Middle East Journal of Applied Volume : 05 | Issue : 04 | Oct.-Dec. 2015 Sciences Pages: 1222-1231 ISSN: 2077-4613

Optimizing Growth Conditions Provoked Ethanol Production by Fungi Grown on Glucose

Sanaa S.H. Sarabana, Khadiga I. M. El-Gabry and Ahmed M. Eldin

Soils, Water & Environment Research Institute, Agricultural Research Centre (ARC), Giza, Egypt.

ABSTRACT

This study was conducted to test nine cellulytic filamentous fungi including: Aspergillus oryzae, Aspergillus versicolor-I, Aspergillus versicolor-II, Fusarium oxysporum, Fusarium oxysporum-I, Mucor indicus, Penicillium citrinum, Phanerochaete chrysosporium and Pleurotus sp., to ferment glucose. They were tested on Mandels medium during 14 days of incubation at 35°C under minor air conditions. The best ethanol producers were F. oxysporum, Ph. chrysosporium and F. oxysporum-I, recording 0.464, 0.360 and 0.325g ethanol/g carbon. Modifications to Mandels medium were done individually to magnify ethanol production and deselect aside lower ethanol producers. Nitrogen sources substitution including ammonium sulfate, ammonium phosphate, casein, peptone, potassium nitrate and yeast extract was done. Different C:N ratios (2:1, 4:1, 6:1 and 8:1) and pH levels (4, 5 and 6) were tested. Glucose concentrations including 100, 200 and 300g/L were also tested. Ph. chrysosporium was selected for its best production recording 0.847 g ethanol/g carbon corresponding to 33.9 g/L, utilizing yeast extract as N source, at C:N ratio of 2:1, pH=5 and initial glucose level of 100g/L. The ethanol yield was improved 2.4 times (from 14% to 33.9%). However, either pH=4 or 300g/L glucose was repressor for ethanol production.

Key words: Ethanol production, glucose, Aspergillus oryzae, Aspergillus versicolor, Fusarium oxysporum, Mucor indicus, Penicillium citrinum, Phanerochaete chrysosporium, Pleurotus sp.

Introduction

In recent years, growing attention has been devoted to the conversion of biomass into ethanol, considering the liquid fuel alternative to fossil fuels (Lin and Tanaka, 2005). Ethanol does not increase atmospheric net-CO2, thus has no contribution to global warming. Combustion of ethanol results in relatively low emissions of volatile organic compounds, carbon monoxide and nitrogen oxides (Park et al., 2010). Besides, ethanol is a clean- burning renewable resource that can be produced from fermented cellulosic biomass (Masud et al., 2012). A number of basidiomycetes produce alcohol dehydrogenase, and therefore it is possible to produce alcohols using hexoses (Okamura et al., 2000, 2001). Kenealy and Dietrich (2004) found that Ph. chrysosporium under oxygen depletion can ferment glucose to ethanol. However, some studies showed that a few white-rot basidiomycetes, including Phanerochaete chrysosporium were capable of producing ethanol from hexose sugars (Kenealy and Dietrich, 2004; Mizuno et al., 2009 a, b and Okamoto et al., 2010). Aspergillus oryzae is a fungus with high potential for the secretary production of various enzymes and is commonly used in traditional Japanese fermentation industries (Machida et al., 2008). Kamei et al. (2012) reported that the white rot fungus Phlebia sp. was able to completely assimilate glucose, mannose, galactose, fructose, and xylose to give ethanol yields of 0.44, 0.41, 0.40, 0.41, and 0.33 g ethanol/g carbon of sugar, respectively. Recently, Hossain (2013) reported the optimization of direct ethanol production using A. oryzae. F. oxysporum has an efficient system able to ferment hexose sugars to ethanol under anaerobic or microaerobic conditions (Anasontzis and Christakopoulos, 2014). Okamoto et al., (2014) characterized Trametes versicolor that was capable of efficiently converting hexose sugars to ethanol. Saccharomyces cerevisiae has been used through thousands of years for its ability to grow on different types of glucose-rich hydrolysates in the production of different types of alcoholic beverages. The probability of another organism that can compete with yeast in the fermentation efficiency is quite low. Even so, in order for filamentous fungi, such as F. oxysporum, to become advantageous in a realistic sense, a number of improvements would have to be made, either through genetic modification or evolutionary engineering, in combination with process development and optimization (Anasontzis and Christakopoulos, 2014). Enzymatic hydrolysis and hexoses fermentation can run together, in a same reactor, as simultaneous saccharification and fermentation which was found to be faster and presented a low cost process since only one reactor is necessary and the glucose formed is simultaneously fermented to ethanol. The risk of contamination is lower due to the presence of ethanol, the anaerobic conditions and the continuous withdrawal of glucose (Castro and Pereira, 2010; Soccol et al., 2010). In the Consolidated Bio Processing (CBP) a single microbial community

