Morphologically favorable mutant of Trichoderma reesei for low viscosity cellulase production.

Metabolite production by filamentous fungi hampered because of high viscosity generated during growth. Low viscosity fermentation by mold is one of the preferred ways of large scale enzymes production. Cellulolytic enzymes play a key role during the process of lignocellulosic biomass conversion. In this study, a mutant RC-23-1 was isolated through mutagenesis (diethyl sulfate followed by UV) of Trichoderma reesei RUT-C30. RCRC-23-1 not only gave higher cellulase production but also generated lower viscosity during enzyme production. Viscosity of mutant growth was more than three times lower than parent strain. RC-23-1 shows unique, yeast-like colony morphology on solid media and small pellet-like growth in liquid media. This mutant did not spread like mold on solid media. This mutant produces cellulases constitutively when grown in sugars. Using only glucose, the cellulase production was 4.1 FPU/ml. Among polysaccharides (avicel, xylan, and pectin), avicel gave maximum of 6.2 FPU/ml and pretreated biomass (rice straw, wheat straw and sugarcane bagasse) produced 5.1-5.8 FPU/ml. At 7 L scale reactor, fed-batch process was designed for cellulase production using different carbon and nitrogen sources. Maximum yield of cellulases was 182 FPU/g of lactose consumed was observed in fed-batch process. The produced enzyme used for hydrolysis of acid pretreated rice straw (20% solid loading) and maximum of 60% glucan conversion was observed. RC-23-1 mutant is good candidate for large scale cellulase production and could be a model strain to study mold to yeast-like transformation.

Bioethanol production by a xylan fermenting thermophilic isolate Clostridium strain DBT-IOC-DC21.

To overcome the challenges associated with combined bioprocessing of lignocellulosic biomass to biofuel, finding good organisms is essential. An ethanol producing bacteria DBT-IOC-DC21 was isolated from a compost site via preliminary enrichment culture on a pure hemicellulosic substrate and identified as a Clostridium strain by 16S rRNA analysis. This strain presented broad substrate spectrum with ethanol, acetate, lactate, and hydrogen as the primary metabolic end products. The optimum conditions for ethanol production were found to be an initial pH of 7.0, a temperature of 70 °C and an L-G ratio of 0.67. Strain presented preferential hemicellulose fermentation when compared to various substrates and maximum ethanol concentration of 26.61 mM and 43.63 mM was produced from xylan and xylose, respectively. During the fermentation of varying concentration of xylan, a substantial amount of ethanol ranging from 25.27 mM to 67.29 mM was produced. An increased ethanol concentration of 40.22 mM was produced from a mixture of cellulose and xylan, with a significant effect observed on metabolic flux distribution. The optimum conditions were used to produce ethanol from 28 g L-1 rice straw biomass (RSB) (equivalent to 5.7 g L-1 of the xylose equivalents) in which 19.48 mM ethanol production was achieved. Thus, Clostridium strain DBT-IOC-DC21 has the potential to perform direct microbial conversion of untreated RSB to ethanol at a yield comparative to xylan fermentation.

Extraction, separation and purification of fatty acid ethyl esters for biodiesel and DHA from Thraustochytrid biomass.

A novel approach for in situ transesterification, extraction, separation, and purification of fatty acid ethyl esters (FAEE) for biodiesel and docosahexaenoic acid (DHA) from Thraustochytrid biomass has been developed. The downstream processing of Thraustochytrids oil necessitates optimization, considering the higher content of polyunsaturated fatty acids (PUFA). While two-step methods are commonly employed for extracting and transesterifying oil from oleaginous microbes, this may result in oxidation/epoxidation of omega-3 oil due to prolonged exposure to heat and oxygen. To address this issue, a rapid single-step method was devised for in situ transesterification of Thraustochytrid oil. Through further process optimization, a 50% reduction in solvent requirement was achieved without significantly impacting fatty acid recovery or composition. Scale-up studies in a 4 L reactor demonstrated complete FAEE recovery (99.98% of total oil) from biomass, concurrently enhancing DHA yield from 16% to nearly 22%. The decolorization of FAEE oil with fuller's earth effectively removed impurities such as pigments, secondary metabolites, and waxes, resulting in a clear, shiny appearance. High-performance liquid chromatography (HPLC) analysis indicated that the eluted DHA was over 94.5% pure, as corroborated by GC-FID analysis.

Enzyme systems of thermophilic anaerobic bacteria for lignocellulosic biomass conversion.

Economic production of lignocellulose degrading enzymes for biofuel industries is of considerable interest to the biotechnology community. While these enzymes are widely distributed in fungi, their industrial production from other sources, particularly by thermophilic anaerobic bacteria (growth Topt ≥ 60 °C), is an emerging field. Thermophilic anaerobic bacteria produce a large number of lignocellulolytic enzymes having unique structural features and employ different schemes for biomass degradation, which can be classified into four systems namely; 'free enzyme system', 'cell anchored enzymes', 'complex cellulosome system', and 'multifunctional multimodular enzyme system'. Such enzymes exhibit high specific activity and have a natural ability to withstand harsh bioprocessing conditions. However, achieving a higher production of these thermostable enzymes at current bioprocessing targets is challenging. In this review, the research opportunities for these distinct enzyme systems in the biofuel industry and the associated technological challenges are discussed. The current status of research findings is highlighted along with a detailed description of the categorization of the different enzyme production schemes. It is anticipated that high temperature-based bioprocessing will become an integral part of sustainable bioenergy production in the near future.

