Centre for Advanced Bioenergy Research

The world of sensors is diverse and is advancing at a rapid pace due to the fact of its high demand and constant technological improvements. Electrochemical sensors provide a low-cost and convenient solution for the detection of variable analytes and are widely utilized in agriculture, food, and oil industries as well as in environmental and biomedical applications. The popularity of electrochemical sensing stems from two main advantages: the variability of the reporting signals, such as the voltage, current, overall power output, or electrochemical impedance, and the low theoretical detection limits that originate from the differences in the Faradaic and nonFaradaic currents. This review article attempts to cover the latest advances and applications of electrochemical sensors in different industries. The role of nanomaterials in electrochemical sensor research and advancements is also examined. We believe the information presented here will encourage further efforts on the understanding and progress of electrochemical sensors.

Biopolymers are a leading class of functional material suitable for high-value applications and are of great interest to researchers and professionals across various disciplines. Interdisciplinary research is important to understand the basic and applied aspects of biopolymers to address several complex problems associated with good health and well-being. To reduce the environmental impact and dependence on fossil fuels, a lot of effort has gone into replacing synthetic polymers with biodegradable materials, especially those derived from natural resources. In this regard, many types of natural or biopolymers have been developed to meet the needs of ever-expanding applications. These biopolymers are currently used in food applications and are expanding their use in the pharmaceutical and medical industries due to their unique properties. This review focuses on the various uses of biopolymers in the food and medical industry and provides a future outlook for the biopolymer industry.

3D printing by fused deposition modeling (FDM) is an advanced additive manufacturing technology for making thermoplastic-based structures. Several studies have recently investigated 3D printing of polylactic acid (PLA) with biomass resources like cellulose, hemicellulose, lignin and whole biomass. Such biodegradable composites are better for the environment and can be used to replace non-biodegradable composites in a variety of applications. Therefore, a deep understanding of printing such biocomposites is needed for supporting such manufacturing. Recent developments focused on FDM printing of PLA filled with biomass resources have been critically reviewed to reveal the intricate aspects of manufacturing of such materials and characterization of the changes caused by biomass-based fillers. Properties of high molecular weight PLA, essentials of printing with PLA and conditions for filament extrusion and printing of biocomposites are discussed. Characterization results from mechanical testing, thermal analysis, viscoelastic properties, imaging and spectroscopy are reviewed for understanding the impact of filling biomass resources in PLA by printing. The latter sections discuss applications, upcycling & recycling and future opportunities for biorefineries.

Abstract The surplus availability of rice straw, its limited usage, and environmental pollution caused by its inefficient burning has fostered research for its valorization to biofuels. This review elucidates the current status of rice straw potential around the globe along with recent advances in revealing the critical factors responsible for its recalcitrance and chemical properties. The role and accumulation of high silica content in rice straw has been elucidated with its impact on enzymatic hydrolysis in a biorefinery environment. The correlation of different pre‐treatment approaches in modifying the physiochemical properties of rice straw and improving the enzymatic accessibility has also been discussed. This study highlights new challenges, resolutions, and opportunities for rice straw based biorefineries. © 2017 Society of Chemical Industry and John Wiley & Sons, Ltd

Recent advances in metabolic engineering enable the production of high-value chemicals via expressing complex biosynthetic pathways in a single microbial host. However, many engineered strains suffer from poor product yields due to redox imbalance and excess metabolic burden, and require compartmentalization of the pathway for optimal function. To address this problem, significant developments have been made towards co-cultivation of more than one engineered microbial strains to distribute metabolic burden between the co-cultivation partners and improve the product yield. In this emerging approach, metabolic pathway modules can be optimized separately in suitable hosts that will then be combined to enable optimal functionality of the complete pathway. This modular approach broadens the possibilities to fine tune sophisticated production platforms and thus achieve the biosynthesis of very complex compounds. Here, we review the different applications and the overall potential of natural and artificial co-cultivation systems in metabolic engineering in order to improve bioproduction/bioconversion. In addition to the several advantages over monocultures, major challenges and opportunities associated with co-cultivation are also discussed in this review.

