS in ABR1, ABR2 because growth of the methanogenic Archeae was and ABR3 were, respectively, 43.2, 34.2 and 32%, and a inhibited under such conditions. similar tendency was observed for COD transformation The proportion of methane produced in ABR1 with efficiencies of 39.5, 33.6 and 16.6%, respectively increased progressively throughout the experimental (Table 3). These findings show that the efficiencies of the period and attained a value of 37% after 160 days of reactors were inversely proportional to the temperature. digestion. The percentage of methane formed in ABR2 The total Kjeldahl nitrogen (TKN) in the raw substrate exhibited a similar profile until day 140 (30%). In ABR3, was 0.63 g.L-1 with the concentration of ammonia no methane was detected in the generated biogas until accounting for 16.6% of this value. In the stabilized day 100, after which the percentage increased steadily substrates, TKN values were in the range 0.45 - 0.56 g L- and attained 5% at day 153. These results demonstrate 1 and ammoniacal nitrogen was roughly 0.1 g L-1. In the that the activities of methane-producing microorganisms raw substrate, the ratio of total organic carbon (TOC) to were inhibited at the higher temperature, thereby TKN (the C/N ratio) was 18.78, a value that was within reducing the efficiency of ABR3. In contrast, Lianhua et the range of approximately 20-30 recommended for al. (2010) obtained biogas containing the equivalent of anaerobic biostabilization. Owing to the insignificant 65, 62.5 and 59.1% of methane by digestion of rice straw conversion of TKN into other forms of nitrogen during slurry containing 75 g L-1 of TS in reactors operating at anaerobic digestion, the reduction in the C/N ratio in the 25, 35 and 55°C, respectively. stabilized substrates was minimal and ranged from The reduced volumes of methane produced in ABR2 11.5% in ABR2 to 23.5% in ABR3. and ABR3 were likely associated with the lack of initial According to Weiland (2006) and Bouallagui et al. adaptation of the gas-producing organisms to high (2009), the C/N ratio between of 20/1 to 30/1 is good for temperatures (40 and 50°C). Similar results have been performance of anaerobic digestion of the vegetable solid reported by Komemoto et al. (2009) following experi- waste. On the other hand, works realized by Guermoud ments involving biogas production from food substrates et al. (2009) and Lee et al. (2009) showed that the best at different temperatures. These authors found that,
506 Afr. J. Biotechnol.
Figure 2. Accumulation of biogas as a function of process time in anaerobic batch reactors (ABR) operating at different temperatures.
Figure 3. Accumulation of methane as a function of process time in anaerobic batch reactors (ABR) operating at different temperatures.
Menezes et al. 507
Table 4. Volume of biogas and methane accumulated in anaerobic batch reactors operating under different temperatures after 160 days of digestion.
Mean accumulated volume (L) Reactor Biogas Methane ABR1 (25.5°C) 23.85A 9.04A ABR2 (40°C) 21.24A 7.11A ABR3 (50°C) 20.54A 1.11B
In each column, values followed by dissimilar uppercase capital letters indicate significant differences according to Tukey test (P < 0.05).
Table 5. Theoretical and experimental values for methane production in anaerobic batch reactors (ABR) operating under different temperatures calculated in terms of total organic carbon (TOC) transformed.
Volume of methane (L) Reactor Theoretical value Experimental value Conversion efficiency (%) ABR1 (25.5°C) 9.48 9.05 95.5 ABR2 (40°C) 7.52 7.11 94.6 ABR3 (50°C) 7.18 1.19 16.5
although temperatures in the thermophilic range promoted decisive factor for the performance of this type of process. the solubilization of substrates, the production of biogas was reduced in comparison with other systems employing Conflict of interests lower temperatures owing to the inhibition of gas-producing microorganisms. Authors did not declare any conflict of interest. As shown in Table 5, the experimental yields of methane generated in ABR1 and ABR2 (95.5 and 94.6%, respec- tively) were close to the theoretical values, while in ABR3, ACKNOWLEDGEMENTS the experimental yield was less than 20% of the theore- The authors wish to thank Coordenação de tical value. Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support to this study.
Conclusions REFERENCES
Anaerobic digestion at ambient temperature (25.5°C) American Public Health Association (2005). Standard Methods for the st provided the most efficient conditions for the production Examination of Water and Wastewater. 21 edn. APHA, Washington. of biogas and methane from a plant-derived organic Bouallagui H, Cheikh RB, Marouani L, Hamdi M (2003). Mesophilic -1 biogas production from fruit and vegetable waste in tubular digester. sludge with a TS concentration of 36.2 g L . Under these Bioresour. Technol. 86:85-89. conditions, the total amounts of biogas and methane Bouallagui H, Rachdi B, Gannoun H, Hamdi M (2009). Mesophilic and produced were 23.85 and 9.05 L, respectively, and these thermophilic anaerobic co-digestion of abattoir wastewater and fruit and vegetable wastein anaerobic sequencing batch reactors. yields were significantly higher compared with those Biodegradation. 20:401-409. produced when a process temperature of 55°C was Curry N, Pillay P (2012). Biogas prediction and design of a food waste applied. Anaerobic digestion at ambient temperature to energy system for the urban environment. Renewable Energy. represents an economical and environmentally viable 41:200-209. De La Rubia MA, Romero LI, Sales D (2005). Temperature conversion strategy for the disposal of municipal solid wastes. (mesophilic to thermophilic) in anaerobic sludge digestion. Aiche J. The best performance of the ABR1, monitored with 5(9):2581-2586. temperature of 25.5°C, should be associated with the Deublin D, Steinhauser A (2008). Biogas from Waste and Renewable inoculum used. The reactor that was used to collect the Resources: An Introduction. Wiley-VCH, Weinheim. El-Mashad HM, Zeeman G, Loon WKP, Bot GPA, Lettinga G (2004). inoculum was operated at the same temperature. In Effect of temperature fluctuation on thermophilic anaerobic digestion conclusion, the adaptation temperature of inoculum is a of cattle manure. Bioresour. Technol. 95:191-201.
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Vol. 14(6), pp. 509-518, 11 February, 2015 DOI: 10.5897/AJB2014.14310 Article Number: AC3B84650368 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Understanding the efficacy of influent waste water on microbial community structure of activated sludge process
Shah M. P.
Industrial Waste Water Research Laboratory Division of Applied and Environmental Microbiology Environ Technology Limited Ankleshwar-393002 Gujarat, India.
Received 11 November, 2014; Accepted 9 February, 2015
The assembling of microbial consortia in wastewater treatment facilities is a significance of environmental conditions. In the present research work, activated sludge from different wastewater treatment plants (WWTPs) were exploited at a molecular level to determine the influence of the complexity of the influent composition on the species structure and the diversity of bacterial consortia. The community fingerprints and technological data were subjected to the canonical correspondence and correlation analyses. The number of separated biological processes realized in the treatment line and the presence of industrial wastewater in the influent were the key factors determining the species structure of total and ammonia- oxidizing bacteria in biomass. The N2O-reducers community composition depended significantly on the design of the facility; the highest species richness of denitrifiers was noted in the WWTPs with separated denitrification tanks. The contribution of industrial streams to the inflow affected the diversity of total and denitrifying bacterial consortia and diminished the diversity of ammonia oxidizers. The obtained data are valuable for engineers since they revealed the main factors, including the design of wastewater treatment plant, influencing the microbial groups critical for the stability of purification processes.
Key words: Denitrification, waste water treatment plant, bacteria, influent.
INTRODUCTION
Rapid industrialization has necessitated the manufacture textile industry effluents because of their high biochemical and use of different chemicals in day to day life (Shah, oxygen demand, chemical oxygen demand, heat, color, 2014). Approximately, 10,000 different dyes and pigments pH and the presence of metal ions (Shah, 2014). are used industrially and over 0.7 million tons of synthetic Activated sludge constitutes a crucial tool in the biodegra- dyes are produced annually, worldwide (Shah, 2014). dation of organic materials, transformation of toxic Pollution due to textile industry effluent has increased compounds into harmless products and nutrient removal during recent years. Moreover, it is very difficult to treat in wastewater treatment plants (WWTPs). It contains a
*Corresponding author. E-mail: [email protected]. Tel: +91-9099965504.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 510 Afr. J. Biotechnol.
highly complex mixture of microbial populations whose was a robust statistical analysis of the dependence composition has been intensively studied in the past between the presence of the particular tanks and decades. By applying culture-dependent methods many processes in the biological treatment line of WWTPs, the species have been isolated from activated sludge (Dias influent characteristics and the community structure of and Bhat, 1964; Prakasam and Dondero, 1967; Benedict total, nitrifying and denitrifying bacteria in the activated and Carlson, 1971). sludge. The relations between the microbial assemblages However, a great majority cannot be obtained by and the technological data were investigated using the conventional techniques (Wagner et al., 2002) and, canonical correspondence analysis and correlation consequently, current molecular techniques such as matrix. sequence analysis of 16S rRNA gene clone libraries The main purpose of carry out this study is mainly (Snaidr et al., 1997), fingerprinting methods such as because several chemical industry’s raw waste water is denaturing gradient gel electrophoresis (DGGE) (Boon et coming to the common effluent treatment plant and as a al., 2002), thermal gradient gel electrophoresis (TGGE) result of treatment, what exactly these effluent affects is (Eichner et al., 1999) and terminal restriction fragment the microbial diversity of activated sludge present in the length polymorphism (Saikaly et al., 2005) along with biological system of common effluent treatment plant. fluorescence in situ hybridization (FISH) have been employed in wastewater microbiology to analyse and compare the microbial structure of activated sludge. MATERIALS AND METHODS Recently, PCR-based 454 pyrosequencing has been applied to investigate the microbial populations of Sampling and DNA extraction activated sludge in different WWTPs as well as in full- scale bioreactors (Sanapareddy et al., 2009; Kwon et al., Biomass samples were collected from Effluent Treatment Plant from Aeration Tank, Secondary Clarifier and Recycle Sump. The 2010; Kim et al., 2011; Ye et al., 2011; Zhang et al., bioreactor consists of anaerobic, anoxic, and aerobic basins for 2011a; b), greatly expanding our knowledge on activated enhanced removals of nitrogen and phosphorus as well as organic sludge biodiversity. An important process in WWTPs is matter. In the aerobic basin, a fixed synthetic mesh made from acryl nitrification, in which ammonium is removed by and polyether was incorporated. Grab samples were collected from converting it first into nitrite and then to nitrate. Different the aerobic basin. A bucket-type sampler was used for the suspended microorganisms, whereas a sterilized scissor was used to cut off a bacterial species involved in this process have been part of the fixed synthetic mesh, after lifting the mesh, for the characterized by means of clone library analysis in attached microorganisms. Samples were immediately frozen before addition to FISH (Juretschko et al., 1998; Purkhold et al., being transported to the laboratory. Genomic DNA was extracted 2000; Daims et al., 2001; Zhang et al., 2011b). Several from 1.5 ml of suspended microorganisms and 1.5 g of fixed ammonia-oxidizing and nitrite oxidizing bacterial synthetic mesh, in duplicate, using the Ultra Clean soil extraction kit populations belonging to the phylum Nitrospira and to following the manufacturer’s protocol. Five of the WWTPs, 1, 2, 3, 4, 5 received industry and domestic wastewater. The remaining Beta- and Gammaproteobacteria have been identified as WWTPs, 6, 7, 8, 9 treated domestic wastewater. key members in this process, such as the genera Nitrosomonas, Nitrobacter, Nitrospira and Nitrosococcus (Wagner et al., 2002; Zhang et al., 2011b). PCR amplifications Nevertheless, most studies of microbial diversity in WWTPs refer to freshwater plants, either domestic or Bacterial 16S rRNA gene fragments were PCR-amplified using primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 518R (5'- industrial, and yet very little is known about plants that ATTACCGCGGCTGCTGG-3'). Each 50 μl PCR reaction included utilize seawater for their operation, mainly because there 1× EF-Taq buffer (Bangalore Genei, India), 2.5 units of EFTaq are still very few of these running in the world. Their polymerase, 0.2 mM dNTP mix, 0.1 μM of each primer, and 100 ng utilization responds to the deficiency in hydric resources of template DNA. The PCR temperatures were as follows: 95°C for prevailing in their locations and their use will probably 10 min; 35 cycles consisting of 94°C for 45 s, 55°C for 1 min, and increase in the near future due to water shortage 72°C for 1 min; and a final extension at 72°C for 10 min. The duplicate PCR products were pooled and purified using the QIA associated to global warming as many areas are quick gel extraction kit (Bangalore Genei, India). The purified PCR experiencing today (Barnett et al., 2005). As a conse- products were used for the following cloning and Sanger quence, knowledge of the microbial diversity becomes Sequencing. crucial to identify the key players in these systems. Many studies have focused on the analyzing the relations between the bacterial communities, especially Cloning and Sanger sequencing nitrifiers, in biomass, and a type of wastewater (Limpiyakorn et al., 2011; Whang et al., 2009) in different The purified PCR products were cloned using the pGEM-T Easy bioreactors (Wan et al., 2011; Ye et al., 2011). There is Vector System (Promega, Madison, USA) according to the manufacturer’s instructions. Randomly picked transformed scarcity of literature on the influence of the full-scale Escherichia coli clones were transferred to 96-well plates and wastewater treatment plant organization on the bacterial sequenced using the T-7 primer on ABI 3730XL sequencers consortia in biomass. Therefore, the goal of the study (Applied Biosystems, USA) by Macrogen. Shah 511
Table 1. Primers used and denaturing gradient gel electrophoresis condition.