Corresponding Author: Ahmed M. Eldin, Soils, Water & Environment Research Institute, Agricultural Research Centre (ARC), Giza, Egypt, E-mail: [email protected]

1222 Middle East J. Appl. Sci., 5(5): 1222-1231, 2015 ISSN 2077-4613 produced all the required enzymes and converted sugars into ethanol in a single reactor, lowering overall costs (Lynd, 1996). Studies suggested that CBP may be feasible and the researches have focused on the development of new microorganisms adapted to this process, which has been a key challenge (Lynd et al., 2002). The aim of this study was to establish a relationship between optimizing nutritional type and balance for the best ethanol production and predicting fermentative behavior of tested cellulytic fungi with glucose representing most aldohexoses in saccharified cellulosic debris. This will be a preliminary study preceding simultaneous saccharification and fermentation processes carried out by the same microorganism or more in the same reactor vessel.

Materials and Methods

Fungal isolates and strains:

Cellulytic fungi including six isolates and three strains were tested for their ability to ferment glucose, as the main hexoaldoses sugar in cellulosic agricultural debris, in producing considerable amounts of ethanol. The six fungal isolates were previously identified as Fusarium oxysporum-I, Mucor indicus, Aspergillus versicolor-I, Aspergillus versicolor-II, Aspergillus oryzae and Penicillium citrinum by Plant Pathology department (A.R.C), Giza, Egypt (Sarabana et al., 2014 and Abou El-Khair et al., 2014). On the other hand, the three fungal strains including Fusarium oxysporum, Phanerochaete chrysosporium and Pleurotus sp. were generously offered by Microbiology department (A.R.C), Giza, Egypt.

Starter medium:

All strains were enriched in Mandels medium (Mandels et al., 1974) on orbital shaker incubator at 35˚C/125 rpm for 5 days. The medium contained the following ingredients (g/l): urea, 0.3; glucose, 10; MgSO4.7H2O, 0.3; KH2PO4, 2; CaCl2.2H2O, 0.3; (NH4)2SO4, 1.4; Bactopeptone, 1; Tween 80, 0.1; trace elements: FeSO4.7H2O, 5mg; MnSO4.H2O, 16mg; ZnCl2.2H2O, 17mg; CoCl2.6H2O, 2 mg. The medium, trace elements and glucose were autoclaved separately.

Fermentation medium (FM):

The enriched cultures from all fungal strains were maintained to 0.5 % dry biomass (w/v) and were used for inoculating (5%, v/v corresponding to 0.1g dry weight/20ml) the FM volume of 150ml medium in 200 ml firmly closed bottles to maintain minimized aeration conditions for fermentation process (Kenealy and Dietrich, 2004). The FM structure was based on modified Mandels medium (Fatma et al., 2010) with further modifications as follows: glucose 100g/L as the sole C source to supply 40% Carbon (w/v) and (NH4)2SO4 47g/L as the sole N source to supply10% nitrogen (w/v), 0.1% yeast extract and initial pH was adjusted at 5. Inoculated fermentation bottles allocated in a complete randomized design with three replicates were statically incubated at 35˚C for 14 days and samples were collected at two days intervals for further studies.

