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Nivedita Sharma et al., IJSID, 2012, 2 (4), 84-95
International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
84
BIOETHANOL-GREEN AND CLEAN ENERGY FROM LIGNOCELLULOSIC BIOMASS
Nivedita Sharma1*, Neha Gautam1 and Sanjeev Kumar1
1 Department of Basic Science, Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan (HP), India
INTRODUCTION
INTRODUCTION
ISSN:2249-5347
IJSID
International Journal of Science Innovations and Discoveries An International peerReview Journal for Science
Review Article Available online through www.ijsidonline.info
Received: 05-06-2012
Accepted: 19-07-2012
*Corresponding Author
Address:
Name:
Nivedita Sharma
Place:
Himachal Pradesh, India.
E-mail:
ABSTRACT
Bioethanol, the liquid combustible fuel has become the most promising
alternative substitute for gasoline. Lignocelluloses have great potential as a biomas
source for bioethanol production. Extracellular enzymes secreted by cellulolytic and
hemicellulolytic microorganisms degrade complex lignocellulosic biomass to simple
sugars. The pretreatment of inert lignocellulosic material by suitable physical, chemica
or biological method is a pre-requisite to increase its accessibility to degrading enzymes
Sugars so formed in turn are converted to ethanol by employing suitable
native/genetically microorganisms. Different fermentation processes like Separate
hydrolysis and fermentation process (SHF), simultaneous hydrolysis, fermentation
processes (SSF) and co-fermentation of pentose and hexose sugars (SSCF) have been
evaluated for the bioethanol production. The feasibility of bioconversion process o
bioethanol production from lignocellulosic biomass at commercial scale has been
explored.
Key Words: Lignocellulosic waste, pretreatments, Bioethanol, Cellulase and Xylanase
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INTRODUCTION
Energy is the life line of the global economy. Diminishing fossil fuel reserves, ever escalating fuel prices, increased
concerned over environment pollution and most importantly quest for energy independence and energy security promoted
the global research community to accelerate the need to look for renewable and environmentally sustainable energy resource
[1]. Therefore there is an urgent need for introduction of alternative energy resources into biomass conversion system. Biofuels
are only renewable carbon dioxide sources of energy. Lignocellulosics have been identified as a primary source of bioethano[2]. Use of lignocellulosic biomass for the production of biofuels is unavoidable if liquid fossil fuels are to be replaced by
renewable and sustainable alternatives. Among them, ethanol accounts for the majority of the biofuels worldwide [3]. Thus
quest of generating renewable energy sources has shifted on plant biomass and its effective utilization through advances in
technology development. Annual production of plant biomass has been estimated to be 163 X 109 tons[4]. The plant biomass
mainly consists of lignocelluloses i.e. cellulose: hemicellulose: lignin generally in the ratio of 6: 2: 2 [5]. Out of which cellulose
and hemicellulose can serve as an alternative raw material for its bioconversion into ethanol. Enzymatic hydrolysis of cellulose
and hemicellulose in the biomass converts them in simple sugars by the cheapest chemical factories i.e. microbial cells. Sugars
formed in turn can be fermented to liquid fuel- ethanol by employing suitable microorganisms[6] thus ultimately resulting in
the production of the value added product like ethanol from sheer waste. Thus, cellulosic plant biomass can act as potentia
substrate for production of alcohol through its efficient biodegradation by bioconversion [7]. Ethanol production from plant
waste is of special importance because of the controversy generated worldwide due to direct use of items of food chain like
corn and sugarcane for it. Ethanol is presently the most well developed possible liquid fuel substitute for conventional fuel. It
is possible to mix it with petroleum in different proportions (gasohols) without making great changes in currently used
engines. In many countries all the vehicles are run with gasohol (mixture of petrol and ethanol) which not only increases the
efficiency of engine but also lowers the air pollution significantly [8]. Recently, Govt. of India has also introduced similar
programmes of blending petrol with ethanol in nine of its state and is seriously thinking for expanding it further which can
only be possible if limited supply of ethanol is compensated with its enhanced and cheaper production at large scale. In this
review, nature of lignocellulosic biomass, its capability for saccharification, further exploitation to ferment alcohol and
feasibility of commercial process for becoming the base of new industries in the near future has been explored.
Degradation of lignocellulosic biomass:
Cellulose is the principal constituent of the cell wall of most terrestrial plants. The source of cellulose in plants is found
in microfibrils (2-20nm in diameter and 100-40,000 mm long) [9]. These form the structurally strong frame work in the cel
wall. Plant residues contain 15-60 % cellulose, 10- 30 % hemicellulose and 5-30 % lignin [10]. The intricate structure of a wood
cell wall and distribution of cellulose and other biopolymers in it are schematically depicted in Fig. 1. The different
lignocellulosic waste for liquid biofuel production are forest biomass hardwood and softwood [11, 12], herbaceous grasses like
switch grass, bermuda grass, alfa-alfa fibre and reed canary grass [12, 13, 14] and agricultural residue [15,12] which can be degraded
down by hydrolytic enzymes to monomeric sugars.