PCR product Denaturant percentage; Amplicon Specificity Primer set Reference References length (bp) gradient of gel (%) V3 region within the 30-60 For This 16S rDNA 341F/515R ~230 Muyzer et al. (1993) bacterial 16S rDNA 6 research
Nitrous oxide Kloos et al. (2001) 40–70 For This nosZ gene NosZ1/NosZ2 ~500 reducing bacteria Throba¨ck et al. (2004) 7 research
301F/302R ~700 Cydzik- Ammonia oxidizing Norton et al. (2002) 30–60 amoA gene amoA-1F/ Kwiatkowska bacteria Rotthauwe et al. (1997) 6 amoA-2R ~500 et al. (2011)
Table 2. Numerical values assigned to express the number of separate biological processes and the presence of the tanks favouring denitrification in WWTP treatment line.
Parameter Process/ denitrification tanks Numerical value Nitrification; carbon removal 1 Nitrification; denitrification; carbon removal 2 Process Phosphorus removal; nitrification; carbon removal 2 Phosphorus removal; simultaneous nitrification/denitrification; carbon removal 2.5 Phosphorus removal; nitrification;denitrification; carbon removal
Lack of separate denitrification tank 0 Pre-denitrification tank 0.5
Post-denitrification tank 0.5 Denitrification tanks Simultaneous nitrification/denitrifiction tank 0.75 Seperated denitrification tank 1
Polymerase chain reaction–denaturing gradient gel min, and a final elongation at 72°C for 5 min. The thermal profile for electrophoresis the nosZ gene amplification was: 94°C for 5 min, 6 cycles of touchdown PCR (denaturation 94°C for 30 s, annealing for 1 min The biomass from Effluent Treatment Plant was sampled twice from with an 1°C for two cycles decrement at temperature 61°C, aerobic tanks and was frozen in -20°C prior to molecular analysis. extension at 72°C for 1 min), followed by 25 cycles of 94°C for 30 s, DNA was extracted from approximately 400 mg of centrifuged 58°C for 1 min, 72°C for 1 min and a final elongation for 10 min at sample using a Fast DNA® SPIN Kit®. The working solutions with 72°C. The presence of the PCR products was confirmed by the DNA concentration of 50 ng/lL were prepared and the agarose electrophoresis. The amplified products were resolved on concentration of the DNA was measured spectrophotometrically DGGE gels using a dCode System (Bio- Rad, USA). The using Bio Photometer (Eppendorf, Germany). The PCRs were electrophoresis conditions are presented in Table 1. Denaturing performed in an Eppendorf® Mastercycler Gradient (Eppendorf). gradient gel electrophoresis gels were stained with SYBR Gold and The determination of the total bacteria diversity was based on 16S digitalized using Kodak 1D 3.6 Image Analysis Software. The amoA rDNA analysis with a primer set 341F/515R. The first-phase amplicons that were clear and had a high intensity were excised nitrifiers’ diversity was based on the analyses of amoA gene from the DGGE gel, reamplified and sequenced at Bangalore (301F/302R and amoA-1F/amoA-2R primer sets) that codes for the Genei, India. ammonium monooxygenase involved in the ammonium oxidation to nitrite. The denitrifying bacteria diversity was assessed based on the presence of nosZ gene (NosZ1/NosZ2 primer set), which codes Statistics for the nitrous oxide reductase responsible for the last step of denitrification (the gene is present in bacteria conducting full In all tests, the significant effects were those with p value <0.05. For denitrification). The PCR mixture contained 1.7 ng/lL of extracted the calculations, the numerical values were assigned to express the DNA, 0.5 lM of each primer, 100 lM of deoxynucleoside number of separate biological processes realized in the biological triphosphate mixture, 1.5 U of GoTaq® DNA Polymerase, 6 lL of 10 treatment line of WWTPs and the presence of the tanks favouring 9 reaction buffer supplied with polymerase, 1.5 mM MgCl2 and denitrification (Table 2). The canonical correspondence analysis sterile water to a final volume of 30 lL. The amplification of the (CCA) was performed on the relative DGGE band intensities with amoA gene was performed as a nested-PCR (Cydzik-Kwiatkowska the Monte Carlo method. To the CCA analysis, next to metadata and Wojnowska-Baryła, 2011). The thermal profile for the 16S obtained by DGGE, the technological data such as the COD/N and rDNA amplification was: 94°C for 5 min, 35 cycles of: denaturation BOD/COD ratios, TKN and COD in the influent, the presence (IN+) at 94°C for 45 s, annealing at 62°C for 45 s, extension at 72°C for 1 or absence (IN-) of industrial wastewater in the influent, the 512 Afr. J. Biotechnol.
Table 3. Numerical values expressing denitrification tank presence and the number of processes realized in WWTPs taken for the CCA.
Waste water treatment plant DT PR 1 1 2 2 0 2 3 1.25 2.5 4 1 2 5 0.75 2.5 6 1.5 3 7 0 1 8 1 2 9 1 3
Figure 1. The DGGE analysis of PCR amplifications of the partial amoA gene. The abbreviations above each lane represent the WWTP the activated sludge samples were taken from. The bands that were sequenced are marked with capital letters.
presence of the denitrification tanks (DT) and the number of RESULTS processes designed to occur in the biological treatment line of WWTP (PR) were taken. The values of DT and PR for each WWTP are presented in Table 3. The analyses were carried out using the In the present research, to analyze the bacterial CANOCO for Windows ver. 4.51 and CANODRAW. The correla- assemblages, the DNA isolated from the biomass was tions between the bacterial diversity and the technological amplified using the specific primer sets and the obtained parameters were analyzed using the correlation matrix in the products were separated in DGGE. In general, a higher Statistica 10.0. The statistical analysis assumed that the values number of bands indicate greater diversity of the from 0.9 to 1.0 point out to almost full correlation, very high analyzed microbial consortia. The DGGE separation of correlation reaches the values from 0.7 to 0.9, strong—from 0.5 to 0.7, the medium correlation from 0.3 to 0.5, while weak from 0.1 to the amoA PCR products is presented in Figure 1, the 0.3 (Stanisz, 2000). DGGE separations of the 16S rDNA and nosZ gene are Shah 513
Figure 2. Phylogenetic tree showing the position within members of the class b-Proteobacteria of partial 16S rDNA sequences recovered from Buttermere lake sediments by using primers designed to amplify sequences from the b- subgroup ammonia-oxidizing bacteria.
presented in Figure 4. The sequencing of the amoA the points represented the total bacterial consortia in bands and the phylogenetic analysis (Figure 2) showed different WWTPs while the environmental variables were that most of the sequenced bands (A, B, C, D) were represented by arrows. The bacterial community structure related to the Nitrosospira sp. The band E was closely differed between the WWTPs and no separate clusters related to Nitrosomonas eutropha. The canonical were observed in the diagram. The first axis of a biplot correspondence analysis is designed for relating the explained the 24% of the species-environment relation, species composition of communities to their environment while the summary variation explained by the two- and can provide an insight into the impact of waste water dimensional diagram was 42%. The first and the second treatment plant design and the operational parameters on eigen values equaled 0.31 and 0.23, respectively. The the bacterial assemblages. The data analysis can be species-environment correlations of the first two axes used in an explanatory way and it leads to an ordination were very high (above 99%) showing that the measured diagram of samples, species and environmental environmental variables were sufficient to explain the variables, which optimally displays how the community major variations among the analyzed WWTPs. The composition varies with the environment. When used in a correlation coefficients showed that the first axis was a confirmatory way, it leads to statistical tests of the effects presence or an absence of industrial waste water in the of particular environmental variables on the community WWTP influent (R2 = ± 0.71). The discrete variable IN composition taking into account the effect of other had the highest power to explain the patterns in the variables (ter Braak and Smilauer, 2002). In Figure 3a, species data and the significance of the explanatory 514 Afr. J. Biotechnol.
