Lignin is a rigid part, responsible to hold the cellulose and hemicellulose together. It also prevents the lignocellulosic material to swell up. Lignin contents resist the enzymatic digestibility by reducing the access of enzyme to cellulose. Delignification increases the enzymatic hydrolysis however; in some cases hemicellulose part is also hydrolyzed which means delignification does not play a sole effect [128]. The hydrolysis of lignin depends upon its type as in soft and hardwoods. In some cases, lignin inhibits the swelling of cellulose. Swelling can be achieved without removing the lignin but in this way hydrolysis does not
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increase [130, 131]. The lignin content and its distribution may influence the enzymatic hydrolysis in two major ways.
1) Lignin prevents enzymes from effective binding to the cellulose.
2) Lignin irreversibly adsorbs the cellulase enzymes, preventing their reaction with substrate. The removal of lignin leaves the cellulose more accessible and more open to swelling on contact with cellulase [130-131]. For example, higher enzymatic conversion of cellulose has been achieved from extensive delignified softwood kraft pulp, containing 4.0% lignin or delignified mechanical pulp, containing 8% lignin; while partial lignin removal (with a final lignin content of 32.0-36.0%) has resulted in lower hydrolysis yield [132]. The extent to which lignin adsorbs enzyme depends very much on nature of the lignin itself [133, 134].
2.2.1.5 Effect of hemicellulose
Enzymatic attack is protected by hemicellulose which is a physical barrier that surrounds the cellulose. Enzymatic hydrolysis can be improved by removing the hemicellulose contents. The method used for the removal of hemicellulose also removes the lignin content that is why these methods are not more beneficial. Hemicellulase enzyme is used for the hydrolysis of hemicellulose. Dilute acid hydrolysis method is also beneficial for the digestion of hemicellulose.
2.2.2Pretreatment methods
A number of pretreatment methods are in practice which vary with the nature of biomass with different reaction conditions and catalyst such as mechanical pre-treatment [128], steam explosion [135], ammonia fiber explosion [136] , supercritical CO2 treatment [137] , alkali or acid pretreatment [138, 139] , ozone pretreatment [140] , and biological pretreatment [141]. Various pretreatment methods, their effect on the biomass structure and area of interest are given in table 2.1 & 2.2.
2.2.2.1Physical methods
Physical pretreatment methods decrease the degree of polymerization and crystallinity hence increase the pore size and accessible surface area.
23 Table 2.1: Pretreatment type, structural change and their outcomes [115b]
Pretreatment Technique
Catalyst and process Biomass structural changes Applications Benefits/Drawbacks
P h y si c a l P r e tr e a tm e n t Milling: Ball milling Two-roll milling Hammer milling Colloid milling Vibro energy milling
Irradiation: Gamma-ray irradiation Electron-beam irradiation Microwave irradiation Others: Hydrothermal
High pressure steaming Expansion
Extrusion Pyrolysis
Increase in accessible surface area and pore size
Decrease in cellulose crystallinity Decrease in degrees of polymerization Ethanol Ethanol and biogas Ethanol and biogas
- Most of the methods are highly energy-
demanding
- Most of them cannot remove the
lignin
- It is preferable not to use these methods
for industrial applications
-No chemicals are generally required for
these methods B io lo g ic a l P r e tr e a tm e n t Fungi Actinomycete Delignification Reduction in degree of polymerization of cellulose Partial hydrolysis of Hemicellulose Ethanol and Biogas
No chemical, low energy requirement, resulting low treatment rate
Mild environmental Conditions Not viable for commercial application
24 Table 2.2: Pretreatment type, structural change and their outcomes
Pretreatment Technique
Catalyst and process Biomass structural changes Applications Benefits/Drawbacks
C h e m ic a l P r e tr e a tm e n t Explosion: Steam explosion
Ammonia fiber explosion (AFEX) CO2 explosion SO2 explosion Alkali: Sodium hydroxide Ammonia Ammonium Sulfite Acid: Sulfuric acid Hydrochloric acid Phosphoric acid Maleic acid Nitric acid Gas: Chlorine dioxide Nitrogen dioxide Sulfur dioxide Oxidizing agents: Hydrogen peroxide Wet oxidation Ozone
Solvent extraction of lignin:
Ethanol-water extraction Benzene-water extraction Ethylene glycol extraction Butanol-water extraction Swelling agents Increase in accessible surface area Partial or nearly complete delignification Decrease in cellulose crystallinity and hydrolysis of hemicellulose Decrease in degrees of polymerization Partial or complete hydrolysis of hemicelluloses
Ethanol, lactic acid and biogas
These methods are among the most effective and include
the most promising processes for industrial
applications
- Usually rapid treatment rate
- Typically need harsh Conditions
- There are chemical Requirement
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Different physical methods were used for the improvement of biodegradability and the enzymatic hydrolysis of lignocelluloses waste materials.
