1270 Chiang Mai J. Sci. 2017; 44(4)

Chiang Mai J. Sci. 2017; 44(4) : 1270-1278 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

A Newly Isolated Green Alga, acuminatus, from Thailand with Efficient Hydrogen Production Yuwalee Unpaprom [a], Rameshprabu Ramaraj [b, c] and Kanda Whangchai* [d] [a] Biotechnology Program, Faculty of Science, Maejo University, Chiang Mai, Thailand. [b] School of Renewable Energy, Maejo University, Chiang Mai, Thailand. [c] Energy Research Center, Maejo University, Chiang Mai, Thailand. [d] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand. *Author for correspondence; e-mail: [email protected]; [email protected]

Received: 3 March 2015 Accepted: 11 September 2015

ABSTRACT

Biological H2 production is considered the most environmentally friendly route of producing H2, fullling the goals of recycling renewable resources and producing clean energy. It has attracted global attention because of its potential to become an inexhaustible, low cost, renewable source of clean energy and appears as an alternative fuel. This paper presents laboratory results of biological production of hydrogen by green alga was isolated from freshwater fish pond in Sansai, Chiang Mai province, Thailand. Under light microscope, this green alga was identified as belonging to the genus Scenedesmus and species S. acuminatus. The successful culture was established and grown in poultry litter effluent medium (PLEM) under a light intensity of 37.5 μmol-1m2 sec-1 and a temperature of 25°C. The nutrient requirements and process conditions that encourage the growth of dense and healthy algal cultures were explored. The highest H2 was produced when cultivated cells in PLEM for 21 hours under light and then incubated under anaerobic adaptation for 4 hours.

Keywords: , poultry litter effluent, biohydrogen

1. INTRODUCTION

Algae, the major biomass of living one of the best strategies of CO2 fixation organisms in marine and freshwater [1], are and biomass production [4]. There are the most important CO2 fixer, the primary several reasons for this approach: (i) the best producer in aquatic ecosystem through growth rate among the plants, (ii) low impacts the photosynthesis into biomass to fulfill on world’s food supply, (iii) specificity for the responsible duty of CO2 [2, 3], and play CO2 sequestration without gas separation to a crucial niche of CO2 bio-fixation of the save over 70% of total cost, (iv) excellent ecosystem [3]. treatment for combustion gas exhausted At present algae is broadly recognized as with NOx and SOx, (v) high value of algae Chiang Mai J. Sci. 2017; 44(4) 1271

biomass including of feed, food, ammonium nitrogen, nitrate, nitrite and which nutrition, pharmaceutical chemicals, are suitable for growing algae. In addition, fertilizer, aquaculture, biofuel, etc [5-7]. pathogens in the effluent were eliminated by As efficient photosynthetic organisms, anaerobic digestion particular after two-stage microalgae have unique advantages in (thermophilic and mesophilic) digestion [17]. capturing solar energy to generate reducing Therefore, digested effluent as medium of equivalents and converting atmospheric algal culture could be an alternative to

CO2 to organic molecules [9]. Microalgae using costly mineral medium which is not also have special advantages in ability to environmentally sustainable. adapt to various stressful environments, In addition, microalgae grown under non-requirement of agricultural land and heterotrophic condition (using organic carbon) so on [4]. Microalgae’s high ability to could have more potential to produce use inorganic nutrients (nitrogen and hydrogen than in autotrophic condition phosphorous) from wastewater makes them (inorganic carbon). In heterotrophic condition, a useful bioremediation tool in waste water high biomass is achieved. Moreover, in treatment process. Many green microalgae heterotrophic mode of cultivation organic species are commonly used in the wastewater wastes, as cheaper carbon sources can be treatment system due to their high tolerance used. Unlike autotrophic condition, light is to soluble organic compounds [10-12]. also not required in heterotrophic cultivation Algae can be successfully cultivated in [18]. Therefore, heterotrophic cultivation wastewaters. It can be potentially a sustainable can be cheaper than autotrophic cultivation. growth medium for the algal feed stock One of the advantages of [12, 13]. Use of microalgae in treatment and for producing hydrogen is their ability recycling of waste water has attracted a great to grow under photoautotrophic and deal of interest because of excessive biomass photoheterotrophic condition. There is little generation at cheaper cost without extra input information available for undertaking of nutrients such as inorganic fertilizer intensive algal production by using digested (chemical medium) [14]. Microalgae can use poultry effluent as a nutrient re-source. inorganic carbon (CO2) and organic carbon The present article reports simultaneous algae source (glucose, mannitol, acetate, sucrose). growth, biomass production and waste Cultivation of microalgae in swine wastes, recycling with the green microalga S. acuminatus dairy manure, and other animal residues has from the poultry litter waste water. been reported by several authors [14-16]. At present, the potential of microalgae More importantly, there were no bacteria as a source of renewable energy has received and pathogens found on aerated manure considerable interested in biofuel such as when algae are grown. The algae culture biodiesel, bioethanol, biogas, biohydrogen carried out in different types of reactors, and bio-oils [17, 18]. H2 is a valuable energy flasks and plastic bags are common practice carrier, an important feedstock to the chemical with mineral medium as nutrient sources. industry, and useful in detoxifying a wide Kumar et al. [17] stated digested piggery range of water pollutants. Compared to fossil effluent could be an alternative nutrient source fuels as traditional energy sources, hydrogen for mass algal production since anaerobically is a promising candidate as a clean energy treated animal waste contains nutrients such carrier in the future because of its higher as phosphorous, nitrogen species including, heating values 141.6 MJ kg-1, or 12.6 MJ m-3 1272 Chiang Mai J. Sci. 2017; 44(4)

