SUMMARY OF THE THESIS BIOCONVERSION OF RICE STRAW TO ETHANOL AHMED ABD EL ...

17 Pages·2016·0.74 MB·English

SUMMARY OF THE THESIS

BIOCONVERSION OF RICE STRAW TO ETHANOL

By

AHMED ABD EL-MONEM ABD ALLAH
B. Sc. Agric. Sci., (Biochemistry and Microbiology), Fac. Agric., Cairo Univ.,
2008

THESIS
Submitted in Partial Fulfillment of the
Requirements for the Degree of

MASTER OF SCIENCE

In

Agricultural Sciences
(Agricultural Microbiology)

Department of Agricultural Microbiology
Faculty of Agriculture
Cairo University
EGYPT
2016

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ABSTRACT
Egypt faces a high population growth rate nowadays, which demands for an increase
in agricultural production efficiency. Consequently, agricultural field residues will increase.
Rice straw is one of the main agriculture residues in Egypt. So this study was performed on
rice straw as a resource for production of bioethanol. In a series of laboratory experiments,
rice straw was pretreated with sodium hydroxide followed by biological treatments for
bioethanol production. Both Aspergillusniger NRRL-3 and Trichodermareesei NRRL-11460
were successfully grown on either cellulose, hemicellulose or holocellulose, byproducts of
rice straw hydrolysis. Appreciable amounts of some extracellular enzymes were produced in
these very special cultivating media. Among tested enzymes, xylanase was produced in the
highest quantity (40.54 IU ml-1), while carboxymethylcellulase ranked second (3.35IU ml-1).
Holocellulose seemed the pioneer byproduct supporting the enzyme production followed by
cellulose, while hemicellulose was the inferior in this respect. Apart from substrate and
enzyme type, the fungus T. ressei overcame A. niger for the enzyme formation. The
enzymatic activity of introduced inocula obviously reflected on producing sugar pools.
Compared to others, T. reesei acted more actively, where the produced reducing sugars could
be arranged in the descending order: 2.62 mg ml-1(T. ressei) > 2.52 mg ml-1 (A. niger + T.
ressei) > 1.10 mg ml-1 (A. niger).The optimal level of reducing sugars was scored at 8th day-
interval in hollocellulose -amended fungal culture medium, the respective quantities of 22.90,
20.30 and 13.22 were produced in the presence of T. reesei, mixed inoculum and A. niger.
Raising the ammonium sulphate over the recommended level in fungal culture medium
significantly stimulated sugar production.Separate, Hydrolysis and Fermentation (SHF) was
pretreating lignocellulosic material in a first unit degraded to monomeric sugars by cellulases
and thereafter fermented to ethanol in a second, separate unit.Simultaneous Saccharification
and Fermentation (SSF) of cellulose by Saccharomyces cerevisiae and cellulases was
evaluated in basal media. The impact of some SSF and SHF conditions on ethanol production
was discussed i.e. cellulose concentration, enzyme quantity, incubation period and shaking
rate. The results clearly show that the SSF technique is better than the SHF technique in
ethanol production.
Key words: Rice straw, Aspergillusniger, Trichodermareesei, Saccharomyces cerevisiae,
Saccharification,Ethanol , SHF, SSF.