These methods are given below
1) Milling (ball milling, two roll- milling, hammer milling, colloid milling and vibro energy milling)
2) Irradiation ( gamma rays, electron beam and microwaves)
2.2.2.2Milling
Degree of crystallinity and ultrastructure of lignocellulose can be changed by applying milling, and make it more acquiescent to enzymes. Milling process is different for wet and dry materials. The wet material can be milled through colloid mill and dissolver, e.g. wet paper and paper pulp and for dry waste materials extruder, hammer mill, cryogenic mill and roller mill are in practice, e.g. the waste paper can be grind with hammer mill [142- 144]. In milling process, the size and degree of crystallinity decrease and material becomes more susceptible for enzymatic hydrolysis [120]. Corn stover with sizes 425-710 µm was 1.5 times less productive than corn stover with smaller sizes of 53-75 µm without any pretreatment [145]. Mild hydrolytic conditions result in saccharification of more than 50.0% of the straw cellulose with minimum degradation of glucose [146]. The crystallinity index can be reduced from 74.9 to 4.9% through ball milling [120]. Hydrolysis process can be improved by performing the milling process, mass transport and enzymatic hydrolysis simultaneously. Ball milling process utilizes enormous amount of energy hence it is costly. To decrease energy cost, a continuous stirred tank reactor is used which is placed between hollow fiber cartridge and ball mill. Milling process is unable to remove lignin which is another disadvantage of this technique [147]. 2.2.3Chemical pretreatments
2.2.3.1 Acidic pretreatment
The most common method in chemical pretreatment is the acid hydrolysis. Acid pretreatment process uses either dilute or concentrated acid for the de-crystallization of the lignocellulose which enhances the hydrolysis yield. Dilute acid pretreatment is one of the most studied and widely used processes for the treatment of lignocellulosic material [148, 149]. Acid
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hydrolysis can be done by two methods, either low temperature and high acid concentration and high temperature with low acid concentration. Variety of acids is used during treatment such as sulphuric acid [150], hydrochloric acid [151], peracetic acid [152], nitric acid [153] and phosphoric acid. The most effective acid is sulfuric acid. Two types of dilute acid pretreatment methods were in practice; one is continuous flow process with low solid loading generally 5.0 – 10.0% at low temperature and second is batch process at high temperature with high solid loading usually 10.0 – 40.0% (w/v) [152]. High acid concentration is dangerous and extremely corrosive. Therefore need to use expensive alloys or non- metallic constructions. In economical point of view, the concentrated acid process requires the recovery of acid which is an energy demanding process. O n the other hand large amount of gypsum is produced during neutralization process. It requires high maintenance costs and high investment therefore it is not preferred at commercial level [125, 153]. In dilute acid process, different types of reactors are used for the pretreatment of lignocellulose such as batch reactors, countercurrent, plug flow, percolation and shrinking-bed reactors. Cellulose hydrolysis can be improved significantly by treating the material at low acid concentration (0.1-1.0% sulfuric acid) and at elevated temperature (140-190oC). Complete hemicellulose can be removed by dilute acid pretreatment method. Dilute acid pretreatment also degraded the lignin which is not much effective; but it increases the susceptibility of the enzyme for cellulose hydrolysis [153]. The main disadvantage of the acid pretreatment at low pH is the formation of some inhibitors such as furans, carboxylic acids and phenolic compounds. These compounds inhibit the microbial growth and fermentation but the enzymatic hydrolysis is not affected b y this method, whereas the yield and productivity of biogas or ethanol is reduced. The formation of these inhibitors should be less if materials are treated at high pH [140].
