Middle East Journal of Applied
Sciences
ISSN: 2077-4613
Volume : 05 | Issue : 04 | Oct.-Dec. 2015
Pages: 1222-1231
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 cleanburning 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]
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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.
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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
Phanerochaete chrysosporium
Pleurotus sp
Aspergillus oryzae
Aspergillus versicolor-II
0.500
0.450
Aspergillus versicolor-I
Fusarium oxysporum
Fusarium oxysporum-I
Mucor indicus
g Ethanol / g Carbon
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
2
4
6
8
Days
10
12
14
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
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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
g Ethanol/g Carbon
0.700
Amm. Sulfate
0.600
0.500
Casein
0.400
Peptone
0.300
Pot. Nitrate
0.200
Yeast extract
0.100
0.000
2
4
6
8
10
12
14
Days
Phanerochaete chrysosporium
0.700
Amm. Phosphate
g Ethanol/g Carbon
0.600
Amm. Sulfate
0.500
0.400
Casein
0.300
Peptone
0.200
Pot. Nitrate
0.100
Yeast extract
0.000
2
4
6
8
10
12
14
Days
Fusarium oxysporum
0.600
Amm. Phosphate
g Ethanol/g Carbon
0.500
Amm. Sulfate
0.400
Casein
0.300
Peptone
0.200
Pot. Nitrate
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
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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.
g Ethanol / g Carbon
Phanerochaete chrysosporium
1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
2
4
6
8
2
4
6
8
10
12
14
Days
g Ethanol / g Carbon
Fusarium oxysporum-I
1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
2
4
6
8
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).
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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.
g Ethanol / g Carbon
Phanerochaete chrysosporium
1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
4
5
2
4
6
8
10
12
14
Days
g Ethanol / g Carbon
Fusarium oxysporum-I
1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
4
5
6
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
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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:
Mycelia wt g/20ml
1.500
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
Mycelia wt
1.300
pH
1.100
0.900
0.700
0.500
0.300
2
4
6
8
10
12
g Ethanol / g Carbon
A
1.700
pH
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).
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
14
A
0.611
0.527
0.348
0.065
0.000
2
4
6
Mycelia wt g/20ml
1.500
Mycelia wt
pH
1.100
0.900
0.700
0.500
0.300
2
4
6
8
10
12
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
0.191
4
0.236
0.142
6
0.900
Mycelia wt
0.700
pH
0.500
0.300
8
8
10
12
14
10
12
10
12
14
C
g Ethanol / g Carbon
1.100
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
pH
Mycelia wt g/20ml
1.300
6
0.419
Days
1.500
4
14
0.512
0.433
2
14
C
2
12
0.768
Days
1.700
10
B
g Ethanol / g Carbon
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
pH
B
1.300
8
Days
Days
1.700
0.847
0.833
14
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
2
4
6
8
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
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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
Initial glucose
g/L
A
B
C
100
200
300
Glucose concentration after 14 days
Residual
Consumed
Consumption
g/L
g/L
%
47
53
53
75
125
62.5
74
226
75.3
Maximum ethanol produced
g ethanol/g
g ethanol
Yield %
carbon
/L
0.847 (day14)
33.88
33.9
0.768 (day 10)
61.44
30
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
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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.
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