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INTRODUCTION
Among the cereals, rice is the world’s second largest crop after wheat,
however, it produces unlimited amounts of residues. According to the Egyptian
Environmental Affairs Agency (EEAA, 2008), more than 2 million acres are
cultivated in the country with an average production of ca. 6.12 million
tons/year. Processing of rice yields extraordinary quantities of straw as residue.
Not less than 20 % is used for paper and fertilizers production as well as fodder
and the remaining part is left in the open fields for burning along a period that
may extend to > 30 days to get rid of leftover debris. The resulting emission
obviously contributes to the air pollution known as the “Black Cloud” (Dina,
2015).
It is well established that, plant cell walls are the most abundant renewable
source of fermentable sugars on earth and are the major reservoir of fixed
carbon in nature. The main components of plant cell walls are cellulose,
hemicellulose and lignin, with cellulose being the most abundant (Yang et al.,
2007). Cellulase enzymes can hydrolyze cellulose forming glucose and other
commodity chemicals. Researchers have strong interests in cellulases because
of their applications in industries of starch processing, grain alcohol
fermentation, malting and brewing, extraction of fruit and vegetable juices, pulp
and paper industry as well as textile industry (Zhou et al., 2008). One of the
potential applications of cellulases is the production of ethanol fuel from
lignocellulosic biomass which is a good substitute for gasoline in internal
combustion engines. The most promising technology for the conversion of the
lignocellulosic biomass to ethanol is based on the enzymatic breakdown of
cellulose by cellulase enzymes (Ahamed and Vermette, 2008). The enzymatic
hydrolysis step is often in close collaboration with the following fermentation
step in the ethanol production. The layout of this process can be designed in
several ways; either by having separate hydrolysis and fermentation step
(separate hydrolysis and fermentation, SHF) or by combining these two in one
step (simultaneous saccharification and fermentation, SSF). Each process
having its own pros and cons (Galbe and Zacchi, 2002, Erdei et al., 2013)
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Many fungal strains secrete higher amounts of cellulases than bacterial ones,
Trichoderma reesei is a model fungus for studying cellulase production
(Kubicek et al., 2009) and is a unique industrial source of cellulase.
BesidesTrichoderma reesei, other fungi such as Aspergillus niger (Ong et al.,
2004) have been employed in cellulase production. The application of pure
cultures dominates in biological processes, but through adopting mixed cultures,
their combined metabolism results in considerable effects of higher enzymes
production. Special attention has been given as well to mixed cultures used for
increasing enzyme production, such as cellulase (Ahmed et al., 2010) and
xylanase (Tame Juhasz et al., 2003). Fermentation time, substrate concentration
and pH of the fermentation medium were all optimized by mixed culture of
Trichoderma viride and Aspergillus niger (Kavitha and Nagarajan, 2011).
Saccharomyces cerevisiae is used extensively in batch fermentations to convert
sugars to ethanol for the production of beverages and biofuels. Despite the
obvious importance of this process, the physiological constraints which limit the
rate of glycolysis and ethanol production are not fully understood.
Identification of these constraints represents an important step toward the
development of improved organisms and process conditions for more rapid
ethanol production. Such improvements could result in an increase in the
ethanol production capacity of existing fermentation plants and a reduction in
the cost of future facilities.(nd Boles, 2012).
The present work is one of the on-going research attempts to improve
enzyme and ethanol production by Trichoderma reesei, Aspergillus niger and
Saccharomyces cerevisiae from rice straw and some intermediate products of
rice straw pretreatment. Optimization of both solid state fermentation (SSF) and
separate hydrolysis and fermentation (SHF) conditions for ethanol production
by Saccharomyces cerevisiae and enzymes was among the targets of this study.
Aim OF WORK
The present study concerns with three main objectives regarding the
use of rice straw as a waste raw material and fungal strains as a bioagent in
cellulases and ethanol production (Figure, 1):
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1. Fractionation of rice straw into holocellulose, cellulose and
hemicellulose.
2. Production of cellulose-decomposing enzymes using Aspergillus niger
NRRL-3 and Trichoderma reesei NRRL-11460.
3. Production of ethanol from rice straw fraction by cellulases and
Saccharomyces cerevisiae NRRL Y-12632.
Figure 1. Schematic diagram for conversion of rice straw into
cellulases and ethanol.