Enhanced cellulosic ethanol production via consolidated bioprocessing by Clostridium thermocellum ATCC 31924☆.

The production of bioethanol was studied by the cultivation of Clostridium thermocellum ATCC 31924 in MTC medium including crystalline cellulose as the sole substrate. The effects of key operational parameters that affect bioethanol production from microcrystalline cellulose were optimized. Under optimum conditions (pH 8.0, temperature 55 °C, inoculum size 4% (v/v) and 0.5% (w/v) substrate concentration), a maximum ethanol yield of 0.30 g ethanol/g cellulose consumed and 95.32% cellulose conversion was obtained. An inclusion of modest acetate concentration in the medium showed that carbon flux shifted away from lactate accompanied by 20% increase in ethanol production. It suggests that strain ATCC 31924 differed in its cellulose conversion efficacy and optimum pH requirements compared to the other reported strains of Clostridium thermocellum. The purified cellulosome of strain ATCC 31924 found to be rich in both cellulase and xylanase enzymes emphasizing the importance of this strain for the degradation of lignocellulosic biomass.

Cellulosic ethanol production via consolidated bioprocessing by a novel thermophilic anaerobic bacterium isolated from a Himalayan hot spring.

BACKGROUND: Cellulose-degrading thermophilic anaerobic bacterium as a suitable host for consolidated bioprocessing (CBP) has been proposed as an economically suited platform for the production of second-generation biofuels. To recognize the overall objective of CBP, fermentation using co-culture of different cellulolytic and sugar-fermenting thermophilic anaerobic bacteria has been widely studied as an approach to achieving improved ethanol production. We assessed monoculture and co-culture fermentation of novel thermophilic anaerobic bacterium for ethanol production from real substrates under controlled conditions. RESULTS: In this study, Clostridium sp. DBT-IOC-C19, a cellulose-degrading thermophilic anaerobic bacterium, was isolated from the cellulolytic enrichment cultures obtained from a Himalayan hot spring. Strain DBT-IOC-C19 exhibited a broad substrate spectrum and presented single-step conversion of various cellulosic and hemicellulosic substrates to ethanol, acetate, and lactate with ethanol being the major fermentation product. Additionally, the effect of varying cellulose concentrations on the fermentation performance of the strain was studied, indicating a maximum cellulose utilization ability of 10 g L-1 cellulose. Avicel degradation kinetics of the strain DBT-IOC-C19 displayed 94.6% degradation at 5 g L-1 and 82.74% degradation at 10 g L-1 avicel concentration within 96 h of fermentation. In a comparative study with Clostridium thermocellum DSM 1313, the ethanol and total product concentrations were higher by the newly isolated strain on pretreated rice straw at an equivalent substrate loading. Three different co-culture combinations were used on various substrates that presented two-fold yield improvement than the monoculture during batch fermentation. CONCLUSIONS: This study demonstrated the direct fermentation ability of the novel thermophilic anaerobic bacteria on various cellulosic and hemicellulosic substrates into ethanol without the aid of any exogenous enzymes, representing CBP-based fermentation approach. Here, the broad substrate utilization spectrum of isolated cellulolytic thermophilic anaerobic bacterium was shown to be of potential utility. We demonstrated that the co-culture strategy involving novel strains is efficient in improving ethanol production from real substrate.

Exploring omega-3 fatty acids, enzymes and biodiesel producing thraustochytrids from Australian and Indian marine biodiversity.

The marine environment harbours a vast diversity of microorganisms, many of which are unique, and have potential to produce commercially useful materials. Therefore, marine biodiversity from Australian and Indian habitat has been explored to produce novel bioactives, and enzymes. Among these, thraustochytrids collected from Indian habitats were shown to be rich in saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), together constituting 51-76% of total fatty acids (TFA). Indian and Australian thraustochytrids occupy separate positions in the dendrogram, showing significant differences exist in the fatty acid profiles in these two sets of thraustochytrid strains. In general, Australian strains had a higher docosahexaenoic acid (DHA) content than Indian strains with DHA at 17-31% of TFA. A range of enzyme activities were observed in the strains, with Australian strains showing overall higher levels of enzyme activity, with the exception of one Indian strain (DBTIOC-1). Comparative analysis of the fatty acid profile of 34 strains revealed that Indian thraustochytrids are more suitable for biodiesel production since these strains have higher fatty acids content for biodiesel (FAB, 76%) production than Australian thraustochytrids, while the Australian strains are more suitable for omega-3 (40%) production.

Characterization of a new zeaxanthin producing strain of Chlorella saccharophila isolated from New Zealand marine waters.

A fast growing strain of Chlorella saccharophila was isolated from the marine water of New Zealand and grown in heterotrophic conditions using glucose or glycerol as a carbon source. Biomass production was found to be higher in culture fed with glucose (2.14±0.08 g L(-1)) as compared to glycerol (0.378±0.04 g L(-1)). Lipid accumulation was similar for both carbon sources, at approximately 22% of dry cell weight. However, carotenoid yield was higher with glycerol (0.406±0.0125 mg g(-1)) than with glucose (0.21±0.034 mg g(-1)). Further optimization of the growth of the isolate gave maximal carotenoid production of 16.39±1.19 mg g(-1) total carotenoid, containing 11.32±0.64 mg g(-1) zeaxanthin and 5.07±0.55 mg g(-1) ß-carotene. Comparison of various chemical and physical carotenoid extraction methods showed that ultrasonication was required for maximum extraction yields. The new strain has potential for biofuel, with carotenoid co-production.