Role of Hydrolytic Enzymes:
Hydrolytic enzymes responsible for saccharification of biomass are cellulase and hemicellulase which are generally
extracellular in nature and these are produced from potential cellulolytic and hemicellulolytic microorganisms
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Biochemistry of Cellulase:
The complete degradation of cellulose is achieved by the synergistic action of three enzymes i.e. Endoglucanase (EC
3.2.1.3.4, endo O-1, 4- glucanase) which act internally on the chain of the cellulose cleaving -linked bonds liberating non
reducing ends, while Exoglucanase (EC 3.2.1.91,1,4--Cellobiohydrolase) removes cellobiose from the non-reducing end o
the cellulose chain. Finally -glucosidase (EC 3.2.1.21) completes the saccharification by splitting cellobiose into glucose
molecules[16].
Biochemistry of Hemicellulase
Xylan, after cellulose, is the most abundant polysaccharide present in wood, agricultural and several agro-industrial
wastes. This complex hetero-polysaccharide consists of a main chain of 1,4-- D-xylose monomers containing differen
substituents or ramification [17, 18]. Several hydrolytic enzymes are involved in complete breakdown of a branched acetyl xylan.
Among them xylanase is most predominant enzyme for lignocellulose degradation. Different studies have been done by
various authors in an attempt to maximize the yield of cellulase and xylanase for scaling up the degradation of biomass.
Production and optimization of enzymes:
The environmental factors play a vital role in enhancing the enzyme production by different microorganisms. Among
these, media for growth of microorganisms [19], pH [20, 21], temperature [22, 23, 24], and incubation time [25] etc. are some of the
important parameters to maximize the yield. Different studies have been done by researchers in an attempt to increase the
production of the cellulase and xylanase by optimizing above mentioned the environmental parameters. In another approach
enzymes/recombinant genes in microorganisms have been made a target with an attempt to modify these strains through
genetic engineering and to escalate the titres of cellulase and xylanase. Metabolic engineering is used as improvement in the
formation of products through modifications of specific biochemical reactions through recombinant DNA technologies [44, 45]
Earlier studies described the sources and properties of microbial -glucosidases, yeast - glucosidases, thermostable fungal
- glucosidase, and the physiological functions, characteristics, and catalytic action of native -glucosidases from microbia
sources. Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of theenzymes. Their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-
function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic
properties and applications[46].
Pretreatment of lignocellulosic biomass
Lignocellulosic residue is not readily degraded by enzymes in its native form due to crystalline nature of cellulose,
lignin shield around cellulose and smaller pore structure of substrate which hinders the action of hydrolytic enzymes [47]
Therefore pretreatment seems to be a prerequisite to enhance the saccharification of biomass [27]. Pretreatment helps to break
down the wood structure and enlarges the pore size thus, making biomass accessible for penetration of the hydrolytic
enzymes [5]. A number of pretreatment methods have been proposed which disrupt the highly complexed cellulose structure
and lignin carbohydrate complex, remove lignin, increase surface area and increase the rate and extent of hydrolysis of
cellulose in various pretreated lignocellulosic residue[48,49]. However many physical, chemical and microbial pretreatment
methods for enhancing bioconversion of lignocellulosic materials have been reported [27,50,51] . Among physical pretreatments
the use of mechanical chopping, hammer milling, grind milling, roll milling, vibrating milling and ball milling have proved
success as a low cost pretreatment strategy [52].Gamma-irradiation, microwave irradiation and thermal methods like steam
explosion, CO2 explosion and hot water treatment have been found most successful in processing of lignocellulosics [53,54,55]
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Chemical pretreatments such as alkali or acid pretreatment have been widely used to remove lignin content of
lignocelluloses[56].Castic swelling is a common chemical method that has the effect of increasing the surface area of the
lignocellulose residue due to the swelling and disruption of lignin. Compared with acid or oxidative reagents, alkali treatments
appears to be most effective method in breaking the ester bonds between lignin, hemicelluloses and cellulose and avoiding
fragmentation of the hemicelluloses polymer [57] Disadvantage of chemical pretreatments for lignin removal include the need
for corrosion resistant apparatus, an effective washing strategy, and the capability for the safe disposal of used chemicals. In
biological pretreatments, white rot fungi is commonly used for biological pretreatments of lignocellulosics which is claimed to
be on ecofriendy method for enhancing saccharification of plant biomass. White rot fungi such as Phanerochaete
chrysosporium, Trametes versicolor and Bjerkandera adusta have the ability to degrade lignin and can be used as an effective
biological pretreatment[5]. This is a cheap and effective method of delignification. But biological pretreatments require a long
time period in comparison to other tried and tested physical and chemical methods. A period of two to five weeks may be
required for sufficient delignification. The direct application of lignolytic enzymes has also been investigated in order to
reduce the length of treatment period, but the direct use of enzymes for delignification is expensive and suffers from poor
enzyme activity on lignocellulosic material[59].