Review
Among the cereals, rice is the world’s second largest crop after wheat,
however, it produces unlimited amounts of residues. According to the Egyptian
Environmental Affairs Agency (EEAA, 2008), more than 2 million acres are
cultivated in the country with an average production of ca. 6.12 million
tons/year. Processing of rice yields extraordinary quantities of straw as residue.
Not less than 20 % is used for paper and fertilizers production as well as fodder
and the remaining part is left in the open fields for burning along a period that
may extend to > 30 days to get rid of leftover debris. The resulting emission
obviously contributes to the air pollution known as the “Black Cloud” (Dina,
2015).
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Summery
1. Raw material (rice Straw)
Sun-dried rice straw samples collected from private fields at Moshtohor-
Kalioubia governorate during the harvesting season of 2012-2013 were milled
to pass through a 40 mesh screen and used as a source of lignocellulosic
biomass. The chemical profile of the agricultural waste was determined i.e.
cellulose, lignin, ash, protein, reducing sugars, total nitrogen, fats, waxes and
glucose.
2. Microbiota
Two fungal strains recommended for cellulose hydrolyzing enzymes
production from organic wastes were used. Those are Aspergillus niger NRRL-
3 and Trichoderma reesei NRRL-11460 obtained from the Northern Regional
Research Laboratory, Peoria, Illinois, USA. Microorganisms were cultivated
and maintained on potato dextrose agar (PDA) (Oxoid, 1982). Besides, one
Saccharomyces cerevisiae NRRL Y-12632 strain characterized by high ethanol
production efficiency was obtained from the Northern Regional Research
Laboratory, Peoria, Illinois, USA. The candidate was grown on basal medium
for ethanol production (BMEP) (Ooshima et al., 1986). Cultures were monthly
sub-cultured, incubated at 30 °C for 48 h. and 7 days for yeast and fungal
strain, respectively and subsequently stored at 4 °C for inocula preparation.
3. Rice straw pretreatment
The pretreatment of rice straw with acidified sodium chlorite was
carried out for fractionation of rice straw into holocelluloses, cellulose and
hemicellulose (Figure, 2) according to the methods described by Siduh and
Sanduh (1980) and Chen and Anderson (1980) as well asthe modified
procedure of Hubble and Ragauskas (2010).
Holocellulose, cellulose and hemicellulose fractions were obtained from
rice straw as follows:
a. For holocellulose separation the finely ground straw (100 g) which
was suspended in 3200 ml of hot water in Erlemeneyer flask and acidified with
10 ml of acetic acid. Then 30 g of sodium chlorite were added. The flask was
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heated in a water bath at 70 °C for one hour. The mixture was filtered through a
cheese cloth. The residue (Holocellulose) was washed several times with tap
water, air dried and weighed.
b. For cellulose separation the holocellulose was soaked in 1N NaOH at
a ratio of 1:20 (w/v) for 24 h at room temperature then filtered. The residue
(cellulose) was thoroughly washed with tap water until being free from NaOH,
air dried and weighed.
c. The alkali solution obtained after separation of cellulose was dialyzed
through a cellophane bag until being sodium hydroxide free. Then 3 volumes
of ethanol were added and the suspension was allowed to stand overnight at 4
°C. The precipitated hemicellulose was separated by centrifugation and dried.
Figure 2. The fractionation technique.
4. Production of enzymes
Production of cellulases by the fungal strains T. reesei and A. niger as
well as their mixed culture was examined in batches using basal medium for
cellulase production.