Optimization of fermentation medium:

To improve ethanol production, FM contents were substituted individually. Nitrogen source content in control (designated as ammonium sulfate) was substituted with ammonium phosphate, casein, peptone, potassium nitrate and yeast extract, fulfilling the same total N% (Pasha et al., 2012). Afterwards, the best N source was validated in different C: N ratios test; 2:1, 4:1, 6:1 and 8:1, followed by testing the best C: N ratio at different initial pH values of 4, 5 and 6, adjusted by HCl/NaCl solutions (0.1N). Based on best former parameters, carbon level test was conducted at levels of 100, 200 and 300 g/L glucose and with fixed inoculums dry weight and size.

Analysis:

Biomass was determined gravimetrically (DW) in the collected samples. The fungal mycelia were harvested every 48 h during fermentation, separated by filtration through a pre-dried and weighed filter paper (Whatman No.1), repeatedly washed with distilled water, dried at 70°C overnight and dry weight was calculated (Srivastava et al., 2011). Glucose utilization was periodically measured as total reducing sugars by 3, 5-Dinitro salicylic acid method according to Miller (1959). Spectrophotometric determination of ethanol was according to dichromate method (Caputi et al., 1968). Ethanol production was expressed as g ethanol/g carbon added in the FM.

1223 Middle East J. Appl. Sci., 5(5): 1222-1231, 2015 ISSN 2077-4613

Ethanol (g/L) x 100 Ethanol production yield = Initial Glucose (g/L)

Ethanol (g/L) x100 Ethanol production efficiency: Consumed Glucose (g/L) x100 Statistical analysis:

All results data were accomplished in triplicates and statistically evaluated by least significant differences (LSD) in one way completely randomized analysis of variance (ANOVA) at 5% significance calculated using CoStat (6.311) software (Maruthai et al., 2012).

Results and Discussion

I. Selecting best ethanol producer among fungal species:

The ability of three fungal strains and six fungal isolates to metabolize glucose, as the major carbon sole in FM under minimized aeration condition, during the fermentation process was investigated as demonstrated in Fig. (1). Glucose metabolism was aimed to produce maximum ethanol than to support mycelial growth. Fusarium oxysporum and Phanerochaete chrysosporium maximally produced 0.464 and 0.360 g ethanol/g carbon after 10 and 14 days, respectively, exceeding other fungal species statistically at LSD = 0.038. Following them, Fusarium oxysporum-I produced 0.325 g ethanol/g carbon after 14 days.

Penicillium citrinum Aspergillus versicolor-I Phanerochaete chrysosporium Fusarium oxysporum Pleurotus sp Fusarium oxysporum-I 0.500 Aspergillus oryzae Mucor indicus 0.450 Aspergillus versicolor-II 0.400 0.350 0.300 0.250 0.200 0.150 g Ethanol / g Carbon g Ethanol 0.100 0.050 0.000 2 4 6 8 10 12 14 Days

Fig. 1: Efficiency of nine fungal species in glucose fermentation to ethanol

II. Optimization of FM for maximizing ethanol production

1- N source effect:

F. oxysporum-I, Ph. chrysosporium and F. oxysporum, chosen as the best ethanol producers among the nine fungal species, were tested for their maximum ethanol production using optimum N source. The nitrogen sources including mainly urea beside ammonium sulfate, peptone, yeast extract (all designated as ammonium sulfate) in FM were substituted by other nitrogen sources on the bases of nitrogen content that reached 10% (w/v) of the medium (Pasha et al., 2012). Yeast extract was the optimum nitrogen source for F. oxysporum-I, Ph. chrysosporium and F. oxysporum, as they gave their maximum ethanol production of 0.693, 0.626 and 0.512 g ethanol/g carbon, respectively. The ethanol production peaks with yeast extract were characterized by two maximum points gapped by 2 days drop, recognizably earlier by 2 days than those maximum points achieved on non modified Mandels medium used. Statistically at LSD of 0.058, F. oxysporum-I was leading the

1224 Middle East J. Appl. Sci., 5(5): 1222-1231, 2015 ISSN 2077-4613 best production after 10 days with a previous minor one after 4 days, followed by Ph. chrysosporium after 10 days with a minor one after 6 days (Fig. 2).