Figure 3. The CCA of a, total b, N2O-reducing and c ammonia-oxidizing bacteria communities; the discrete (triangle) (IN ± = the presence/ absence of industrial waste water in the influent) and the continuous variables (right arrow) (the COD/N and BOD/COD ratios of the influent, COD and TKN in the influent; DT, presence of the denitrification tanks; PR, the number of the processes realized in the biological treatment line of WWTP).
effect was statistically important. 1, 2 and 5 (Figure 3c). The first axis of a biplot explained The correlations of the second axis showed a contrast the 30% of the species-environment relation, while the between the WWTPs with the different number of summary variation explained by the two-dimensional processes realized in the treatment line (R2 = 0.50) and diagram was high and equalled 53%. The first and the different TKN and COD concentrations in the influent (R2 second eigen values equalled 0.57 and 0.45, = 0.48). The length of an arrow representing an respectively. The species-environment correlations of the environmental variable is a measure of how much the first two axes were above 99%. The correlation total bacteria community structure differs along that coefficients showed that the first axis is concentration of environmental variable. Since the PR arrow was the TKN in the influent (R2 = 0.67) and that this factor was longest one, the number of processes realized in the the most important continuous environmental variable. WWTPs was the most important continuous The second axis was the discrete variable namely the environmental variable influencing the total bacteria presence/absence of industrial waste water in the influent community structure in activated sludge from all (IN; R2 = ± 0.65). Figure 4 presents the microbial diversity analyzed. From Figure 3b it can be concluded that the expressed as the average number of amplicons in the points representing the N2O-reducing bacteria DGGE patterns. The number of 16S rDNA bands varied communities in 2, 7, 5 and 3 were similar and grouped depending on the WWTP. The highest number of together in the diagram. The first and the second axes amplicons (47) was in 1, the WWTP with a technological explained 36 and 20% of the species-environment line ensuring the removal of carbon, nitrogen and relation with the respective eigen values of 0.41 and 0.24. phosphorus with the respective efficiency of 96, 86 and The species-environment correlations for the first axis 95%. The lowest number of 16S rDNA amplicons was were 0.98 while for the second one it equalled 0.99. The obtained in 6. Three facilities (2, 8, 6) operated without correlation coefficients showed that the first axis is DT in the separated denitrification tanks had the lowest the WWTP treatment line (R2 =-0.73). diversity of denitrifiers (13, 16 and 15 nosZ bands, This environmental continuous variable had the highest respectively) in activated sludge. power to explain the DGGE patterns of denitrifiers. The The highest diversity of denitrifiers (from 26 to 32 nosZ correlations of the second axis showed that the bands) was obtained in activated sludge from 3, 1 and 4. BOD/COD ratio of the influent was the second The highest diversity of AOB (ca. 27 bands) was noted environmental variable influencing mostly the N2O- for the biomass from 7 and 9. The lowest ammonia- reducing bacteria communities in activated sludge from oxidizers diversity characterized activated sludge from 6. the analyzed WWTPs (R2 = -0.58). The CCA analysis of The correlation matrix was constructed to find the the DGGE patterns characterizing the AOB communities dependences between the diversity of particular groups showed that the most similar assemblages were in 6, 3, of bacteria (total, N2O-reducing and ammonia-oxidizing)
Shah 515
Figure 4. The average number of 16S rDNA, nosZ, amoA bands in the DGGE patterns characterizing activated sludge communities from analyzed WWTPs; the averages of two different measurements, standard deviations are given.
and the investigated WWTP design and the influent deciding about the structure of total bacteria composition. It was observed that the total bacteria assemblages. The complexity of the treatment line diversity was strongly correlated with the COD/N ratio positively affected the overall diversity of bacteria in the (R2= 0.84) and the number of processes realized in the biomass. The highest number of different bacterial facility (R2 = 0.79). We also obtained a positive strong species was noted for TY with a complex, many-stage correlation (R2 = 0.50) between the 16S rDNA band technological line. Reversely, the lowest number of 16S number and the presence of denitrification tank. As to the rDNA bands characterized activated sludge community in facility design, the presence of anoxic tanks had the 6, the smallest of all analyzed WWTPs (PE = 2,405) with greatest impact on the diversity of N2O-utilizing denitrifiers the simplest biological treatment line consisting only of (R2 = 0.57). Analyzing the wastewater characteristics, the aeration tank. It can be concluded that the presence both the presence of the industrial stream and the high of many tanks with the different oxic conditions in the COD/N ratio in the WWTP influent influenced the species treatment line favors the growth of multispecies microbial richness of denitrifiers (R = 0.51 and R = 0.48, consortia thus promoting the stability of the purification respectively). The AOB diversity was weakly positively processes. As to the wastewater composition, the correlated with the TKN concentration in the influent (R2 = statistically significant influence of the industrial waste 0.25) and negatively influenced by the presence of water presence on both the structure and the diversity of industrial waste water in the influent (R2= -0.25). the total bacteria communities were proven. Since the origin of industrial waste water in the analyzed treatment facilities was broad, it was difficult to claim which DISCUSSION components of the sewage promoted the biodiversity. Our results, however, pointed out that the presence of the Activated sludge from nine WWTPs was investigated to industrial stream favoured species richness of total determine the influence of the complexity of the biological bacteria in activated sludge due to the accessibility of a treatment line and the influent characteristics on the broader range of substrates as compared to that present structure and diversity of microbial consortia. For the in typical domestic waste water. statistical analysis, the CCA and correlation matrix were The WWTPs are one of the major sources of organics. applied. The species composition of the total bacterial Many parameters such as a substrate concentration, a communities in activated sludge was influenced by both C/N ratio, a type of carbon source, a nitrite accumulation the design of the treatment line and the waste water and an NO concentration influence the N2O production characteristics. The CCA analysis showed that the (Adouani et al., 2010). The knowledge about the links number of processes realized in the WWTPs was the between the waste water treatment line design and the most important of all continuous environmental variables structure of N2O-utilizing bacteria communities in the 516 Afr. J. Biotechnol.
biomass would allow engineers to apply the purification of industrial influent (except from 6) were grouped strategy favouring the effective reduction of the together in a separate cluster. The coexistence of various greenhouse gas emission. The research proved that the nitrifiers in WWTP is an evidence of a functional redun- number of separated denitrification tanks in the WWTPs dancy, a feature that may help in maintaining the stability was the major factor deciding about the species composi- of the system for nitrification. Wang et al. (2010) investi- tion and the diversity of the N2O reducing microorganisms gated the communities of microorganisms in activated in activated sludge. In three facilities (2, 8, 6) operated sludge of eight waste water treatment systems and without the separated denitrification tanks, the lowest suggested the negative impact of the presence of industrial species richness of denitrifiers in the biomass was noted. waste water in the influent on the nitrifiers’ diversity. Our In 2, the tank with the simultaneous nitrification/denitri- observations pointed out to a weak negative correlation fication was exploited meaning that nitrifiers and denitri- between the industrial waste water presence and the fiers co-existed in activated sludge; however, the alter- species richness of AOB (R = -0.25), nevertheless, the nating oxic conditions did not favor the full denitrification highest diversity of these bacteria was noted in biomass toN2 since the activity of bacteria that possess the nosZ from 7 and 9 that received only domestic wastewater. genes is inhibited in the presence of even low oxygen The lowest AOB diversity characterized activated sludge concentrations (Hochstein et al., 1984). For comparison, from 6, the WWTP with the simplest technological line in 5, nitrogen removal was realized by SND in a carrousel- consisting of only an aeration tank. It can be concluded type bioreactor; however there was an additional tank for that in this system, under a stable oxygen concentration, pre-denitrification favouring the growth of more diverse the microorganisms underwent a strong selective assemblage of the N2O-utilizing denitrifiers. The highest pressure favouring the growth of only a few best adapted diversity of denitrifiers was obtained in activated sludge species. from 3, 1 and 4. These WWTPs were characterized by Whang et al. (2009) evaluated the nitrifying community both the presence of separate anoxic tanks and the and the nitrification performance of the full-scale highest ratio of COD/N(12–14) that was also proven to municipal (20 mg N/L) and swine (220 mg N/L) WWTPs. positively influence the species richness of investigated Authors observed dissimilar nitrifying populations bacteria (R = 0.48). prevailing in these two plants and related this fact to The microorganisms capable of denitrification belong to different input nitrogen concentrations. In our research, in a broad variety of groups and encompass a wide range of the analyzed range of the influent TKN (62 ± 14–129 ± 18 the physiological traits. Most denitrifiers are the aerobic mg/L), only a weak correlation between this parameter heterotrophic organisms that transfer redox equivalents and the AOB diversity was proven. In general, the from the oxidation of a carbon source to N oxides under members of the Nitrosospira spp. or/and the Nitrosomonas the anaerobic conditions. The ability to reduce N2O by the oligotropha clusters are the dominant AOB in the nitrifying and phosphorus accumulating bacteria is also ammonia-low environments, whereas the members of the documented (Zumft, 1997). This can explain the tendency N. europaea–Nitrosococcus mobilis cluster comprise the observed in the current research that more processes majority of AOB in the ammonia-rich environments realized separately in the treatment line favoured the (Limpiyakorn et al., 2005). The sequencing of the chosen diversity of N2O-reducers. Siripong and Rittman (2007) amoA bands and the phylogenetic analysis showed that investigated the AOB communities in activated sludge in the AOB in the analyzed activated sludge samples were seven typical single stage municipal plants. Among the related to both Nitrosomonas sp. and Nitrosospira sp. analyzed factors (flow rate, influent and effluent BOD and (Figure 2). An interesting observation can be made on TKN, effluent ammonia, nitrite and nitrate, pH, and band E, obtained from N. eutropha. This species was sewage temperature), only the seasonal temperature present in all the WWTPs except from 3, in 6 only a weak variations seemed to change the nitrifying community, band was observed. N. eutropha is commonly found in especially the balance between Nitrosospira sp. and strongly eutrophic environments such as the municipal Nitrosomonas sp., although both genera coexisted in and industrial sewage disposal systems. This winter and summer samples. Lydmark et al. (2007) microorganism has a high tolerance for both elevated showed that ammonium concentration was an important ammonia concentrations and the fluctuating conditions structuring factor for an AOB community. In the present (especially oxic/anoxic cycles) (Koops et al., 1991; Stein research, the CCA proved that the main variable et al., 2007). The low number of N. eutropha in 6 (DGGE influencing the AOB consortia in activated sludge was the is a semi-quantitative technique so such a statement is presence of the industrial stream in the WWTPs influent. justified) can be explained by the simplicity of the In fact, the IN variable was the only one that had a biological treatment line in this WWTP—it consisted of statistically significant impact on the species composition only an aeration tank. The stable oxic conditions and a of the AOB. These results are well depicted in Figure 3c. lack of environmental fluctuations did not favour the The DGGE patterns characterizing activated sludge growth of the analyzed species. The 3 WWTP, on the from6, 3, 1, JE and 5 facilities with the lower values of other hand, received waste water from a wood industry COD and TKN in the influent (except from 1) and the that did not use water as an input for the manufacturing presence processes but generated several waste water streams Shah 517
after washing/cleaning procedures. In the production of Nitrosomonas nitrosa sp. nov., Nitrosomonas eutropha sp. nov., wood-based floors and wood-laminates, small volumes of Nitrosomonas oligotropha sp. nov. J. Gen. Microbiol. 13:1689-1699. Kwon S, Kim T-S, Yu GH, Jung JH, Park HD (2010). Bacterial highly polluted waste waters with the high contents of community composition and diversity of a full-scale integrated fixed- formaldehyde and COD are generated (for example film activated sludge system as investigated by pyrosequencing. J. cleaning of machines that are used to apply urea– Microbiol. Biotechnol. 20:1717-1723. formaldehyde resins onto wood-fiber boards) (Kaczala et Limpiyakorn T, Shinohara Y, Kurisu F, Yagi O (2005). Communities of ammonia-oxidizing bacteria in activated sludge of various sewage al., 2010). Waste water with formaldehyde could have treatment plants in Tokyo. FEMS Microbiol. Ecol. 54:205-217 negatively influenced the AOB community in 3, especially Limpiyakorn T, Sonthiphand P, Rongsayamanont C, Polprasert C N. eutropha, since its genome lacks genes for urease (2011). Abundance of amoA genes of ammonia-oxidizing archaea metabolism (Stein et al., 2007). and bacteria in activated sludge of full-scale wastewater treatment plants. Bioresour. Technol. 102:3694-3701 Lydmark P, Almstrand R, Samuelsson K, Mattsson A, So¨rensson F, Lindgren PE, Hermansson M (2007). Effects of environmental Conflict of interests conditions on the nitrifying population dynamics in a pilot wastewater treatment plant. Environ. Microbiol. 9(9):2220-2233 Shah MP (2014). Exploring the Strength of Pseudomonas putida ETL-7 The author did not declare any conflict of interest. in Microbial Degradation and Decolorization of Remazol Black-B. Int. J. Environ. Bioremediat. Biodegrad. (USA). 2(1):12-17. Shah MP (2014). 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Vol. 14(6), pp. 519-524, 11 February, 2015 DOI: 10.5897/AJB2014.14330 Article Number: 2AA965850411 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Ethanol production using hemicellulosic hydrolyzate and sugarcane juice with yeasts that converts pentoses and hexoses
Juliana Pelegrini Roviero1, Priscila Vaz de Arruda2, Maria das Graças de Almeida Felipe2 and Márcia Justino Rossini Mutton1*
1Faculdade de Ciências Agrárias e Veterinárias, UNESP – Univ Estadual Paulista. Via de Acesso Prof. Paulo Donato Castellane s/n 14884-900 - Jaboticabal, SP, Brasil. 2Escola de Engenharia de Lorena, USP - Universidade de São Paulo. Estrada Municipal do Campinho s/n, CEP 12602- 810, Lorena, SP, Brasil.