2.2.3.2 Alkaline pretreatment
Alkali solution such as sodium hydroxide, ammonia and calcium hydroxide is used as a pretreatment catalyst for the removal of lignin and hemicellulose which increases the surface area of the cellulose by swelling and decreasing the degree of crystallinity [154]. Saccharification is sharply increased after alkali pretreatment [155]. In alkaline pretreatment method, high concentration of base is used for long time at low temperature. When ammonia liquor (10.0%) is used at room temperature for the soaking of soybean straw for 24 hours, the
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lignin and hemicellulose decrease by 30.16 and 41.45% respectively. Wood material is less affected than agricultural residues by alkaline pretreatment method [154]. Different substrates such as wheat straw, poplar wood, switch grass and corn stover were pretreated with calcium hydroxide at different temperatures for varying times, which removed the lignin, acetyl groups and uronic acid substitutions for maximum hydrolysis of cellulose and hemicellulose [156].
2.2.3.3 Alkaline peroxide
For the pretreatment of biomass alkaline peroxide is an effective process. In this process the pH was adjusted with water, containing H2O2 and NaOH, for 5-6 hour at room temperature which softens the lignocelluloses materials. Delignification process can improve the enzymatic hydrolysis.
2.2.3.4 Organosolv process
Lignocellulosic materials can also be treated through organosolv process with variety of organic and aqueous- organic solvents at temperature range of 150-200oC, with or without catalyst certifying the maximum removal of lignin, some part of hemicellulose and maximizing the enzymatic hydrolysis [157 – 159]. The organic solvents used in this process are alcohols, glycols, organic acids, esters, ketones, ethers and phenols. The price and recovery factor of the solvent should also be considered. To reduce the operational costs solvent should be separated through evaporation and recycling. Usually the solvents inhibit the digestion of the action of enzymes and hydrolysis, therefore the removal of solvent from the pretreated cellulose is necessary. Lignin and hemicellulose can be separated in a two stage progression by organosolv and acid. Lignin can be extracted from the solvent for e.g. generation of electricity, process heat, lignin-based adhesives and other products, due to its high purity and low molecular weight [160].
2.2.3.5 Wet oxidation
For the production of biogas and ethanol wet oxidation is used as a pretreatment method. In this process, the air or oxygen along with water is used for the pretreatment of materials at temperature above 120oC for 30 minutes [161]. In wet oxidation oxygen pressure, temperature and reaction time are the most important parameters. The wet oxidation is an
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exothermic process; there is no need to supply heat when the reaction is initiated [162]. In this method the hemicellulose and lignin fraction can be separated from cellulose. Oxygen plays an important role in degradation reaction at low temperature which enhances the production of organic acids. During wet oxidation, rate of reaction is too high which generate heat creating problem in temperature control of the reactor. In this process all the three contents of the lignocellulosic materials are affected. The lignin undergoes both cleavage and oxidation; the hemicelluloses are cleaved to monomeric sugars; and the cellulose is partly degraded. The cellulose becomes more accessible to the enzymatic hydrolysis [163].