Fusarium oxysporum-I 0.800 Amm. Phosphate 0.700 Amm. Sulfate 0.600 0.500 Casein 0.400 Peptone 0.300 Pot. Nitrate 0.200 g Ethanol/g Carbon g Ethanol/g Yeast extract 0.100 0.000 2 4 6 8 10 12 14

Days Phanerochaete chrysosporium 0.700 Amm. Phosphate 0.600 Amm. Sulfate 0.500

0.400 Casein

0.300 Peptone

0.200 Pot. Nitrate g Ethanol/g Carbon g Ethanol/g 0.100 Yeast extract 0.000 2 4 6 8 10 12 14 Days Fusarium oxysporum 0.600 Amm. Phosphate 0.500 Amm. Sulfate 0.400 Casein 0.300 Peptone 0.200 Pot. Nitrate g Ethanol/g Carbon g Ethanol/g 0.100 Yeast extract 0.000 2 4 6 8 10 12 14 Days

Fig. 2: Effect of different nitrogen sources on ethanol production by F. oxysporum-I, Ph. chrysosporium and F. oxysporum

Yeast extract proved to posses many important vitamins, growth promoters, short chain peptides and free amino acids that aid microorganism growth (Valle-Rodriguez et al., 2012). Most of filamentous fungi under study favored yeast extract because of free amino acids it possessed to maximize their ethanol production than other nitrogen sources lacking free amino acids. On the same track, Sharma and Pandy (2010) stated that F. oxysporum maximum growth was achieved on medium supplemented with yeast extract more than other media, insisting on the importance of this nitrogen source for F. oxysporum metabolic activity and growth. Also many studies stated that different amino acids improved fermentation capability of different yeast strains, as asparagine and glutamine with Koloeckera africana and argenine with S. cerevisiae (Valle-Rodriguez et al., 2012). Consequently, yeast extract containing free amino acids was optimum for S. cerevisiae growth and

1225 Middle East J. Appl. Sci., 5(5): 1222-1231, 2015 ISSN 2077-4613 during fermentation of glucose (Taylor et al., 1995) and starch digest (Manikandan and Viruthagiri, 2010) for maximum ethanol production.

2- C:N ratio effect:

Ph. chrysosporium and F. oxysporum-I were chosen for their highest ethanol production on yeast extract for further optimization by testing different C:N ratios below and above that used in the control (1:4). The capability of both fungi to increase their ethanol production was conducted to narrow C:N ratio, as shown in Fig. (3). Statistically, at LSD=0.122, best ethanol production was achieved by Ph. chrysosporium at C:N ratio of 2:1 to be 0.864 g ethanol/g carbon after 10 days of fermentation preceded by 0.495 after 6 days. The same fungi gave 0.575 g ethanol/g carbon after 10 days at C:N ratio of 4:1 (control). On the other hand, maximum ethanol production of 0.545 g ethanol/g carbon was achieved by F. oxysporum-I after 10 days at C:N ratio of 4:1. Increasing C:N ratio decreased ethanol production by both fungal species. Mostly, with both fungal species grown on all C:N ratios a noted decrease in ethanol production for 48hr took place between 6th and 8th days.

Phanerochaete chrysosporium 1.000 0.900 0.800 0.700 2 0.600 0.500 4 0.400 6 0.300 g Ethanol / g Carbon g Ethanol 8 0.200 0.100 0.000 2 4 6 8 10 12 14 Days

Fusarium oxysporum-I 1.000 0.900 0.800 0.700 2 0.600 0.500 4 0.400 6 0.300 8 g Ethanol / g Carbon g Ethanol 0.200 0.100 0.000 2 4 6 8 10 12 14 Days

Fig. 3: Effect of C:N ratio on Ph. chrysosporium and F. oxysporum-I ethanol production.

Sorensen and Giese (2013) stated that three strains of Fusarium avenacium proved to be affected by carbon sources and levels, as they regulated secondary metabolites through activating respective genes in response to abiotic components such as aeration, pH and temperature. Mucor indicus produced maximum ethanol by fermenting glucose in the presence of yeast extract at narrow C:N ratio of 8:1 (Asachi et al., 2011). On the contrary, Kenealy and Dietrich (2004) stated that Ph. chrysosporium, fermenting glucose throughout 12 days under limited oxygen conditions, maximized ethanol production with wider C:N ratio of 13:1. Also, S. cerevisiae fermented tapioca starch digest (Manikandan and Viruthagiri, 2010) and glucose (Taylor et al., 1995) to produce maximum ethanol in presence of yeast extract at very wide C: N ratios of 35:1 and 33:1, respectively. The high need of Ph. chrysosporium to a narrow C:N ratio for growth (increase nitrogen conc.) decreased as it didn't grow fermentatively but survived transient oxygen limitation by fermentation producing more ethanol than with wide C:N ratio (Kenealy and Dietrich, 2004).