[8, 9]. Heat and water are the only products 10 days with cool white fluorescent light of combustion of H2 with no releasing using the same light intensity. of CO2 into the atmosphere [19, 20]. The single colonies on agar were picked Thus, hydrogen is a clean, renewable, and up and cultured in liquid BBM medium, non-polluting fuel. Previous reports showed and the streaking and inoculation procedure that photosynthetic microorganisms, was repeated until pure cultures were cyanobacteria and green algae, can generate obtained. The purity of the culture was hydrogen from solar energy and water monitored by regular observation under [17-20]. microscope. The isolated microalgae were The algal cultivation process was focused identified microscopically using light on in this study for reducing the cost. microscope with standard manual for algae Cultivation of microalgae in swine wastes, [23,24]. dairy manure, and other animal residues has been reported by several literatures 2.2 Inoculums Preparation [10-17, 20, 21]; by using Scenedesmus sp., which Isolated and purified microalgae is part of our ecosystem and is very accessible, (S. acuminatus) were inoculated in 250-ml a more environmental friendly and renewal Erlenmeyer flasks containing 125 ml culture fuel can be produced. In this study, we medium (BBM). Flasks were placed on a examined a newly isolated species of green reciprocating shaker at 120 rpm for 7 d at microalga S. acuminatus for biohydrogen room temperature of 25±1 °C. Light was production applied with inexpensive poultry provided by cool white fluorescent lamps at litter effluent medium (PLEM). an intensity of 37.5 μmol-1m2 sec-1. The algae culture was then transferred to 500-ml 2. MATERIALS AND METHODS Erlenmeyer flasks containing 450 ml. 2.1 Isolation and Identification of Microalgae 2.3 Preparation of Poultry Litter Effluent Microalgae were collected by plankton Medium net (20-μm pore size) from freshwater fish The anaerobically digested poultry litter pond (18°55′4.2′′N; 99°0′41.1′′E) at a effluent (PLEM) raw was collected from the location near Maejo University, Sansai, chicken farm at Mae Fak Mai, Chiang Mai Thailand. The collected samples were province, Thailand (18°97′20.01′′N; samples of about 5 ml were inoculated into 98° 97′99.41′′E) and transported to the 5-ml autoclaved Bold Basal Medium (BBM) Maejo University laboratories in 10 L plastic [22] in 20-ml test tubes and cultured at room containers and stored in a cold room temperature (25 °C) under 37.5 μmol-1m2 maintained at 4°C. Pre-treatment was sec-1 intensity with 16:8 h photoperiod for carried out by sedimentation and filtration 10 days. After incubation, individual colonies with a filter cloth to remove large, non-soluble were picked and transferred to the same particulate solids. After filtration the substrate media for purification in 250 mL conical was autoclaved for 20 min at 121°C, after flask. The culture broth was shaken manually which the liquid was stored at 4 °C for for five to six times a day. The pre-cultured 2 days for settling any visible particulate samples were streaked on BBM medium- solids and the supernatant was used for enriched agar plates and cultured for another microalgae growth studies. Chiang Mai J. Sci. 2017; 44(4) 1273

