bioethanol-green and clean energy from lignocellulosic biomass

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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

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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:

[email protected]

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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