1226 Middle East J. Appl. Sci., 5(5): 1222-1231, 2015 ISSN 2077-4613

On the other hand, under aerobic conditions F. oxysporum consumed glucose so rapidly during exponential growth phase (Srivastava et al., 2011) and that was not favorable in fermentation under minimized aeration conditions. This was emphasized by (Kenealy and Dietrich, 2004) who found that alcohol dehydrogenase activity inside Ph. chrysosporium cells was detectable during ethanol production but not under aerobic conditions, indicating its crucial role in ethanol formation under minor aeration or anaerobic conditions. According to these emphases, it seemed in present work that minimum aeration available for either Ph. chrysopsporium and F. oxysporum-I pushed more growth at narrow C:N than wide C:N ratios leading to increase in mycelial growth. As the grown fungus could still ferment under limited aeration the glucose to ethanol, but not efficiently as with anaerobic condition, this was the main cause of accumulating more ethanol with narrow C:N than wide C:N ratios. This can be deduced from reverse relationship between ethanol production and C:N ratio as shown in Fig (3).

3- Effect of pH value:

As both Ph. chrysosporium and F. oxysporum-I achieved the best production with C:N of 2:1, respectively, the effect of FM different initial pH values of 4, 5 and 6 on ethanol production by either species was established. FM initial pH value proved to affect both fungal species, as they shifted their successful ethanol production between pH values 5 and 6, with pH 5 being the best, as shown in Fig. (4). Statistically at LSD=0.039 and after 10 days, Ph. chrysosporium and F. oxysporum-I produced their maximum ethanol recording 0.755 and 0.574 g ethanol/g carbon, respectively. Both species kept their characterized decline in ethanol production between the 6th and the 8th days, as the decrease reached 16% with the former and 21% with the later.

Phanerochaete chrysosporium 1.000 0.900 0.800 0.700 0.600 4 0.500 0.400 5

g Ethanol / g Carbon g Ethanol 0.300 0.200 0.100 0.000 2 4 6 8 10 12 14 Days

Fusarium oxysporum-I 1.000 0.900 0.800 0.700 0.600 0.500 4 0.400 5 g Ethanol / g Carbon g Ethanol 0.300 0.200 6 0.100 0.000 2 4 6 8 10 12 14 Days

Fig. 4: Effect of initial pH value on Ph. chrysosporium and F. oxysporum-I ethanol production

Sorensen and Giese (2013) stated that secondary metabolites of three strains of Fusarium avenacium were influenced by regulators that activate the respective genes in response to pH as an abiotic component. Manikandan and Viruthagiri (2010) mentioned that saccharified tapioca starch fermentation by S. cerevisiae was optimum at pH 5.5. In a continuous stirred bioreactor, fermentation of saccharified wheat straw by F. oxysporum gave its highest ethanol yield at initial pH 6 but declined drastically by increasing acidity down to pH 4.5 (Hossain et al., 2012). Nevertheless, Khilare and Ahmed (2012) found that pH range from 4.5 to 8 was

1227 Middle East J. Appl. Sci., 5(5): 1222-1231, 2015 ISSN 2077-4613 suitable for the growth of F. oxysporum, being optimally attained at pH 6. Bhattacharya et al. (2013) maintained best conditions for solid state fermentation of hydrolyzed water hyacinth at pH 5 for ethanol production by Pichia stipitis, Candida shehatae and S. cerevisiae.

4- Carbon leveling effect:

According to the previous optimization trials, Ph. chrysosporium maximum ethanol production was achieved collectively using yeast extract as N source, C:N ratio of 2:1 and pH= 5. The impact of changing initial glucose concentration on the fermentation process carried out by Ph. chrysosporium was evaluated by ethanol production magnitude, biomass propagation and pH values determined during 14 days of fermentation, as shown in Fig. (5).