2.4 Growth Conditions and Measurements electron impact, EI, 70 eV with HP 5973 mass The isolated green alga was grown in selective detector and a molecular sieve 450 ml Erlenmeyer flasks each containing 5A 60/80 mesh packed column using a 450 ml of poultry litter effluent medium thermal conductivity detector). The injector (PLEM) for the heterotrophic growth and detector temperatures were kept at condition. It was cultivated at room 100°C whereas the oven temperature was temperature for 3 days under fluorescent maintained at 50°C. Argon gas was used as a light illumination for 18 hours per day with a carrier gas during hydrogen analysis. shaking speed of 120 rpm. All experiments Hydrogen production was calculated as were carried out in triplicate. Optical a term of hydrogen evolved per chlorophyll μ density at 730 nm was measured using a content per time (nmolH2/ g chl/h). spectrophotometer (HACH, DR/4000U) Hydrogen evolution of cells at 6, 12, 18, 24 every 3 days interval for 18 days. and 36 hours of cultivation was measured The cell density and growth was under light and dark conditions. In addition, determined by measuring the chlorophyll algae cells were harvested and incubated concentration; the algal culture was under anaerobic condition for 2, 4, 6, 8 and measured every 3 hours of cultivation by 24 hours in darkness before measuring optical density measurement at wavelength hydrogen evolution. 750 nm. The chlorophyll of cell suspension was extracted with 90% methanol and the 2.7 Statistical Analysis total chlorophyll concentration was calculated Data are reported as mean ± SE from by the method of Kosourov et al. [25]. triplicate observations. Significant differences between means were analyzed. All statistical 2.5 Analytical Methods analyses were performed using SPSS Version All the indices including pH, chemical 20.0. oxygen demand (COD), total nitrogen (TN), total phosphorous (TP) were continuously 3. RESULTS AND DISCUSSION monitored throughout the study, following 3.1 Species Identification the standard protocols of APHA [20]. The green alga isolated from a fresh water fish pond, was identified as S. acuminatus.

2.6 Measurement of H2 Production Under light microscope, single cells, four One hundred ml of cell culture was cells surrounded by transparent sheath, harvested by centrifugation at 5000×g for and truncated transparent sheaths can be 10 min at 4°C. The cell pellet was washed observed. Figure 1 shows the morphology with PLEM medium and the cell resuspension of S. acuminatus observed under a light was transferred into a 10 ml glass vessel and microscope. sealed with a rubber stopper. The anaerobic adaptation was performed by purging Systematic classification: Argon gas to the cell suspension for 5 min Phylum: under dark condition and the cells were Class: incubated at room temperature for 2h. Order: Hydrogen was determined by analyzing Family: gas phase by a gas chromatography (Agilent Genus: Scenedesmus 6890 gas chromatography coupled to Species: Scenedesmus acuminatus 1274 Chiang Mai J. Sci. 2017; 44(4)

S. acuminatus is an example of colonial Figure 2. It was found that the cells stayed in green algae. It is more often found floating the lag phase period for the first 9 hours of in the creek water and not attached to rocks cultivation. After that cells grew rapidly and on the bottom. It grew well in water from entered the log phase period until reaching a wastewater contaminated creek and the stationary phase at about 12-27 hours of dominated the primary ponds. Unlike most growth. Consequently, anaerobically digested of the colonial green algae that form long poultry litter effluent medium was revealed filaments, S. acuminatus forms small chains of that feasible algae biomass production. four cells. The ends of the colony possess Furthermore the biomass is projected as a cell wall extensions that have a spike like virtually eternal raw material for H2 appearance. production.

3.3 Hydrogen Production by S. acuminatus in Photoheterotrophic Condition Biohydrogen is a renewable biofuel produced from biorenewable feedstocks by a variety of methods, including chemical, thermochemical, biological, biochemical, and biophotolytical methods [26]. Biological hydrogen production processes are found to be more environment friendly and less energy intensive as compared to thermochemical and Figure 1. Cell morphology of cells of S. electrochemical processes. Furthermore, acuminatus observed under a light microscope. biohydrogen produced from biological processes has the potential for renewable 3.2 Algae Growth Medium, Biomass biofuel to replace current unsustainable Production and Characterization hydrogen production technologies, which Algal growth was monitored by utilizing rely on nonrenewable fossil fuels through the optical properties of the culture to thermochemical processes [26]. The basic measure either its chlorophyll content / optical advantages of biological hydrogen production density. Growth of S. acuminatus was examined over other “green” energy sources are that it when grown in PLEM medium, S. acuminatus does not compete for agricultural land use, showed a fast growth. PLEM medium was and it does not pollute, as water is the only identified as the most suitable for S. acuminatus by-product of the combustion. These since high cells density. The nutritious characteristics make hydrogen a suitable characteristics of PLEM presented in Table fuel for the future. Among several 1. The PLEM had high concentrations of biotechnological approaches, photobiological nitrogen (1890 mg L-1), phosphorus (187 mg hydrogen production carried out by green L-1), potassium (1749 mg L-1) and other microalgae has been intensively investigated micronutrients (Table 1), which were in recent years. One of the promising signicantly higher than recommended for algae biohydrogen production approaches is cultivation. Algae growth was presented in conversion from microalgae, which is Chiang Mai J. Sci. 2017; 44(4) 1275