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a. Inocula preparation
Inocula of T. reesei, A. niger and their mixture were prepared by
transferring spores from a 7-10-day old Agar slant culture to 50 ml basal
medium (Mandels and Weber, 1969) containing 2 % glucose (w/v) in 250 ml
Erlenmeyer flask. The inoculated flask was agitated at 150 rpm on a rotary
shaker (Labline) for 24 h at 30°C.
b. Factors affecting production of cellulases
The effects of different carbon sources, fermentation periods and
changes in the basal medium components on growth of the two fungal strains
and their mixed culture as well as their enzyme production were examined in a
set of batch fermentations.
1. Effect of carbon source
The type of the carbon source is one of the factors affecting growth of
the examined microorganisms and thus affecting their enzyme production. Raw
rice straw, holocellulose or cellulose were applied as the sole carbon source
(1% w/v) in the basal growth medium. One hundred ml aliquots of the basal
medium containing one of the examined carbon sources in 250 ml Erlenmeyer
flask were autoclaved at 121 ºC for 20 minutes. The medium was inoculated
with 5 ml seed culture inoculum and incubated at 150 rpm on a rotary shaker
(Labline) for 7 days at 30 °C. The cultures were sampled and filtrated through a
cheese cloth and the filtrate was subjected to enzyme assay and estimation of
hydrolytic value of the produced enzymes.
2. Fermentation period (time course)
A set of fermentation batches were conducted to examine the influence
of different fermentation periods prolonged from 2-12 days on the enzyme
production by the examined microorganisms from holocellulose. The basal
growth medium containing holocellulose as the carbon source was inoculated
with 5 ml inocula of the seed cultures and incubated at 150 rpm on a rotary
shaker (Labline) for periods ranged from 2 to 12 days at 30 ºC. Cultures were
sampled after the different incubation periods, filtrated through a cheese cloth
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and the filtrate was subjected to enzyme assay and estimation of hydrolytic
value of the produced enzymes.
3- Optimizing the growth medium composition for cellulase
production
Production of cellulases by either pure or mixed cultures of the
examined fungal strains was studied in basal media with different
concentrations of one of each of these components (NH ) SO , urea, KH PO
4 2 4 2 4
and holocellulose individually; while other components remain unchanged. For
(NH ) SO , the applied concentrations were 1.2, 1.3, 1.4 and 1.5 g ml-1.
4 2 4
Meanwhile urea concentrations were 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 g ml-1.
Whereas KH PO concentrationswere1, 2, 3, 4, 5 and 6 g ml-1. Holocellulose
2 4
concentrations were 0.75, 1.0, 1.25, 1.5, 1.75 and 2.0 g ml-1. All batches were
carried out in 250 ml Erlenmeyer flasks containing 100 ml of the production
medium inoculated with 5 ml of seed culture and incubated at 150 rpm on a
rotary shaker for 7 days at 30 ºC. The cultures were sampled and filtrated
through a cheese cloth and the filtrate was subjected to enzyme assay and
estimation of hydrolytic value of the produced enzymes.
c. Estimation of the hydrolytic value of the produced enzymes
At the end of each fermentation batch, the filtrate was used for the
determination of the hydrolytic value of the produced enzyme according to
Chahal (1985).One ml culture filtrate was mixed with 50 mg of the cellulose
fraction and incubated at 50 ºC for 60 minutes followed by centrifugation at
6000 rpm for 20 min. The supernatant was used for reducing sugar and glucose
determination after adequate dilution. The hydrolytic value was expressed as
mg of reducing sugar per ml enzyme (mg ml-1).
d. Separation of crude enzymes in a powder
For crude enzyme production, a set of fermentation batches were run
using a mixed culture of T. reesei and A. niger in 250 ml conical flasks
containing 100 ml of a modified basal medium according to the obtained
results from the previous experiment on optimizing the medium components
for enzyme production. Flasks containing the modified basal medium were
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supplemented with 1.75 % holocellulose as a carbon source and incubated at
150 rpm at 30 °C for 8 days. At the end of the fermentation period, a total
amount of 1 liter of culture was filtrated through cheese cloth for separation of
the fungal mycelia and spores. The crude enzymes precipitation in the culture
filtrate was examined using ammonium sulfate solutions with different
concentrations ranged from 0-66 % (w/v) (Table, 1) according to the methods
described by Richard (2009), dehydrated with a solvent i.e. cold acetone
(Kristyna Pospiskova and Ivo Safarik, 2015).The protein content of dissolved
enzyme and its enzyme activity were determined (Lowry et al., 1951).
Table 1. Ammonium Sulfate Saturation
Ammonium Sulfate
Concentration Concentration
Saturation Saturation
g / % ( w / v) g / % ( w / v)
(%) (%)
0 - 10 0 – 5.6 60 - 70 36.3 – 43.2
10 – 20 5.6 – 11.3 70 - 80 43.2 - 50.4
20 - 30 11.3 – 17.2 80 - 90 50.4 – 58.1
30 - 40 17.2 – 23.4 90 - 100 58.1 – 66.0
40 - 50 23.4 – 29.7 40 - 50 23.4 – 29.7
50 - 60 29.7 – 36.3 60 - 70 36.3 – 43.2
4. Fermentation Batches for ethanol production
a. Inocula preparation
Seed inocula yeast (Saccharomyces cerevisiae NRRL Y-12632) cultures
were prepared in 50 ml conical flasks contained 10 ml basal media
supplemented with glucose (20 g/l). After sterilization, flasks were inoculated
with a loop full of 48 h old yeast culture. Inoculated flasks were incubated on a
rotary shaker (150 rpm) for 24h at 30 °C. The growing yeast was used as a seed
culture to inoculate the ethanol fermentation bio-reactor with 10 % (V/V).
b. Enzyme preparation for saccharification
T The cellulase solution was prepared by dissolving (4.55 g) enzyme powder
in100 ml acetate buffer (0.1 M, pH 4.8) and filter-sterilized through a micro
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