Anju Arora

Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.

Novel perspectives for evolving enzyme cocktails for lignocellulose hydrolysis in biorefineries

The unstable and uncertain availability of petroleum sources as well as rising cost of fuels have shifted global efforts to utilize renewable resources for the production of greener energy and a replacement which can also meet the high energy demand of the world. Bioenergy routes suggest that atmospheric carbon can be cycled through biofuels in carefully designed systems for sustainability. Significant potential exists for bioconversion of biomass, the most abundant and also the most renewable biomaterial on our planet. However, the requirements of enzyme complexes which act synergistically to unlock and saccharify polysaccharides from the lignocellulose complex to fermentable sugars incur major costs in the overall process and present a great challenge. Currently available cellulase preparations are subject to tight induction and regulation systems and also suffer inhibition from various end products. Therefore, more potent and efficient enzyme preparations need to be developed for the enzymatic saccharification process to be more economical. Approaches like enzyme engineering, reconstitution of enzyme mixtures and bioprospecting for superior enzymes are gaining importance. The current scenario, however, also warrants the need for research and development of integrated biomass production and conversion systems.

Pretreatment of paddy straw with Trametes hirsuta for improved enzymatic saccharification

Delignification of paddy straw with the white-rot fungus, Trametes hirsuta under solid state fermentation, for enhanced sugar recovery by enzymatic saccharification was studied. T. hirsuta MTCC136 showed high ligninase and low cellulase activities. Solid state fermentation of paddy straw with T. hirsuta enhanced carbohydrate content by 11.1% within 10 days of incubation. Alkali extracts of Trametes pretreated paddy straw showed high absorbance at 205 nm indicating high lignin break down. The amount of value-added lignin recovered from the Trametes pretreated paddy straw was much higher than controls. Enzymatic hydrolysis of the Trametes pretreated paddy straw yielded much higher sugars than controls and yields increased till 120 h of incubation. Saccharification efficiency of the biologically pretreated paddy straw with Accelerase®1500 was 52.69% within 72 h and was higher than controls. Thus, the study brings out the delignification potential of T. hirsuta for pretreatment of lignocellulosic substrate and facilitating efficient enzymatic digestibility of cellulose.

Comparison of biomass productivity and nitrogen fixing potential of Azolla SPP

Study was conducted on six different Azolla species, available in the germplasm collection of NCCUBGA, IARI, New Delhi namely A. filiculoides, A. mexicana, A. microphylla, A. pinnata, A. rubra and A. caroliniana in a polyhouse to assess their growth potential by determining their maximal biomass productivity, doubling time and relative growth rates. Their nitrogen fixing potential was assessed by acetylene reduction assay. Among them Azolla microphylla gave highest biomass production and relative growth rate followed by Azolla caroliniana. Both these had high nitrogenase activity also. Peak nitrogenase activity of these strains was found on 14th day of growth and it declined on further incubation. Azolla microphylla and Azolla rubra were more tolerant to salinity than others. On the other hand Azolla pinnata, which is endemic species found in India, exhibited low biomass production, relative growth rate and lower nitrogenase activity compared to other species. It was unable to sustain growth in saline medium. Under polyhouse conditions, A. microphylla was found to perform better than other cultures in terms of biomass productivity, N fixing ability and salt tolerance. Hence it is taken up for mass production.

Streptomyces griseorubens mediated delignification of paddy straw for improved enzymatic saccharification yields.

Biological pretreatment of paddy straw was carried out using an actinomycete isolate, identified as Streptomyces griseorubens ssr38, for delignification under solid state fermentation and enhanced sugar recovery by enzymatic saccharification. After 10 days incubation, the inoculated paddy straw was extracted with mild alkali and high absorbance at 205 nm was shown by the extracts indicating the ability of S. griseorubens ssr38 to depolymerize/solubilize lignin to a high extent. Also, almost 25% of depolymerized lignin could be recovered as value-added acid-precipitable polymeric lignin (APPL) as compared to controls. Enrichment in carbohydrate content of inoculated paddy straw following delignification led to a high saccharification efficiency of 97.8% upon enzymatic hydrolysis with Accelerase®1500. The study, therefore, proves the potential of actinomycetes, besides the conventionally used white-rot fungi, for biological pretreatment, in the biomass to bioethanol process, with respect to the high extent of delignification, lignin recovery, cellulose enrichment and very high saccharification efficiency.

Beta-Glucosidase: Key Enzyme in Determining Efficiency of Cellulase and Biomass Hydrolysis

Overall economics of the biomass to ethanol process is largely determined by the efficiency of biomass hydrolysis. Performance of cellulase cocktails used for saccharification of cellulose in biomass is often limited by lower amounts of β-glucosidases present, which catalyse hydrolysis of cellobiose, the product of endo and exocellulases to glucose. Inappropriate ratio of these enzymes in commercial cocktails leads to accumulation of cellobiose which inhibits the activity of cellulases. Thus, this rate limiting enzyme is of crucial importance in determining the efficiency of commercial cellulases. The saprophytic fungus Trichoderma sp., exploited for production of commercial cellulases, produces very minute quantities of β-glucosidases as compared to endo and exocellulases. However, several other organisms are known to produce β-glucosidases in higher quantities, over a broader substrate range. Strategies to get optimal ratio of exocellulases, endocellulases and β-glucosidases to enhance saccharification yields are, therefore, discussed. Appropriate levels of β-glucosidase activity in commercial cocktails have been obtained by supplementing with accessory β-glucosidases, transgenic approaches and by optimizing β-glucosidase production through manipulation of culture conditions. These approaches have resulted in achieving higher β-glucosidase activity in cellulase cocktails, facilitating higher sugar yields and thereby potentially improving enzymatic saccharification of biomass and eventually ethanol production.