Received 21 November, 2014; Accepted 2 February, 2015
The use of vegetable biomass as substrate for ethanol production could reduce the existing usage of fossil fuels, thereby minimizing negative environmental impacts. Due to mechanical harvesting of sugarcane, the amount of pointer and straw has increased in sugarcane fields, becoming inputs of great energy potential. This study aimed to analyze the use of hemicellulosic hydrolyzate produced by sugarcane pointers and leaves compared with that of sugarcane juice fermented by yeasts that unfold hexoses and pentoses in the production of second generation biofuel, ethanol. The substrates used for ethanol production composed of either sugarcane juice (hexoses) or hemicellulosic hydrolyzate from sugarcane leaves and pointers (pentoses and hexoses), and the mixture of these two musts. Fermentation was performed in a laboratory scale using the J10 and FT858 yeast strains using 500 ml Erlenmeyer flasks with 180 ml of must prepared by adjusting the Brix to 16 ± 0.3°; pH 4.5 ± 0.5; 30°C; 107 CFU/ml with constant stirring for 72 h, with four replications. Cell viability, budding, buds viability, and ethanol production were evaluated. Among the yeasts, the cell viability was greater for J10. The use of FT858 + J10 was effective in producing ethanol. The hemicellulosic hydrolyzate had low efficiency in ethanol production compared with sugarcane juice.
Key words: Hydrolysis of sugarcane straw and pointers, sugarcane juice, xylose, cell viability, ethanol.
INTRODUCTION
With the decline in world oil reserves, along with price biofuel production has intensified (Oderich and Filippi, instability and the appeal for the sustainable use of 2013). Brazil stands out as the world's largest producer of natural resources, the search for alternative such as sugarcane, with an estimated production of 25.77 billion
*Corresponding author. E-mail: [email protected]. Tel: +55 16 3209-2675.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 520 Afr. J. Biotechnol.
gallons of ethanol in the 2013/2014 harvest (Conab, (straw, pointers and juice) was collected from a production unit in 2013). In order to increase ethanol production, the the region of Jaboticabal, SP. The straw and pointers were technology of converting lignocellulosic biomass into subjected to hydrolysis process. Before and after this process, these fractions were characterized as cellulose, hemicellulose and fermentable sugars for ethanol production is an lignin (Van Soest and Robertson, 1985). The sugarcane juice was alternative to meet the global demand for fuels (Santos et adjusted and available to fermentative process. al., 2012).
Due to the expansion of energy crops in conjunction Hydrolysis with environmental responsibility measures, an agro- environmental protocol on the cooperation between the In order to obtain the hemicellulosic hydrolyzate, 2 kg of leaves and government and the sugar-energy sector was established pointers previously dried in an aerated-oven at 60°C to constant weight were used. Acid hydrolysis of the hemicellulosic fraction was with the purpose of ending sugarcane burning and performed in a 40 L reactor under the following conditions: expanding mechanized harvesting. Without previous temperature of 121°C, residence time of 20 min, and 105 ml of burning of sugarcane trash, mechanized harvesting results sulfuric acid in 20 L of water. in large amounts of straw and pointers in the field, reaching 5-20 tons per hectare (Foloni et al., 2010). Musts The dry bagasse (which has now been used in cogeneration) and sugarcane trash account for two-thirds To obtain the hemicellulosic hydrolyzate must (HHM), the hydrolysis of the planted area, that is, only one third of the biomass fraction were initially detoxified for the removal of the fermentation inhibitors. The solution pH was adjusted to 7.0 by the addition of in plants is used in the production of ethanol or sugar, but calcium oxide (CaO), followed by an adjustment to pH 4.0 using they have great potential to produce second generation phosphoric acid (H3PO4). Furthermore, the hydrolyzate underwent ethanol (Fugita, 2010). For straw and sugarcane bagasse adsorption using activated carbon (1%) in an incubator at 50°C cellulose content with an average of 39 and 43% (B.O.D) for 30 min. At the end of each pH adjustment step, the respectively, there is a potential ethanol production of hydrolyzate was centrifuged and filtered (Marton, 2002), resulting in about 88/101 billion liters (Nunes et al., 2013). the must to be fermented. To obtain the sugarcane juice must (SJM), the original juice was The ethanol production from lignocellulosic hydrolysates subjected to clarification process for the removal of impurities. This in an economically feasible process, requires micro- process consisted of 300 mg/L of phosphoric acid and pH adjusted organisms that produce ethanol with a high yield from all to (6.0 ± 0.1) with calcium hydroxide (0.76 mol/L) of analytical sugars present (hexoses and pentoses), have high ethanol reagent grade (a.r.). The lime juice was then heated to 100 to productivity and can withstand potential inhibitors; 105°C and was transferred to beakers, and allowed to rest for 20 min for all impurities settling. To promote high settling rate, the furthermore, an integration of fermentation with the rest of beakers contained a polymer (Flomex 9074 – 2 mg/L) that group the process should be investigated (Olsson and Hahn- the small amount of impurities in high molecular weight flocs. After Hägerdal, 1996). that, the juice was filtered through a 14 µm filter paper in order to Several studies have been conducted focusing on viable separate the precipitated impurities, thereby resulting in a clarified and low cost alternatives for the production of biofuel juice. The clarified juice was standardized with distilled water to 16° from biomass (Canilha et al. 2012; Cheng et al., 2008). In Brix (soluble solids), and its pH was adjusted to 4.5 with sulfuric acid ( 0.3) at a temperature of 30°C, resulting in the must. order to allow the release of sugars present in the The third must (HSJM) was obtained by mixing the sugarcane hemicellulosic fraction of the mechanized harvesting juice must and hemicellulosic hydrolysate must in the ratio 1:1 (v/v). residues and to make it available for fermentation using microorganisms, prior hydrolysis of biomass is required. Yeast strains Amongst the available processes, acid hydrolysis provides recovery of up to 90% of fermentable sugars The following yeasts were isolated and mixed at the ratio of (1:1) present in the hemicellulosic fraction (Rodrigues, 2007). (four replications): 1. J10 (Rhodotorula glutinis -xylose metabolizing) obtained from a stock-culture maintained at 4°C provided by the However, this process may generate inhibitors, such as yeast bank of the Laboratory of Sugar and Ethanol Technology of the phenolic compounds which are mainly formed during the Department of Technology - School of Agrarian and Veterinary partial degradation of lignin (Martin et al., 2007), thereby Sciences, UNESP, SP, Jaboticabal, (Guidi, 2000); 2. FT858 inhibiting the fermentation process and resulting in low (Saccharomyces cerevisiae - used for industrial ethanol production) efficiency and low industrial production (Ravaneli et al., with the following characteristics: high-yield fermentation; resistant at low pH; tolerance to higher levels of alcohol; high viability during 2006). cell recycling fermentation; low foam formation; non- The objective of this study was to evaluate the produc- flocculent yeast strain; good fermentation speed (8 h when used in tion of second generation biofuel, the ethanol from a sugarcane industry), and low residual sugar levels in the must hemicellulosic hydrolyzate obtained from sugarcane (Amorim, 2011). leaves and pointers, sugarcane juice and their mixture, The initial cell viability was determined for 72 h using a Neubauer fermented by two different yeasts. cell-counter chamber (Lee et al., 1981), and a cell mass of both strains, containing a sufficient amount of cells to start fermentation 7 (10 CFU/ml), was used. MATERIALS AND METHODS
Raw material Fermentation and ethanol production
The raw material obtained from sugarcane variety RB867515 Fermentation was performed in laboratory scale using 500 ml Roviero et al. 521
Table 1. Cellulose, hemicellulose and lignin from the straw and sugarcane tip before and after hydrolysis of the hemicellulose fraction.
Percentage Cellulose Hemicellulose Lignin Composition before acid hydrolysis 37.32 35.98 5.52 Composition after acid hydrolysis 22.44 28.71 11.80
Values are represented as means.
Table 2. Analytical of pretreated substrates used for ethanol production.
Evaluated parameters SJM HHM HSJM Brix 16.1 16 16.3 pH 4.51 4.24 4.31 Sulfuric Acid Concentration (g/L) 0.73 5.72 2.69 Phenolic Compounds (g/L) 0.17 2.85 1.82 Total Monosaccharides (g/L) 111.7 81 97.5
Values are represented as means. SJM, Sugarcane juice; HHM, hemicellulosic hydrolyzate sugarcane leaves and pointers; HSJM, the mixture of these two substrates.