1.700 A 6.0 A 0.900 5.8 0.847 1.500 0.800 0.833 Mycelia wt 5.6 0.700 ml 1.300

0 5.4

2 pH 0.600 0.611 5.2 1.100 0.500 0.527 5.0 0.900 pH 0.400 4.8 0.348 0.300 0.700 4.6 g Ethanol / g Carbon g Ethanol Mycelia wt g/ Mycelia 4.4 0.200 0.500 0.100 4.2 0.065 0.300 4.0 0.000 0.000 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Days Days

1.700 B 6.0 B 0.900 5.8 1.500 0.800 Mycelia wt 5.6 0.768 0.700 ml 1.300 5.4

20 pH 0.600 5.2 1.100 0.500 0.512 5.0 pH 0.400 0.433 0.419 0.900 4.8 0.300 0.700 4.6 0.236 Mycelia wt g/ Mycelia 4.4 / g Carbon g Ethanol 0.200 0.191 0.500 0.142 4.2 0.100 0.300 4.0 0.000 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Days Days

1.700 C 6.0 C 0.900 5.8 1.500 0.800 5.6 0.700 ml 1.300 5.4

20 0.600 5.2 1.100 0.500 5.0 pH 0.400 0.900 4.8 Mycelia wt 0.300 0.700 4.6 g Ethanol / g Carbon g Ethanol Mycelia wt g/ Mycelia pH 4.4 0.200 0.500 4.2 0.100 0.300 4.0 0.000 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Days Days

Fig. 5: Effect of three glucose levels designated A, B and C for 100, 200 and 300g/L, respectively, on ethanol production, biomass propagation and pH value during 14 days of fermentation by Ph. chrysosporium.

Glucose concentration, as the main carbon source at C:N ratio of 2:1, variably affected Ph. chrysosporium growth and ethanol production. The fermentation process was carried out using three glucose levels designated

1228 Middle East J. Appl. Sci., 5(5): 1222-1231, 2015 ISSN 2077-4613

A, B and C for 100, 200 and 300g/L, respectively. Inoculum size was fixed at 0.1g dry weight/20ml transferred into 150ml fermentation medium. Continuity of ethanol production at its maximum level with the control A conc., recording 0.833 and 0.847 g ethanol/g carbon at day 10 and 14, respectively, was apparently affected as pH started to decrease drastically. Equal statistically, maximum ethanol production was achieved with intermediate B conc. to be 0.768 g ethanol/g carbon after day 10 that soon decreased with the decrease in pH afterwards too. The increase in ethanol production, in both glucose concentrations A and B, was correlated with the fungal exponential phase of growth happening in the first 4 days, afterwards fluctuation in pH value happened as the growth propagate through its stationary phase parallel to increase in ethanol production. Ethanol production apparently began with a high rate then decreased in the same time the pH was dropping fast while the biomass increased again. As with C conc.; obvious steady growth parallel to elevation in pH value was recorded, interrupted with remarkable decrease in both after 6th day with no worthy ethanol production, statistically at LSD=0.099. As shown in Table (1) the residual glucose measured at the end of 14 days in tests A, B and C recorded 47, 75 and 74 g/L proving that Ph. chrysosporium consumed 53, 125 and 226 g/L, corresponding to 53, 62.5 and 75.3% of initial glucose added, respectively. Maximum ethanol production was achieved in both A and B after 14 and 10 days of fermentation recording 0.847 and 0.768 g ethanol/g carbon corresponding to total ethanol production of 33.9 and 61.5 g/L and production yield of 33.9 and 30%, respectively. Noticeably, the efficiency calculated for initial concentration A (100g glucose/L) at day 14 corresponding to maximum ethanol production recorded 64% approx.