Mode of degradation:
Cellulosic biomass is saccharified to a mixture of hexsoses and pentoses either by potential cellulolytic/ hemicellulytic
microorganisms by secreting extracellular cellulase and xylanase or directly adding enzymes in it [80]. Saccharification o
biomass indeed is a key factor for final bioconversion process i.e. ethanol fermentation. Therefore to make the process
commercially viable and more efficient, different methods i.e. submerged fermentation (SmF) / solid state fermentation (SSF)
of plant biomass have been compared to release higher amount of sugars from it [81]. However, SSF holds tremendous potentia
for the enhanced biodegradation of lignocellulosic biomass and has several advantages over submerged fermentation [82]. In
SSF, enzymes produced are many folds more than submerged fermentation and thus has direct impact on biodegradation o
biomass [83].SSF is comparatively simple low technology operation, convenient and economical technique for the degradation
of biomass. The use of solid state fermentation technique to degrade lignocellulosic biomass is gaining interest due to higher
yield obtained [84]. Solid state fermentation has marked advantage over submerged fermentation in terms of productivity
concentration of the product and effluent generation.
Production of ethanol:
Sugars formed after saccharification of biomass are converted to alcohol through a process called fermentation
Maximum fermentation of mixture of hexose & pentose sugars to alcohol is a major challenge worldwide for fermentation
technologists to allure the industrialists for a profit making biofuel industry. Different microbial strains used for fermentation
of sugars to ethanol include Saccharomyces cerevisae, Candida brassicae, Zymomonas mobilis, Pichia stiptis
Schizosaccharomyces pombe, Clostridium sp., and Bacillus macerans etc. The strains used for fermentation depend upon
composition of hydrolysate, temperature tolerance, ethanol tolerance and ability to grow on composition of hydrolysate [85].
Separate hydrolysis and fermentation process (SHF)
Traditionally hydrolysis and fermentation processes are done in separate steps using either a single reactor or no of
bioreactors in series. This process is called as (SHF) i.e. Separate hydrolysis and fermentation process. However, productivity
and yields reported so far are lower SHF as compared other and fermentation processes [86].
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Simultaneous hydrolysis and fermentation processes (SSF)
The simultaneous hydrolysis and fermentation process entails inoculating the enzyme- producing microorganism
(enzyme) and the ethanol producing microorganisms at the same time so that the produced sugars are simultaneously
converted to ethanol. Because the produced sugars are immediately converted to ethanol, the problem of feedback inhibition
is avoided. Contamination risk is also low since the sugar concentration is very low throughout the process. In general, the
productivities and yields reported for this method are higher than those reported for the SHF process 87. Furthermore, less
enzyme is needed because glucose inhibition is avoided. In the simultaneous hydrolysis and fermentation processes, however
recycling of the biocatalysts is very difficult when free cells or free enzymes are [88]. This problem can be avoided by
immobilizing the biocatalysts. Another problem is that the optimal conditions for hydrolysis are different from those for
fermentation. The temperature optima for most hydrolysis enzymes are often much higher than those for most fermenting
microorganisms. The use of thermophilic or thermotolerant strains of fermentation microorganisms can help to overcome this
problem[88].
Simultaneous Co-fermentation process (SSCF)
Degradation of cellulose leads to the formation of hexose sugars while hemicellulose fraction of biomass is rich in five
carbon sugars, which are also called pentoses. Xylose is the most prevalent pentose released by the hemicellulose hydrolysis
reactions and cannot be fermented by native conventional yeast- Saccharomyces cerevisae. Thus co-fermentation of hexoses
and pentoses is suggested by using microbes like Zymomonas mobilis or genetically engineered bacteria. Progress is rapid in
the field of xylose fermentation; a few industrial modified yeast strains have yet shown the demonstrated capability of
fermenting xylose in lignocellulosic hydrolysates efficiently. Arabinose fermentation in SSF has not yet been reported
although arabinose fermenting S.cerevisae strains have recently been constructed. Co-utilizating arabinose and xylose strains
ofZ.mobilis have been developed [89, 81]. Other than conventional yeast, Zymomonas mobilis has emerged as promising
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microorganisms for ethanol production. This organism has several advantages and it is suitable for continuous fermentation
[90,91] over Saccharomyces cerevisiae[92].