Erlenmeyer flasks containing the substrate used for ethanol released and used as a substrate for ethanol production. production (180 mL): SJM, HHM and HSJM. A total cell concen- 7 Lignin has a high calorific value and can be used in tration of 10 CFU/mL of the following strains J10, FT858, and J10 + cogeneration. The values obtained from cellulose, hemi- FT858 was used. The flasks after inoculation with respective cultures at desired cell concentration were incubated at 30± 1°C cellulose and lignin, for the sugar cane bagasse are with continuous stirring for 72 h. Cell viability, budding and buds around 48, 7.8 and 34.5%, respectively. These differences viability were determined at 0, 6, 12, 24, 36, 48, and 72 hof are explained by the straw characteristics and tips of fermentation (Lee et al., 1981). sugarcane used in the study, which are structurally less The concentrations of sugars and ethanol were determined by rigid than the bagasse from sugarcane stalks. HPLC (Waters, Milford, MA) with a Bio Rad Aminex HPX-87H column under the following conditions: column temperature 45°C, The average values of Brix, pH, sulfuric acid concen- eluent: H2SO4, 0.005 mol/L, flow rate of 0.6 ml/min, and an injection tration, total monosaccharides and phenolic compounds volume of 20 μL. of the musts are shown in Table 2. It can be seen that the The aliquots collected at 0, 6, 12, 24, 36, 48, and 72 h of fermen- three musts (SJM, HHM and HSJM) had similar tation, for analysis of sugar consumption and ethanol production, characteristics in terms of pH and Brix. Regarding to total were properly diluted and filtered through a “Sep Pack” C18 filter acidity, highest values were found in the hemicellulosic (Millipore). The eluent was prepared by subjecting it to vacuum filtration using Millipore membrane filter (0.45 pm, Hawp) and was hydrolyzate probably because sulfuric acid (0.5%) was degassed in an ultrasound bath (Microsonic SX-50) for 15 min added in the hydrolysis process. which was subsequently analyzed by HPLC. The concentration process means an increase in the content of sugar and phenolic compounds, which were higher than the values reported in the literature. Phenolic Statistical analysis compounds and other compounds that remain after
The results of cell viability and ethanol production were subjected to detoxification can inhibit fermentation (Polakovic et al., analysis of variance by the F test, and the comparison of the means 1992) directly affecting cell viability and ethanol produc- was performed by the Tukey test (Barbosa and Maldonado, 2011). tion (Ravaneli et al., 2006; Garcia et al., 2010). The presence of toxic compounds may influence fermen- tative organisms to an inefficient use of reducing sugar RESULTS AND DISCUSSION and formation of the product (Mussatto and Roberto, 2004). Martinez et al. (2000) observed a synergistic effect The composition of the cellulose and hemicellulose was when inhibitors compounds combined; including a variety reduced when considering the results reported by Santos of phenolic, aromatic compounds and several types of et al. (2012) (Table 1). After hydrolysis there was a acids, derived from lignin degradation, that ethanol reduction in the percentage of cellulose and hemi- production by E. coli was affected. cellulose, and an increase in lignin concentration. On the other hand, the results of yeast cell viability in The cellulose and hemicellulose have a low calorific the three substrates are given in Table 3, which clearly value and after the hydrolysis process, the sugar were show that the yeast cell viability in the sugarcane juice 522 Afr. J. Biotechnol.
Table 3. Variance analysis and comparison of means by the Tukey test (5% probability) of the microbiological analysis results using the musts composed of sugarcane juice (SJM), hemicellulosic hydrolyzate (HHM), and sugarcane juice + hemicellulosic hydrolyzate (HSJM), with the strains J10, FT858, and J10 + FT858.
Musts and yeats Cell viability (%) Budding (%) Buds viability (%) Musts SJM 88.69A* 7.57A 87.46A HHM 68.89C 3.67B 63.54C HSJM 78.59B 5.77A 77.71B
Yeasts J10 83.77A 6.14A 80.27A FT858 73.84C 5.45A 69.85B J10 + TF858 78.55B 5.42A 78.58A Must X Yeasts 4.66** 15.04** 2.80ns
**Significant at 1% (P<0.01); ns, non-significant (P≥0.05); *Means followed by the same uppercase in letters in a column are not significantly different according to the Tukey Test.
must was 22.33% higher than that in the hemicellulosic hemicellulosic hydrolyzate was the one with values lower hydrolyzate must. It was found that J10 had the best cell than the optimal ones reported in literature (3.67%. on viability, while the worst viability was found for FT858; the average), probably affected by the presence of inhibitor mixture of these two yeasts showed intermediate viability compounds. values. The buds viability yeast cells in the fermentation of the There was a continuous decrease in cell viability after sugarcane juice was also statistically higher, followed by 72 h of fermentation for all strains. This behavior is due to that in the fermentation of the must composed of the the natural metabolism of yeast strains since they trans- mixture of hemicellulosic hydrolyzate and sugarcane juice. form sugar into fermentation products such as ethanol, Among the yeasts, the best performance was found for acids, glycerol and other compounds that accumulate in J10; FT858 produced the lowest performance, and the the culture medium inhibiting their metabolic process, mixture of J10 and FT858 produced intermediate negatively affecting cell viability (Amorim et al., 1996). performance. The fermentation process was evaluated for 72 h, Literature reports suggest that hexoses and pentoses which in an industrial scale is considered a process too were completely consumed in the first few of fermentation long for ethanol production. In the present study, the as glucose is the universal carbon source (Schirmer- fermentation process occurred within the first 10 h, with Michel et al. 2008). Similar results has been reported by cell viability of approximately 90, 86, and 78% for the Cheng and coworkers (2008) in sugarcane bagasse sugarcane juice must, mixture (broth and hydrolyzate), hydrolyzates. Xylose consumption in this study (the main and for the hydrolyzate must , respectively. Very low sugar in the hemicellulosic hydrolyzate), however, was values around 40% were found for the hydrolyzate must not complete (Table 4). at the end of the process due to the combination of Our results are in accordance with the report of Toivari inhibitory compounds which accumulate over time. et al. (2001) wherein a higher concentration of phenolic During sugarcane juice must fermentation, cell viability compounds and acids could be responsible for lower was statistically the highest, followed by the mixture of production of ethanol. Evaluating the effect of the the fermented hemicellulosic hydrolyzate and sugarcane fermentation time (Figure 1) on the musts, it was juice. Among the yeasts, the best performance was found observed that in 24 h of fermentation, the highest for J10 and the mixture of J10 and FT858. The strain concentration of ethanol with the clarified broth of FT858 had shown lowest cell viability. sugarcane juice yield was 70% higher than that of the The bud was the highest in sugarcane juice broth and hemicellulosic hydrolyzate (around 9 g/L) in same time was found to be lowest in the hemicellulosic hydrolyzate. period. The sugarcane juice must produced the highest When strains used in the present study was compared, level of ethanol (33 g/L), followed by the must composed no statistical significant differences was obtained in terms of the mixture of hemicellulosic hydrolyzate and of budding. The optimum budding index of a fermentation sugarcane juice (22 g/L). process should generally range between 5 and 15% The variation in the ethanol production, cell viability, (Amorim et al., 1996); while, in the present report, the budding and buds viability was mainly attributed to the Roviero et al. 523
Table 4. Variance analysis and comparison of means by the Tukey test (5% probability) of the use of xylose by yeasts J10, FT858, and J10 + FT858.
Use of xylose by yeasts Time of fermentation (h) J10 FT858 J10 + FT858 0 49.80A 56.55A 52.51A 6 37.51B 45.91B 42.96B 12 34.49B 35.76C 35.67C 24 24.15C 25.80D 27.03D 36 23.40C 21.49DE 22.24DE 48 22.55C 20.45DE 19.03EF 72 18.09C 18.59E 14.74F Teste F 53.92** 92.26** 81.69**
**Significant at 1% (P<0.01); *Means followed by the same uppercase in letters in a column are not significantly different according to the Tukey Test.
Figure 1. Graphical representation of the unfolding of the musts and yeasts (J10. FT858 and J10 + FT858) over a 72 h period for ethanol production.
composition of the pretreated substrates, which contain for the removal of acids and phenolic compounds, which large concentration of inhibitory compounds; which were negatively influenced the production of ethanol from the not efficiently removed during the detoxification process hemicellulosic hydrolyzate. The detoxification method has and this might have negatively influenced the final result. to be based on concentrations and the degree of microbial Some toxic compounds can stress fermentative organisms inhibition caused by the compounds. To a certain types of to an inefficient utilization of sugar resulting in product compounds, better results can be obtained by combining formation decreases (Silva, 2004). two or more different detoxification method (Silva, 2004). The final ethanol concentration varies according to the In the present investigation, the level of ethanol produced concentration of sugar, nutrients, contaminants, and inhibi- using clarified broth of sugarcane juice, although lower tors presents in the substrate. Accordingly, it was found than most of the literature reports using sugarcane juice, that only one single detoxification process was not sufficient in the present investigation, the level of ethanol produced 524 Afr. J. Biotechnol.
using clarified broth of sugarcane juice was the higher Guidi RH (2000). Characterization, classification and determination of (about 9 g/L) compared with 1.5 g/L reported by Fugita molecular genetic markers strains and contaminants yeast in the fermentation process. Dissertation. Univ. Estadual Paulista. (2010) that used sugarcane bagasse as raw material and Lee SS, Robinson FM, Wong HY (1981). Rapid determination of yeast J10 yeasts. viability. Biotechnology Bioengineering Symposium. In conclusion, we observed highest cell viability and Martin C, Almazán O, Marcet M, Jonsson LJ (2007). A study of three ethanol production in the clarified broth of sugarcane juice strategies for improving the fermentability of sugarcane bagasse hydrolysates for fuel ethanol production. Int. Sugar J. 109(1297):33- using the strain J10. The detoxification process used 35, 37-39. promoted a partial removal of acids and phenolic Martinez A, Rodriguez ME, York SW, Preston J, Ingram LO (2000). compounds. The use of a yeast co-culture produced the Effects of Ca (OH) 2 treatments (“overliming”) on the composition and best performance in ethanol production. The pointer and toxicity of bagasse hemicellulose hydrolysates. Biotechnol. Bioeng. 69(5):526-536. straw cane are an important raw material to be Marton JM (2002). Evaluation of various activated carbons and considered for the ethanol production. adsorption conditions for treating the hydrolyzate hemicellulosic sugar cane bagasse for the biotechnological obtain xylitol. Dissertation. Universidade de São Paulo. Mussatto SI, Roberto IC (2004). Alternatives for detoxification of Conflict of interests diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review. Bioresour. Technol. 93(1):1-10. The authors did not declare any conflict of interest. Nunes MR., Guarda EA, Serra JC V, Martins ÁA (2013). Agroindustrial residues ethanol production potential of second generation in Brazil. Revista Liberato 14(22):135-150. Oderich EH, Filippi EE (2013). The different speeches in the debate ACKNOWLEDGMENTS about biofuels and the options of the Brazilian state. J. Economic Universidade Estadual de Goiás. CAPES is acknowledged for the master scholarship Olsson L, Hahn-Hägerdal B (1996). Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb. Technol. 18(5): granted to the first author. 312-331. Polakovic M, Handriková G, Kosik M (1992). Inhibitory effects of some phenolic compounds on enzymatic hydrolysis of sucrose. Biomass REFERENCES Bioenergy. 3:369-371. Ravaneli GC, Madaleno LL, Presotti LE, Mutton MA, Mutton MJR Amorim HV (2011). How much does select an industrial yeast? V Week (2006). Spittlebug infestation in sugarcane affects ethanolic of Alcoholic Fermentation “Jayme Rocha de Almeida”. Piracicaba: fermentation. Sci. Agric. 63:6 ESALQ / USP. Rodrigues FA (2007). Evaluation of sugarcane bagasse Acid Hydrolysis Amorim HV, Basso LC, Alves DG (1996). 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Vol. 14(6), pp. 525-529, 11 February, 2015 DOI: 10.5897/AJB2014.14136 Article Number: 8965FED50417 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Antitrypanosomal activity of Khaya senegalensis and Anogeissus leiocarpus stem bark on Trypanosoma brucei brucei infected rats
Awobode, Henrietta O.*, Fagbemi, Folasade T. and Afolayan, Funmilayo I. D.