2.2.3.6 Ozonolysis
Due to easy availability of ozone and miscibility with water, it is used as strong oxidant during pretreatment. Hemicellulose and lignin can be broken down by ozone which was obtained from bagasse, peanut, pine oil, wheat, cotton straw [164]. Those compounds which have more electron based functional groups and having high conjugation of double bond (C=C) which immediately oxidize lignin [165]. Pretreatment with ozone was done at room temperature and pressure for lignin removal and it did not give any harmful materials during downstream steps [166]. By using some critical catalyst or giving higher temperature for decomposition of ozone, it can reduce the risk of environmental pollution [167]. If percentage of ozone is taken in higher ratio, then this method could be very costly [127].
2.2.3.7 Ammonia fiber explosion (AFEX)
In this process, high pressure was used to pretreat the lignocellulosic substance in the presence of ammonia and water at high temperature and pressure. This pretreatment can enormously enhance the fermentation of different herbaceous grasses and crops. AFEX process can also be used for pretreatment of bagasse, rice straw, wheat straw & chaff and barley [127]. The effectiveness of AFEX treatment is the production of minute quantity of inhibitors and maximum removal of lignin. Duration of this treatment is short but crystallinity of the cellulosic structure changed remarkably which resulted in enhanced hydrolysis of carbohydrate, due to easy accessibility of enzymes [167].
29 2.2.3.8 Carbon dioxide explosion
The CO2 explosion is very much similar to NH3, which forms the carbonic acid on reaction with water during pretreatment reaction, enhancing the hydrolysis rate. CO2 molecules can enhance the hydrolysis of hemicelluloses and cellulose through formation of carbonic acid when in contact with biomass surfaces. The penetration of CO2 molecules become more effective at high pressure which ultimately enters into crystalline structure of cellulose. As high temperature is not required during CO2 explosion so decomposition of monosaccharide is less, ultimately less quantity of inhibitors are formed, as a result glucan conversion yield was higher than steam explosion or AFEX process [164]. This method is difficult to handle and involves multiple steps but this method is cost effective as compared to ammonia explosion and also environment friendly [115].
2.2.3.9 Hot water
In this method, hemicellulose fraction is removed with hot water under high pressure. No chemical treatment is involved in this method as only hot water is used. Corrosion resistant substances and reduction in raw material size is not necessarily required in hydrolysis reactor [116]. Corn fibers and herbaceous crops pretreatment are generally carried out using this technique [117]. Temperature of water is maintained at around 200-230oC in contact with biomass for 15min which dissolved 38-62% biomass in which complete hemicellulose is removed.
2.2.3.10 Biological pretreatment
For production of biogas, biological pretreatment was also investigated in which microorganisms are used to increase the saccharification efficiency. Biological process is more beneficial for use because lower energy is required; no chemical and other harsh conditions are required [127]. Cellulose molecule is most resistant to biological attack while lignin and hemicelluloses are easily degraded, leaving pure cellulose for production of glucose. Different types of fungi such as brown, white and soft-rot fungi were used but white-rot fungus was favorable for pretreatment of lignocelluloses [127]. Taniguchi et al., 2005, [168] pretreated the rice straw with four types of white-rot fungi named Trametes versicolor, Phanerochaete chrysosporium, Ceriporiopsis subvermispora and Pleurotus ostreatus. They have evaluated the
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structural changes and composition of biomass components in pretreated straw. P. ostreatus potentially degraded the lignin component more and resulted in improved enzymatic saccharification efficiency. K urakake et al., 2007 [151] pretreated the office paper for maximum enzymatic saccharification with two strains of bacteria (Sphingomonas paucimobilis and Bacillus circulans). At optimum conditions during bacterial pretreatment, enzymatic digestibility of the office paper reached up to 94.0%. This pretreatment is not only used for the removal of lignin but also for the removal of antimicrobial substances. Solid state fermentation of orange peels by fungal strains of Sporotrichum, Aspergillus, Fusarium and Penicillum enhanced the availability of sugars and reduction in toxic substances [169]. In a similar work, cultivation of white rot fungi was used to detoxify the olive mill wastewater and improve its digestion [170].