Table 1. Extracellular enzymes of microbial origin used in hydrolysis of biomass
Enzyme Micro-organisms Substrate
Cellulases Aspergillus niger, Trichoderma viridae,Thermoascus aurantiacus
Sporotrichum pulverulentum,T. harzianum, T . resei, A. niger
Sawdust, bagasse and corncob, rice husk,agriculture woody waste, rice straw, wheat
bran, wheat straw , wheat bran[26,27,28,29,30,31]
-glucosidases A. niger, Trichoderma sp., Botritis sp. Radilcle, malt manufacture residue wheatbran + rice straw, spent wheat-bran [32]
Cellulase, Ligninase Strains of Basidiomycetes, Polyporussp.
Bagasse [33]
-glucosidase Thermoascus aurantiacus Agriculture waste[34]
Xylanase Bacillus sp., Bacillus pumilis,Streptomyces sp. Melanocarpus
albomyces Talaromyces emersonii
Rice bran, rice strawwheat/ bagasse, rice
wheat bran, barley straw, oat straw, wheatstraw [35,36,37,38,39,40]
Cellulase and hemicellulase A. niger Lignocellulosics biomass[41]
Cellulase, -glucosidase Acremonium cellulolyticus Cellulosic wastes[42]
-xylosidase A. awamori,Aureobasidium sp.,Thermoascus aurantiacus
Wheat straw, wheat bran, agriculture waste[34,43]
Xylanase, Xylosidase A. fumigates Rice straw, corn hull, corncobs[2]
Economics of bioconversion process
To be competitive, and find acceptance of ethanol bioconversion process at commercial scale, the cost for the
bioconversion of biomass to liquid fuel must be lower than different gasoline prices [17]. The cost of feedstock and cellulolytic
enzymes are two important parameters for low cost of ethanol production. Biomass feedstock represents around 40 %
ethanol production cost[93].
Table 2: Different recommended pretreatment methods for lignocellulosic waste
Pretreatments Substrate
Physical pretreatment
Milling Corn stover, lignocellulosic biomass[60]
Irradiation Bagasse, sawdust, chaff 53,61
Steam explosion Masonite plants, sunflower, Populas nigera, Eucalyptus,forest residue cattle manure [62,63,64,65,66,67]
Ammonia fibre explosion Switch grass [68]
CO2 explosion Hard wood & soft wood [69]
Liquid hot water Corn fibre, sugarcane baggase [70,71]
Chemical pretreatment
Alkaline hydrolysis Soyabean straw, wheat straw [26,27]
Alkaline peroxidase Wheat straw, rice hullswater hyacianth, water lettuce [72,73]
Wet oxidation Wheat straw; yard waste, digested biowaste [74,75]
Ozonolysis Agricultural biomass, olive mill waste[76]Acid hydrolysis Eucalyptus grandis, rice straw and bermoda grass; olive
tree biomass [77,78,56]
Biological pretreatment
White rot fungi (e.g. Ceriporia lacerata , stereum hirsutum) andbrown rot fungi (Gloeophyllum sepiarium, Fomitopsis pinicola,
and Laetiporus sulphureus).
Rice straw, forestry waste [79]
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Employing integrated approaches using the largest industrial facilities by integrated action plan along with cheap feed
stock and potent cellulases and xylanases could make the process more economically viable [70]. The cost economics of ethano
production from the small size lignocellulose refinery with a capacity of 100 tons per day producing approximately 3 million
gallons of ethanol plus co-products. The estimated cost of ethanol was found to be $ 1.00-1.20 per gallon. The consistent
efforts of scientists to use efficiently the components of cellulose and hemicellulose as fuel are further simplifying the technica
difficulties. The chemical complexities of these molecules are legendry but intensive research and advanced technology will
definitely lead to further decreased production cost for its commercial viability.
CONCLUSION
Bioethanol production from the lignocellulosic biomass represents potential alternative source of the fuel. This is
especially important considering the ongoing energy crisis. As the worldwide market share of bioethanol/biofuels rapidly
increases in the coming year, we must seek nonfood resources in order to avoid the food vs fuel conflicts in the agriculture
sectors. Therefore availability and renewability of plant lignocellulosic biomass represents a real advantage over source o
dwindling fossil fuels. Efficient and economically viable technology for sufficient production of ethanol is being developed in
several stages, including low cost pretreatments, highly effective cellulase and hemicellulase, and efficient and robust
fermentative mechanism.
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