Table 1. Elemental composition of PLEM fundamental and practical importance of the (after removing TSS). process. Below is an itemized list of the properties and promise of photosynthetic Elemental analysis PLEM (mgL-1) H -production [28]: Aluminum (Al) 10.55 2 Boron (B) 2.33 ⋅Photosynthesis in green algae can operate Cadmium (Cd) <0.1 with a photon conversion efficiency of > 80%. Calcium (Ca) 210.2 Chromium (Cr) <0.1 ⋅Microalgae can evolve H Copper (Cu) 14.7 2 photosynthetically, with a photon conversion Iron (Fe) 28.71 efficiency of > 80%. Lead (Pb) 1.05 Magnesium (Mg) 57.35 ⋅Molecular O acts as a powerful and Manganese (Mn) 5.99 2 effective switch by which the H2-production Molybdenum (Mo) 1.16 activity is turned off. Nickel (Ni) 1.02 Phosphorus (P) 187 Figure 3 exhibited the H evaluation Potassium (K) 1749 2 which was measured in cells at different time Silicon (Si) 48.07 of cultivation period. The result showed Sodium (Na) 359 that H production was highest with Sulfur (S) 124 2 2.1 nmolH /μg chl/h in cells grown for Zinc (Zn) 10.27 2 21 hours (late-log phase cells) in PLEM. After Nitrate nitrogen (NO -N) 4.811 3 21 hours of cultivation, hydrogen production Ammonia nitrogen (NH4-N) 1384 COD 900 Total nitrogen 1890 Total organic carbon 881

Figure 3. Hydrogen production during cultivation periods.

Figure 2. Algae growth under of cells decreased very sharply. photoheterotrophic condition. It was found that the late-log phase cells or 21-hours old cells could produce abundant, clean, and renewable [26, 27]. the highest hydrogen yield due to the The ability of green algae to enough accumulation of glycogen from the photosynthetically generate molecular H2 fermentation process in the PLEM. In the has captivated the fascination and interest lag-phase cells or 18-hours old cells, under of the scientific community because of the photoheterotrophic condition they need 1276 Chiang Mai J. Sci. 2017; 44(4)

acetic acid for generating energy utilized in have higher optical density than those the cellular metabolism and for dividing under light condition (data not mentioned). cells. The generated energy and reducing It might be explained that cells grown powers are necessarily used for cell growth under dark condition used only acetic acid instead of producing hydrogen. In case of as carbon source for growing and dividing stationary phase cells (24-and 36-hours old cells whereas cells grown under light cells), they were not fit and began to condition could fix CO2 from the atmosphere die because of carbon source starvation. via photosynthetic process, therefore Hence, the results from S. acuminatus verified that 18 to 21 hours are suitable time for

H2 production using biological method.

3.4 Hydrogen Production by S. acuminatus in Anaerobic Adaptation Time, under Light and Dark Condition S. acuminatus was heterotrophically grown in PLEM at room temperature with same the shaking speed and time Figure 4. Hydrogen production under as before mentioned. Algae cells were different anaerobic adaptation time. incubated under anaerobic condition for 2 to 36 hours in darkness before hydrogen requiring more time for the initial growth. production measurement. It was found that The result showed those cells grown hydrogen production was highest, with under light produced hydrogen about μ 0.149 nmolH2/ g chl/h, in cells incubated 4 times higher than cells grown under dark under anaerobic adaptation for 4 hours μ condition (1.99 and 0.45 nmolH2/ g chl/h, (Figure 4). After that hydrogen production respectively); the results presented in of cells was obviously decreased. It might Figure 5. It was suggested that under be explained that during anaerobic light condition the energy in form of ATP adaptation, oxygen, an inhibitor of (adenosine triphosphate) and the reducing hydrogenase enzyme, was decreased powers NADPH (nicotinamide adenine resulting in an increase of hydrogen dinucleotide phosphate) or NADH production in the first 4 hours after anaerobic (nicotinamide adenine dinucleotide) adaptation. Incubation under anaerobic were obtained from the light reaction condition for more than 4 hours did not of photosynthesis, giving their electrons promote the higher hydrogen production to excess protons for hydrogen production. because of the decrease of electron Under dark condition cells produced and proton donors in cells as well as the less ATP and reducing powers, resulting limitation of hydrogenase enzyme. in less hydrogen production. Consequently, S. acuminatus cells were separately the isolated green alga S. acuminatus grown in PLEM either providing light produced the highest hydrogen when illumination of 37.5 μmol-1m2 sec-1 intensity cultivated cells for 21 hours in PLEM or under dark condition. The result showed under light and then incubated cells that cells grown under dark condition under anaerobic adaptation for 4 hours. Chiang Mai J. Sci. 2017; 44(4) 1277

Maejo University, Sansai, Chiang Mai 50290, Thailand) for offered chicken manure, poultry forming support & Poultry litter effluent transportation.

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