Department of Zoology, University of Ibadan, Ibadan, Oyo State, Nigeria.
Received 23 October, 2014; Accepted 12 January, 2015
Trypanosoma brucei brucei, a haemo-protozoan parasite causes African Animal Trypanosomiasis (AAT). Khaya senegalensis (KS) and Anogeissus leiocarpus (AL) are medicinal plants used either individually or in combination by local farmers in Northern Nigeria in the treatment of many diseases including trypanosomiasis. There is however, no information on the efficacy of the plants used in combination. In this study, the antitrypanosomal activity of combined methanolic stem bark extracts of K. senegalensis and A. leiocarpus were determined in vivo using suppressive and repository tests. The combined extracts were administered at 250 mg/kg to T. b. brucei infected rats in ratios 1:4, 2:3, 1:1, 3:2 and 4:1 (K. senegalensis to A. leiocarpus). Diminor® (3.5 mg/kg) was positive control and Tween-80 the negative control. Trypanocidal activity was recorded in all four ratios with the highest in the 4:1 ratio for both tests. All ratios in repository test had varying levels of prophylactic activity which were significantly higher (p<0.05) than the negative control group. Chemo-prophylactic activity in the 4:1 ratio compared (p>0.05) favorably with the positive control. The extracts however had significantly lower (p<0.05) parasite suppressive activity compared to Diminor® (100%). The 1:4 combinations had the lowest activity (4.35%). In the repository test, packed cell volume (PCV) levels varied in the groups with an increase as the quantity of K. senegalensis in the dose increased. The results therefore show that the antitrypanosomal activity and haemolytic effects of the extracts was dependent on the ratio of K. senegalensis to A. leiocarpus. A higher quantity of K. senegalensis provided a more effective prophylaxis and normal PCV. The use of a threefold quantity of K. senegalensis to A. leiocarpus in the local management of animal trypanosomiasis is therefore suggested.
Key words: Antitrypanosoma, suppressive test, repository test, Khaya senegalensis, Anogeissus leiocarpus.
INTRODUCTION
Trypanosoma species are blood or tissue parasites of all important species causing African animal trypanosomiasis classes of vertebrates. In some hosts particularly man (AAT) are Trypanosoma congolense, Trypanosoma vivax and domestic animals, they are highly pathogenic causing and Trypanosoma brucei brucei (CFSPH, 2009). In wild diseases generally known as trypanosomiasis. The most animals, these parasites cause relatively mild infections
*Corresponding author. E mail: [email protected]. Tel: +234 802 682 0100.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 526 Afr. J. Biotechnol.
while in domestic animals they cause a severe, often fatal using Whatmann filter paper 1. The extract was concentrated to dryness using water bath at 60°C. A standard drug Diminazene disease. African animal trypanosomiasis is most ® pronounced in cattle but can cause serious losses in aceturate (Diminor ) used in the treatment of animal trypanosomes was used as the positive control in the study while the vehicle pigs, camels, goats, and sheep. Despite the attempts at (Tween-80) was given as negative control. For oral administration control, trypanosomiasis is still one of the limiting factors of the extract, 30% ethanol was added to 70% Tween-80 and 1 part to livestock industry in sub-Saharan Africa (Kamuanga, of this preparation was mixed with 9 part of distilled water (1:9). 2003). Tween-80 was used to enhance solubility of the extract. Tween- 80 is not toxic when less than 10% of it is administered to rats. (Food Currently, trypanocidal drugs constitute principal method Safety Commission, 2007) of control of trypanosomiasis. Poor clinical efficiency, drug resistance and toxicity are some of the limitations facing programmes targeted at controlling trypanosomiasis Parasite (Onyeyili and Egwu, 1995; Legros et al., 2002). This emphasizes the need for the identification of new Trypanosoma brucei brucei was obtained from Department of therapeutic and prophylactic agents for controlling the Veterinary Pathology, Faculty of Veterinary Medicine University of Ibadan, Ibadan, Nigeria. The parasites were maintained in the disease. Resistance associated with chemotherapeutic 4 agents has necessitated the development of potent drugs laboratory by continuous passage in rats, introducing 1 × 10 parasites intraperitoneally. Blood from the tail was used for the with antitrypanosomal activity from plant source. Plants estimation of parasite density using the “rapid matching” method of have provided the basis for traditional treatment for Herbert and Lumsden (1976). This method involved microscopic different types of diseases and still always offer enormous counting of parasites in pure blood or blood appropriately diluted potential for new chemotherapeutic agents. with buffered phosphate saline. Khaya senegalensis also known as African mahogany has been documented to be active in vitro against T. b. Experimental animals brucei (Wurochekke and Nok, 2004; Atawodi, 2005). Many prescribed recipes for local treatment of Seventy adult albino male rats weighing 150-200 g (177.7±16.18) trypanosomiasis contained K. senegalensis (Atawodi et were obtained from animal facility of Faculty of Veterinary Medicine, al., 2002). Traditional healers in North Eastern Nigeria, University of Ibadan, Nigeria. They were kept and acclimatized in also believe that the bark of Anogeissus leiocarpus also the animal facility of the Department of Zoology, University of known as African Birch is very effective in the treatment Ibadan before commencement of the experiment. The animals were divided into two groups of 35 animals each for the suppressive test of African trypanosomiasis (Bizimana, 1994). In Northern and repository test respectively. The experiments were conducted Nigeria, K. senegalensis and A. leiocarpus are listed in compliance with the international guiding principles for research among the plants used in combination as treatment for involving animals animal trypanosomosis by local farmers and herdsmen (Atawodi, 2002) but there is no information on the Antitrypanosomal studies appropriate combination ratios and activity of the plants in combination. The study was therefore designed to The rats for each test were divided into five experimental groups (A- ascertain the efficacy of K. senegalensis and A. E) and two control groups (F-G). leiocarpus when used in varying combination ratios.
Suppressive test
MATERIALS AND METHODS The Peters’4 day suppressive test was adopted (Peters, 1967). The animals were inoculated intraperitoneally with 1 × 104 parasites in Plant materials 0.2 ml Phosphate buffered saline (PBS) solution and after 2 h the extract was administered orally and subsequently for four days. The stem bark of K. senegalensis and A. leiocarpus were collected Extracts were administered in the following combination ratios of from the Botanical Garden University of Ibadan, Ibadan, Nigeria. K. senegalensis (KS) and A. leiocarpus (AL): 1:4, 2:3, 1:1, 3:2 and Plants were identified and authenticated at the Herbarium of 4:1 at 250 mg/kg (this dose was adopted from the proposed Forestry Research Institute of Nigeria (FRIN), Ibadan. Samples thresholds for in vivo activity of antimalarial extracts Rasoanaivo et deposited in the herbarium were assigned voucher numbers al., 2004) for the experimental groups A-E, respectively. The 109535 (Anogeissus leiocarpus) and 109536 (Khaya senegalensis). positive control group (F) was treated with a single dose 3.5 mg/kg of Diminazene aceturate (Diminor®) while the negative control (G) was given Tween-80. On days 5, 9, 14 and 17 post infection, rats Preparation of plant extracts were tailed and smears prepared for parasite count. Mean parasitaemia was calculated for each group and percentage The stem barks of the plants were rapidly washed under running chemosuppression was determined using formular described by tap water and air-dried in the laboratory at room temperature. The Odeghe et al. (2012). dried barks was ground into coarse particles and then milled to a powdery form. One hundred and fifty grams (150 g) of the resulting hemosuppression - powder was soaked in 500 mL absolute methanol and stirred intermittently for 72 h at room temperature. The plant material was here is the mean parasitaemia for negative contro and is the filtered using clean muslin cloth and the filtrates were further filtered mean parasitaemia of the e perimenta groups positive contro Henrietta et al. 527
Table 1. Repository (Prophylactic) activity of combined stem bark extract of Khaya senegalensis and Anogeissus leiocarpus.
Combination Parasitaemia Prophyla Parasitaemia Propylax Parasitaemia Prophylax Parasitaemia Prophylaxis Parasitaemia Prophylaxis ratios (KS vs AL) Day 7 xis (%) Day 10 is (%) Day 13 is (%) Day 17 (%) Day 21 (%) 1: 4 1.44±0.15b 68.0 3.28±0.11b 59.56 12.06±2.13ab 54.01 31.56±1.94c 41.56 40.39±4.97d 27.45 2:3 1.61±0.06b 64.22 3.00±0.25b 63.01 11.78±1.93ab 55.07 29.17± 0.88c 45.98 31.63±0.57c 43.18 1:1 0.00±0.00a 100 2.22±0.31b 72.63 11.53±1.18 ab 55.84 20.89± 1.34bc 61.32 26.17±3.26bc 53.00 3:2 0.00±0.00a 100 2.06±0.57b 74.60 8.00±0.97a 69.49 16.61±0.87b 69.24 20.17±0.60b 63.77 4:1 0.00±0.00a 100 0.00±0.00a 100 0.00±0.00a 100 0.00± 0.00a 100 0.72±0.72a 98.71 Positive control 0.00±0.00a 100 0.00±000a 100 0.00±0.00a 100 0.00 ±0.00a 100 0.61±0.61a 98.90 Negative control 4.50±0.19c - 8.11±0.72c 26.22±1.24b 54.00±8.54d 55.67±4.09c
Columns with values bearing the same superscript are not significantly different (p<0.05).
Repository (prophylactic) test analysis of variance (ANOVA) followed by Duncan multiple day 9 post infection with highest parasitaemia range test. Values of p<0.05 were considered significant. recorded in the negative control animals. The The method of Abatan and Makinde (1986) was adopted in highest chemo-suppressive activity (68.53%) was the evaluation of the prophylactic potential of the combined however observed in animals administered the 4:1 extract of K. senegalensis and A. leiocarpus. Extracts RESULTS containing KS and AL in ratios 1:4, 2:3, 1:1, 3:2 and 4:1 at ratio. This is a similar trend to that observed in 250 mg/kg were administered to experimental groups A-E, Repository test repository test were an increase in the quantity of respectively, positive control (group F) was treated with a KS gave an increased activity of the extract. The single dose of Diminazene aceturate (Diminor®) at 3.5 The prophylactic activity of the extracts on suppressive activity of the varying ratios of the mg/kg while the negative control (G) was given the vehicle infected rats increased with increasing quantity of combined extract decreased to between 4.35- (Tween-80). All animals were infected with 1 × 104 T. KS in the combination (Table 1). On day 7 post 57.26% by day 17 while the positive control brucei brucei on day 4 post treatment. Rats were tailed and infection, ratios 1:1, 3:2 and 4:1 showed 100% smears prepared for parasite count daily and mean remained at 100%. percentage parasitaemia was calculated for each combina- chemoprophylactic activity. The activity of 4:1 tion ratio. combination ratio remained at 100% until after day 17 and decreased to 98.7% by day 21 post Packed cell volume (PCV) infection. This compared well with the standard Packed cell volume determination (PCV) drug Diminazene aceturate which had 98.9% Packed cell volume of infected rats in both tests is prophylactic activity by day 21 post infection. The presented in Table 3. The highest PCV values The PCV was determined for all animals in both the activity in the other combination ratios decreased were recorded in animals receiving 4:1 dose ratio. repository and suppressive assays. progressively and by day 21 post infection, ratios In the two tests there was no significant difference 1:4, 2;3, 1:1 and 3:2 had reduced to 27.45, 43.18, (p<0.05) between PCV values of negative control 53.00 and 63.77%, respectively (Table 1). animals and animals receiving 1:4 combination Statistical analysis ratio. Only animals in the positive control group,
The Statistical Package for Social Sciences (SPSS) Suppressive test treated with the commercial drug had PCV values software version 16.0 was used for data analysis. The within the normal range (35-57) in rats. In the results were expressed as Mean ± SEM. The statistical Table 2 shows the effect of the combined extract repository test, PCV values increased as the ratio difference between groups was performed using one way on early infection. Parasites were observed on of K. senegalensis increased in the dose. 528 Afr. J. Biotechnol.