Biological delignification of paddy straw and Parthenium sp. using a novel micromycete Myrothecium roridum LG7 for enhanced saccharification.

A new lignolytic micromycete fungus Myrothecium roridum LG7 was isolated and selected for biological delignification of agro residue-paddy straw and herbaceous weed Parthenium sp. Physical and chemical modifications in the biomass following pretreatment with M. roridum LG7 for 7 days in term of structural modification and lignin removal, changes in lignin skeleton, and alteration of cellulose crystallinity was observed through SEM-EDXA, FTIR and XRD analysis, respectively. Colonization of the fungus led to high amount of lignin removal (5.8-6.98mg/gds) from pretreated biomass which could be recovered as a value added product. Enzymatic hydrolysis of M. roridum LG7 pretreated biomass released significantly higher amount of reducing sugars (455.81-509.65 mg/gds) as compared to respective raw biomass within 24h. This study illustrates the promise of M. roridum LG7 for biological pretreatment through structural and chemical alteration of biomass beside creation of alkaline environment which prevent the growth of other contaminants.

Optimization of enzymatic saccharification of alkali pretreated Parthenium sp. using response surface methodology.

Parthenium sp. is a noxious weed which threatens the environment and biodiversity due to its rapid invasion. This lignocellulosic weed was investigated for its potential in biofuel production by subjecting it to mild alkali pretreatment followed by enzymatic saccharification which resulted in significant amount of fermentable sugar yield (76.6%). Optimization of enzymatic hydrolysis variables such as temperature, pH, enzyme, and substrate loading was carried out using central composite design (CCD) in response to surface methodology (RSM) to achieve the maximum saccharification yield. Data obtained from RSM was validated using ANOVA. After the optimization process, a model was proposed with predicted value of 80.08% saccharification yield under optimum conditions which was confirmed by the experimental value of 85.80%. This illustrated a good agreement between predicted and experimental response (saccharification yield). The saccharification yield was enhanced by enzyme loading and reduced by temperature and substrate loading. This study reveals that under optimized condition, sugar yield was significantly increased which was higher than earlier reports and promises the use of Parthenium sp. biomass as a feedstock for bioethanol production.

Deciphering the traits associated with PAH degradation by a novel Serratia marcesencs L-11 strain

Polycyclic aromatic hydrocarbons (PAHs) are wide spread industrial pollutants that are released into the environment from burning of coal, distillation of wood, operation of gas works, oil refineries, vehicular emission, and combustion process. In this study a lipolytic bacterium was isolated from mixed stover compost of Saccharum munja and Brassica campestris. This strain was identified by both classical and 16S ribosomal DNA sequencing method and designated as Serratia marcesencs L-11. HPLC-based quantitation revealed 39- 100% degradation of PAH compounds within seven days. Further its ability to produce catechol 1, 2-dioxygenase (1.118 μM mL−1 h−1) and biosurfactants (0.88 g L−1) during growth in PAH containing medium may be responsible for its PAH-degradation potential. This novel bacterium with an ability to produce lipases, biosurfactant and ring cleavage enzyme can prove to be useful for in-situ degradation of PAH compounds.

Enhanced biodegradation of PAHs by microbial consortium with different amendment and their fate in in-situ condition.

Microbial degradation is a useful tool to prevent chemical pollution in soil. In the present study, in-situ bioremediation of polyaromatic hydrocarbons (PAHs) by microbial consortium consisting of Serratia marcescens L-11, Streptomyces rochei PAH-13 and Phanerochaete chrysosporium VV-18 has been reported. In preliminary studies, the consortium degraded nearly 60-70% of PAHs in broth within 7 days under controlled conditions. The same consortium was evaluated for its competence under natural conditions by amending the soil with ammonium sulphate, paddy straw and compost. Highest microbial activity in terms of dehydrogenase, FDA hydrolase and aryl esterase was recorded on the 5(th) day. The degradation rate of PAHs significantly increased up to 56-98% within 7 days under in-situ however almost complete dissipation (83.50-100%) was observed on the 30(th) day. Among all the co-substrates evaluated, faster degradation of PAHs was observed in compost amended soil wherein fluorene, anthracene, phenanthrene and pyrene degraded with half-life of 1.71, 4.70, 2.04 and 6.14 days respectively. Different degradation products formed were also identified by GC-MS. Besides traces of parent PAHs eleven non-polar and five polar products were identified by direct and silylation reaction respectively. Various products formed indicated that consortium was capable to degrade PAHs by oxidation to mineralization.