Table 1: Effect of initial glucose level in FM on ethanol maximum Glucose concentration after 14 days Maximum ethanol produced Initial glucose Residual Consumed Consumption g ethanol/g g ethanol g/L Yield % g/L g/L % carbon /L A 100 47 53 53 0.847 (day14) 33.88 33.9 B 200 75 125 62.5 0.768 (day 10) 61.44 30 C 300 74 226 75.3 0 0 0

Bak et al. (2009) stated that Ph. chrysosporium produced merely 10 g/L ethanol, with a yield of 62.7% after 4 days of simultaneous saccharification and fermentation of 100 g rice straw. On the other hand, non filamentous fungi as yeasts including S. cerevisiae and Candida tropicalis produced 15.3 and 12 g/L ethanol but with high yield of 89.7% and 75.7%, respectively (Abo-State et al., 2014). Emphasizing the reciprocal effect of glucose level at a certain concentration on ethanol fermentative production, Taylor et al. (1995) clarified the effect of glucose initial level on S. cerevisiae fermentation and ethanol production. It was found that glucose conversion of 100, 100, 95 and 90% were achieved by the yeast with initial glucose concentration of 100, 200, 400 and 600g/L, respectively, through which glucose shared in both cell and ethanol yields, by the aid of continuous fermentation and stripping column technique. On the same trend, in the present work Ph. chrysosporium produced ethanol at a yield of 33.9 and 30% for 100 and 200 g glucose/L while negligible amount was produced with 300 g glucose/L. Apparently with glucose levels of 100 and 200 g/L, the Ph. chrysosporium biomass developed in the first 48hr, same as most filamentous fungi did (Srivastava et al., 2011). It was worthy to notice that the biomass weight increased by increasing glucose to level C concentration, same as stated before by Taylor et al. (1995). Suitability of minor air conditions for more ethanol production was clarified by Kenealy and Dietrich (2004) as they stated that under minor aeration conditions Ph. chrysosporium formed sufficient intracellular alcohol dehydrogenase that shared in the formation of ethanol. Previous studies insisted on the fact that both acidity and ethanol over accumulation negatively affected filamentous fungi and consequently caused fluctuation in ethanol production and mycelia biomass (Paschos et al., 2015 and Gomaa, 2012). Considerable levels of acetate or oxalate were produced when C: N ratio was narrow or wide, respectively, that caused acidic conditions during ethanol production (Kenealy and Dietrich, 2004). Gomaa (2012) stated that Ph. chrysosporium metabolic activity and biomass propagation were negatively affected by ethanol concentration exceeding 10g/L. This stress pushed the fungus to form protective reductive proteins like glutathione parallel to peroxidase increased activity against free radical formation to prevent lipid peroxidation and cell membrane damage that caused cell death. On the same trend, increased acidity caused same response. These facts reasoned the drop in biomass and pH after ethanol production reached 14 and 19 g/L after 6 and 4 days of fermentation with A and B concentration, respectively. Also, the apparent reduction in ethanol production rate between 6 and 8 days in both A and B concentrations happened as the mycelia biomass seemed to be searching for another carbon source substituting glucose consumed. It pushed its metabolic activity to consume accumulated ethanol as a substitutive carbon source instead of consumed glucose, as clarified by Srivastava et al. (2011). Besides, it was possible that organic acid excretion like acetate causing pH

1229 Middle East J. Appl. Sci., 5(5): 1222-1231, 2015 ISSN 2077-4613 to drop was consumed as a carbon source afterwards, which was obvious by rise in pH value again after 8 days parallel to increase in ethanol production rate (Anasontzis and Christakopoulos, 2014). This can be predicted because with the highest glucose concentration, the biomass decrease delayed 48 hr later than with lower glucose concentrations.

Conclusion

As demonstrated previously, cellulytic filamentous fungi can ferment glucose and produce considerable amounts of ethanol under minor aeration condition. The criteria of consuming glucose by cellulytic fungus either for growth or ethanol production could emphasize fungal consumption of glucose released from cellulose during saccharification under aerobic or limited aeration conditions by the same fungus. This can help us predict glucose fate in ethanol production if it is intended to apply simultaneous saccharification and fermentation process gathering specified action of both filamentous fungus and yeast strain, respectively. Optimizing culture conditions improved maximum ethanol production and accumulation by the filamentous fungi Phanerochaete chrysosporium, as it was successfully magnified from 0.360 g ethanol/g carbon to more than 0.800 g ethanol/g carbon after 10 days of fermentation. It is recommended on large scale production to substitute most of yeast extract by wide scale locally available N sources and even to use the same fungal autolysate from the economic point of view.

References

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