Table 2. Suppressive activity of combined stem bark extracts Khaya senegalensis of Anogeissus leiocarpus.
Combination ratios Parasitaemia Suppressi Parasitaemia Suppressio Parasitaemia Suppressi Parasitaemia Suppressi (KS vs AL) Day 5 on (%) Day 9 n (%) Day 14 on (%) Day 17 on (%) 1: 4 0.00±0.00a 100 8.33±1.51c 12.32 25.99±6.23c 8.81 44.00±2.52c 4.35 2:3 0.00±0.00a 100 7.11±1.64bc 25.16 22.88±4.03bc 19.72 41.33±0.66c 10.15 1:1 0.00±0.00a 100 3.22±0.97ab 66.11 22.67±1.76bc 20.46 35.06±3.95c 23.78 3:2 0.00±0.00a 100 3.16±0.17ab 66.74 18.13±1.49bc 36.39 20.94±8.07b 54.49 4:1 0.00±0.00a 100 2.99±0.25ab 68.53 12.57±3.56b 55.90 19.66±4.79b 57.26 Positive control 0.00±0.00a 100 0.00±000a 100 0.00±0.00a 100 0.00±0.00a 100 Negative Control 1.95±0.15b - 9.5±2.29c 28.50±1.89c 46.00±1.16c
Columns with values bearing the same superscript are not significantly different (p<0.05).
Table 3. Packed cell volume (PCV) of Trypanosoma brucei brucei infected rats in repository and suppressive test.
Combination ratios PCV (KS vs AL) Repository test Suppressive Test 1:4 26.50±0.65a 31.33±0.88b 2:3 29.67±1.45ab 24.67±5.04ab 1:1 30.00±2.35abc 16.33±2.33a 3:2 30.25±2.02abc 28.00±2.31b 4:1 33.75±2.78bc 30.50±1.50b Positive Control 36.20±0.97c 42.80±0.86c Negative Control 26.50±1.50a 31.25±3.77b
Columns with values bearing the same superscript are not significantly different (p<0.05).
DISCUSSION standard drug in both repository and suppressive (azaanthraquinone), highly aromatic planar tests. This suggests a high efficacy of the combined quaternary alkaloids, barbarine and harmaine. K. senegalensis and A. leiocarpus are among the extract in parasite clearance. K. senegalensis has In both repository and suppressive tests, the plants used by local farmers in northern Nigeria in been reported to contain saponins, tannins, highest antitrypanosomal activities were observed the treatment of trypanosomiasis. Reports have alkaloids, glycosides, steroids, terpenoids and when KS and AL were combined in ratio 4:1. This shown that K. senegalensis and A. leiocarpus flavonoids (Makut et al., 2008) while A. leiocarpus suggests that the antitrypanosomal activity of the when used singly in treatment of trypanosomes contains alkaloids, glycolsides, phenols, steroids, combined extract was dependent on the quantity infected rats possess antitrypanosomal activity tannins, anthraquinones, saponins and flavonoids of KS in the combination. An increased amount of (Umar et al., 2010; Ibrahim et al., 2008; Wurochekke (Mann et al., 2010; Kaboré et al., 2010). Nok KS, may have resulted in a higher concentration and Anyanwu, 2012). The antitrypanosomal activity (2001) attributed the trypanocidal activity of a of active phytochemical components needed for of the combined extracts compared well with the number of tropical plants to flavonoids parasite clearance. Henrietta et al. 529
Detection of parasites 21 days post infection both in Nigeria savannah plants. Afr. J. Biotechnol. 4(2):177-182. animals treated with 4:1 combination of KS: AL and Atawodi SE, Ameh DA, Ibrahim S, Andrew JN, Nzelibe HC, Onyike EO, Anigo KM, Abu EA, James DB,Njoku GC, Sallau AB (2002). positive control group in the repository test indicates a Indigenous knowledge system for treatment of trypanosomiasis similar efficacy in both treatments. The presence of in Kaduna state of Nigeria. J. Ethnopharmacol. 79:279-282. parasites on day 21 however, indicates the inability of Bizimana N (1994). Traditional Veterinary Practice in Africa. German both the extract and Diminazene aceturate to completely Technical Cooperation. ISBN 3880855021 Centre for Food Security and Public Health (2009). African animal clear the parasites when administered at 250 mg/kg and trypanosomiasis. www.cfsph.iastate.edu. Accessed on April12, 2012. 3.5 mg/kg respectively. This is in consonance with the Food Safety Commission (2007). Evaluation Report of Food Additives findings of Jatau et al. (2010) which reported relapse of Polysorbates (Polysorbates 20, 60, 65 and 80). T. evansi infection in rat treated with 3.5 mg/kg of www.fsc.go.jp/english/evaluationreports/foodadditive/polysorb ate_report. Accessed on May 10, 2012. Diminazene aceturate on day 17 post treatment. They Herbert WJ, Lumsden WH (1976). Trypanosoma brucei: a rapid however reported no relapse of the infection at a higher matching method of estimating the host parasitemia. Exp. Parasitol. dose of 7mg/kg. Mamman et al., (1994) suggested that 40:427-431. the relapse of T. brucei infection could result from the Ibrahim MA, Njoku GC, Sallau AB (2008). In vivo activity of stem barks aqueous extract of Khaya senegalensis. Afr. J. Biotechnol. 7(5):661- inaccessibility of the drugs to privilege sites where 663. trypanosomes were shielded from exposure to curative Jatau ID, Lawal AL, Agbede RIS, Abdurrahman EM (2010). Efficacies of concentration of the drug. Diminazene aceturate and isometamidium chloride in T. evansi In suppressive test, lower parasitaemia observed in the experimentally infected rats. Sokoto J. Vet. Med. 8 (1&2):4-8. Kaboré A, Tamboura HH, Traoré A, Meda R, Kiendrebeogo M, Belem experimental groups in comparison to untreated animals AMG, Sawadogo L (2010). Phytochemical analysis and acute toxicity (negative control) suggests a modulation of infection by of two medicinal plants (Anogeissus leiocarpus and Daniellia oliveri) the extracts. The daily decrease in the suppressive ability used in traditional veterinary medicine in Burkina Faso. Arch. Appl. of the extracts compared to Diminazene aceturate Sci. Res. 2(5):47-52. Kamuanga M (2003). Socio-economic and cultural factors in the suggests a quicker break down of the plant metabolites research and control of trypanosomiasis. Information Division FAO, resulting in a lowering of the antitrypanosomal activity of Rome. pp. 1-10. the extracts. Karori SM, Ngure RM, Wachira FN, Wanyoko JK, Mwangi JN (2008). Anaemia is a constant feature of trypanosome Different types of tea products attenuate inflammation induced in Trypanosoma brucei infected mice. Parasitol. Int. 57:325-333 infections and its severity is linked to the level of Legros D, Legros D, Ollivier G, Gastellu-Etchegorry M, Paquet C, Burri parasitemia (Umar et al., 2000). Karori et al. (2008) C, Jannin J, Büsche P (2002). Treatment of human African reported that trypanosomes generated reactive oxygen trypanosomiasis present situation and needs for research species that attack red blood cells' membranes, inducing development. Lancet Infect. Dis. 2:437-440. Makut MD, Gyar SD, Pennap, GRI, Anthony P (2008). Phytochemical oxidation and subsequently haemolysis. The increased screening and antimicrobial activity of ethanolic and methanolic PCV values recorded with an increase in the quantity of extracts of leaf and bark of Khaya senegalensis. Afri J. Biotechnol. KS in the extract suggest the prophylactic ability of KS 7(9):1216-1219. which protects the animals from anaemia, This improved Mamman M, Moloo SK, Peregrine AS (1994). Relapse of Trypanosoma congolense infection in goats after diminazene aceturate is not PCV in the experimental groups, suggest that the extract a result of invasion of the central nervous system. Ann. Trop. Med. could reduce the severity of T. b. brucei infection if used Parasitol.88:87-88 as prophylaxis. The low PCV observed in suppressive Mann A, Barnabas BB, Daniel II (2010). 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Vol. 14(6), pp. 530-534, 11 February, 2015 DOI: 10.5897/AJB12.558 Article Number: xx ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Fungal infection in freshwater fishes of Andhra Pradesh, India
S. A. Mastan
Matrix-ANU Advanced Aquaculture Research Centre, Acharya Nagarjuna University, Nagarjuna Nagar-522 510, India.
Received 6 February, 2012; Accepted 19 January, 2015
A total of 17 isolates of fungi were isolated from diseased fishes which belong to five species namely Saprolegnia diclina, Saprolegnia ferax, Saprolegnia hypogyana, Saprolegnia parasitica and Achlya americana. All these fungi were isolated from six different species of fresh water fishes viz. Channa stratius, Channa punctatus, Clarias batrachus, Labeo rohita, Heteropneuste fossilis and Mystus cavasius. The parasitic ability of all the fungal species were confirmed by conducting experimental infection under laboratory conditions using healthy fishes of the same species. All the species of fungi were found to be pathogenic to fish.
Key words: Saprolegnia, pathogenicity, fishes.
INTRODUCTION
The saprolegniaceae are responsible for significant infec- Mastan, 2008; and Vinay, 2008; Refai et al., 2010; Rekha tions, involving both living and dead fish and their eggs, and Qureshi, 2012, 2013; Hatai, 2012; Vickie et al., 2013; particularly in aquaculture. Oomycetes are sapro-phytic Zafar Igbal and Reshma, 2013). This paper communicates opportunists multiplying on fish that are physically injured, the fungal infection in freshwater fishes. stressed or infected (Pickering and Willoughby, 1982). Members of this group are generally considered as agents of secondary infections arising from conditions such as MATERIALS AND METHODS bacterial infection, poor husbandry practices, and infesta- tions by parasites and social interactions. However, there Collection of infected fish samples are several reports of Oomy-cetes as infectious agents of fish and their eggs (Scott and O’Bier, 1961; Bhargava Incidences of fungal infection were recorded during the winter et al., 1971; Willoughby, 1978; Srivastava, 1980; Sati and months of 2008 to 2009 from unnamed water bodies of Akividu and Khulbe, 1983; Sati, 1991; Walser and Phelps, 1993; Hatai Bhimavaram divisions of West Godavari Dist, A.P,. A total of 1,270 fishes were screened. The fungal infected fishes were brought to and Hoshiai, 1993; Khulbe et al., 1994; 1995; Kitancharoen the laboratory in the living condition and kept in glass aquarium, of et al., 1995; Qureshi et al.,1995; Kitancharoen and Hatai, the size of 90 × 45 × 45 cm filled with clean fresh water. The dead 1996; Bisht et al., 1996; Rajender and Khulbe, 1998; as well as living fishes were examined grossly for lesions and Qureshi et al., 1999; Vikas et al., 2005; Ramaiah, 2006; ulcerations.
*Corresponding author. E-mail address: [email protected].
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License
Mastan 531
Table 1. Fungi isolated from infected fishes.
Isolate no. Name of fungi Host fish S.d/1 Saprolegnia diclina Channa gachua S.d/2 Saprolegnia diclina Channa striatus S.f/3 Saprolegnia ferax Clarias batrachus S.f/4 Saprolegnia ferax Channa striatus S.f/5 Saprolegnia ferax Channa gachua S.h/6 Saprolegnia hypogyana Channa gachua S.p/7 Saprolegnia parasitica Heteropneustis fossilis S.p/8 Saprolegnia parasitica Heteropneustis fossilis S.p/9 Saprolegnia parasitica Heteropneustis fossilis S.p/10 Saprolegnia parasitica Mystus cavasius S.p/11 Saprolegnia parasitica Mystus cavasius S.p/12 Saprolegnia parasitica Channa punctatus A.m /13 Achlya americana Heteropneustis fossilis A.m14 Achlya americana Heteropneustis fossilis A.m/15 Achlya americana Mystus cavasius A.m/16 Achlya americana Mystus cavasius A.m/17 Achlya americana Labeo rohita
Isolation of fungi from infected fishes each trough. All the experiments were conducted at 20.0 to 25.0°C temperature in triplicate sets. Water samples were collected from Isolation of fungi from infected fishes was carried out by taking infected water bodies for analysis of various Physico-chemical small pieces of muscles about 2 mm in diameter from infected parameters such as Temperature, Conductivity, PH, FCO2, Dissolved portions of the body. They were then washed thoroughly with oxygen, Total alkalinity, Total hardness and Chloride as per methods sterilized distilled water to remove the unwanted micro organisms of APHA (1995). adhered on the surface. These tissues were then inoculated over the plates containing different agar media. Alternatively small pieces of mycelia taken out from infected parts of fish body were RESULTS AND DISCUSSION washed thoroughly with distilled water. They were placed in a Petri dish containing 20 to 30 ml distilled water and baited on different baits viz. Hemp seeds, and Mustered seeds. These Petri dishes In the present study, a total of 17 isolates of fungi were were incubated at 15 to 22°C tem for a week. Pure and bacteria obtained from the fishes investigated. These isolates free cultures were prepared by using the methods of Coker (1923), represent five species and belonged to two genera Johnson (1956) and Scott (1961). Identification of fungi was done namely Saprolegnia and Achlya (Table 1). S. diclina was on the basis of their vegetative and reproductive characters using the monographs of Coker (1923) and Khulbe (1994). isolated twice from infected fishes. One isolate was collected from Channa gachua and one from Channa straitus. A total of three isolates of S. ferax were Artificial infection trials obtained, one was collected from Clarias batrachus, one from C. straitus and one from C. gachua. One isolate of In order to demonstrate the pathogenicity of the isolates obtained S. hypogyana was collected from infected C. gachua. S. from the naturally infected fishes, experimental infection trails were parasitica is the most frequently occurring parasite of fish. conducted in the laboratory. Isolated species of fungi viz. Saprolegnia diclina, Saprolegnia ferax, Saprolegnia hypogyana, Saprolegnia A total of six isolates of this species were obtained from parasitica and Achlya americana were tested separately on the infected fishes. Three isolated from Heteropneuste fingerlings of different species of fishes, having average size and fossilis, two collected from Mystus cavasius and one from weight 8.16 ± 0.13 cm and 12.5 ± 0.28 gm, respectively. The Channa punctatus. A total of five isolates of this species pathogenecity tests were carried out by employing the methodology were obtained, two were collected from H. fossilis, two of Scott and O’ warren (1964). Covered glass troughs (12 x 9) wrapped in aluminum foils were sterilized in hot air oven at 120°C from M. cavasius and one from Labeo rohita. The tem for 24 h. Filtered sterile lake water was filled aseptically in to maximum percentage of infection was recorded to be 4.8 each trough. An aerator was used to aerate the water throughout in the month of December; 2008. While, the minimum the experiment. Six fungal inoculated blocks (1.0 cm2) of SPS agar/ percentage of infection was recorded to be 0.8 in the Potato Dextrose Agar (PDA) medium were placed at different sites month of February, 2009. In case of fish species the in the trough. Six uninoculated blocks of the same agar medium were placed in another trough which was used as control. After 48 highest percentage of infection (1.6) was reported in H. h, when spores developed, experimentally injured fishes were fossilis while lowest infection (0. 8) was reported in L. placed in these troughs. Four fishes of each species were kept in rohita. The experimental infection trails were conducted
532 Afr. J. Biotechnol.
Table 2. Experimental infection trails with various species of fungi isolated from diseased fishes.
No. of fish Mycosis Death S/N Isolate No. Fungi inoculated Experimental fish used Evident (h) (h) 1 S.d/1 Saprolegnia diclina Channa gachua 6 16 24 2 S.d/2 Saprolegnia diclina Channa striatus 6 24 48 3 S.f/3 Saprolegnia ferax Clarias batrachus 6 48 96 4 S.f/4 Saprolegnia ferax Channa striatus 6 36 72 5 S.f/5 Saprolegnia ferax Channa gachua 6 48 72 6 S.h/6 Saprolegnia hypogyana Channa gachua 6 48 72 7 S.p/7 Saprolegnia parasitica Heteropneustis fossilis 6 09 38 8 S.p/8 Saprolegnia parasitica Heteropneustis fossilis 6 08 36 9 S.p/9 Saprolegnia parasitica Heteropneustis fossilis 6 08 36 10 S.p/10 Saprolegnia parasitica Mystus cavasius 6 08 42 11 S.p/11 Saprolegnia parasitica Mystus cavasius 6 08 42 12 S.p/12 Saprolegnia parasitica Channa punctatus 6 09 42 13 A.m/13 Achlya americana Heteropneustis fossilis 6 24 72 14 A.m/14 Achlya americana Heteropneustis fossilis 6 24 76 15 A.m/15 Achlya americana Mystus cavasius 6 18 48 16 A.m/16 Achlya americana Mystus cavasius 6 18 48 17 A.m/17 Achlya americana Labeo rohita 6 18 48
Table 3. Showing Water quality parameters of Hoshiai, 1992; Roberts et al., 1993; Chinnabut et al., 1995; affected water bodies during study period. Willoughby et al., 1995; Khulbe et al., 1995; Mastan, 2008; Rekha and Qureshi, 2012). S/N Parameters Values in range In India the mycological studies were initiated by 1 Water temperature (°C) 17-28 Chidambaram (1942) who observed red patches on the 2 Conductivity (uscm) 260-290 body of Osphronemus gouramy due to Saprolegnia 3 pH 6.8-8.7 species. Tiffney (1939) was the first to demonstrate the
4 FCO2 (mg/l) 1.0-2.6 ability of S. parasitica (Coker) to parasitize a wide range 5 Dissolved oxygen (mg/l) 5.6-8.5 of fishes and amphibians and emphasized the fact that 6 Total Alkalinity (mg/l) 78-193 the injury greatly lowers the resistance of hosts to fungal 7 Total hardness (mg/l) 69-170 infections. Vishniac and Nigrelli (1957) conducted 8 Chloride (mg/l) 10-32 laboratory experiments and demonstrated the parasitic ability of sixteen species of aquatic fungi belonging to seven genera of Saprolegniaceae. Scott (1964) demonstrated that S. parasitica, S. ferax, S. diclina, with fungi isolated from naturally infected fish to test their Saprolegnia monoica, Achyla bisexualis and some non pathogenicity under laboratory conditions. Each isolate fruiting isolates of Saprolegnia could parasitize wounded was tested on that particular species of fish from which it platy fish under controlled conditions. Sati and Khulbe was originally isolated. The results are summarized in (1983) carried out host range studies with S. diclina on Table 2. All the isolates of genus Saprolegnia are found nine species of coldwater fishes such as Barilius to be pathogenic to fish. Hyphal growth of fungi was bendelisis, Carassius auratus, Cyprinus carpio, clearly visible at the site of injured areas of experimental Nemachelius rupicola, Puntius conchonius, Puntius ticto, fishes within 8 to 48 h after inoculation. All the test fishes Schizothorax palgiostomus, Saprolegnia richardsoni and died within 24 to 96 h after catching infection (Table 2). Tor tor. The experimental infection of Saprolegnia on It is observed that although all the isolates has poten- different species of fishes has also been reported by tiality to parasitize the fish but S. parasitica is more Qureshi et al. (2002). Chinabut et al. (1995) and Hatai et vigorous showing infection within 8 h (Table 2). A wide al. (1994) reported the pathogenicity of Aphanomyces range of fluctuations were noticed in various water quality species on Dwarf gourami. Kitanchroen and Hatai (1996) parameters of affected water bodies (Table 3). Fungal have conducted experimental infection trials with infection in fish was first reported during mid – eighteen Saprolegnia sp. on Rainbow trout eggs. century (Arderon, 1748). Later, other workers reported In the present study mycological examination of several pathogenic fungi from different species of fish infected fishes revealed the presence of sixteen isolates and fish eggs. (Sati, 1982; Fraser et al., 1992; Hatai and of five species viz. S. diclina, S. ferax, S. hypogyana,
Mastan 533
S. parasitica, A. americana. All the species of Saprolegnia Kitancharoen N, Hatai K (1996). Experimental infection of are found to be virulent for fishes. This observation is in Saprolegniasis Spp. in Rainbow trout eggs. Fish Pathol. 31(1):49-50. Kitancharoen N, Hatai K, Ogihara R, Aya DNN (1995). A new record of agreement with the finding of Scott and O.Bier (1962) Achlya klebsiana from snakehead, Channa striatus with fungal who have reported that the species of fungi, S. parasitica infection in Myanmar. Mycoscience 36:325-238. is found to be the most destructive. This finding confirms Mastan SA (2008) Incidences of Dermatomycosis in fishes of Larpur with the reports of Hatai and Hoshiai (1992) who have reserviour, Bhopal, (M.P). J Herbal Med. Toxicol. 2(1):37- 40. Pickering AD, Willoughby LG (1982). Saprolegnia infection of salmonid reported that the infection caused by S. parasitica in fish. In: 50th Annual Report, Institutes of freshwater Ecology. salmon resulted mass mortality. 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