The use of tailored medication delivery in cancer treatment has the potential to increase efficacy while decreasing unfavourable side effects. For researchers looking to improve clinical outcomes, chemotherapy for cancer continues to be the most challenging topic. Cancer is one of the worst illnesses despite the limits of current cancer therapies. New anticancer medications are therefore required to treat cancer. Nanotechnology has revolutionized medical research with new and improved materials for biomedical applications, with a particular focus on therapy and diagnostics. In cancer research, the application of metal nanoparticles as substitute chemotherapy drugs is growing. Metals exhibit inherent or surface-induced anticancer properties, making metallic nanoparticles extremely useful. The development of metal nanoparticles is proceeding rapidly and in many directions, offering alternative therapeutic strategies and improving outcomes for many cancer treatments. This review aimed to present the most commonly used nanoparticles for cancer applications.

Multiple enzymes are required for efficient saccharification of lignocellulosic biomass and no wild type organism is capable of producing all enzymes in desired levels. In this study, steam explosion of wheat straw was carried out at pilot scale and a synthetic enzyme mixture (EnzMix) was developed by partially replacing the cellulase with critical dosages of commercially available accessory enzymes (β-glucosidase, xylanase and laccase) through central composite design. Highest degree of synergism (DS) was observed with β-glucosidase (1.68) followed by xylanase (1.36). Finally, benchmarking of EnzMix (Celluclast, β-glucosidase and xylanase in a protein ratio of 20.40: 38.43: 41.16, respectively) and other leading commercial enzymes was carried out. Interestingly, saccharification improved by 75% at 6h and 30% at 24h, respectively in comparison of control. By this approach, 25% reduction in enzyme dosage was observed for obtaining the same saccharification yield. Thus development of enzyme cocktail is an effective and sustainable approach for high saccharification efficiency.

Novel and sustainable pretreatment approaches are desired to improve the techno-commercial feasibility of biorefineries in the future. In this study, 10 renewable deep eutectic solvents (DESs) were evaluated for their pretreatment efficiency at facile conditions with sugar cane bagasse as substrate and compared with conventional pretreatment approaches (dilute alkali, dilute acid, and ionic liquid (IL)) for lignin removal, saccharification yield, cellulose accessibility, crystallinity, and physiochemical properties. Although, the highest delignification was obtained with dilute alkali (59.7%) and choline chloride:lactic acid or ChCl:LA (50.6%), the maximum enzymatic conversion of 98.0% and 90.4% was observed with IL (1-butyl-3-methylimidazolium acetate) and ChCl:LA, respectively. uclear magnetic resonance analysis of ChCl:LA-derived lignin showed selective removal of guaiacyl lignin without condensation structure formation observed. Interestingly, unlike IL, the lignin was substantially depolymerized after ChCl:LA pretreatment as determined by gel permeation chromatography. Further, high compatibility of ChCl:LA with cellulase in comparison of IL with easy recyclability and recycling showed that DESs synthesized from a renewable resource are promising “green” solvents for future biorefinery operations.

Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.

The quest for cheaper and better enzymes needed for the efficient hydrolysis of lignocellulosic biomass has placed filamentous fungi in the limelight for bioprospecting research. In our search for efficient biomass degraders, we identified a strain of Penicillium funiculosum whose secretome demonstrates high saccharification capabilities. Our probe into the secretome of the fungus through qualitative and label-free quantitative mass spectrometry based proteomics studies revealed a high abundance of inducible CAZymes and several nonhydrolytic accessory proteins. The preferential association of these proteins and the attending differential biomass hydrolysis gives an insight into their interactions and clues about possible roles of novel hydrolytic and nonhydrolytic proteins in the synergistic deconstruction of lignocellulosic biomass. Our study thus provides the first comprehensive insight into the repertoire of proteins present in a high-performing secretome of a hypercellulolytic Penicillium funiculosum, their relative abundance in the secretome, and the interaction dynamics of the various protein groups in the secretome. The gleanings from the stoichiometry of these interactions hold a prospect as templates in the design of cost-effective synthetic cocktails for the optimal hydrolysis of biomass.

There is an urgent requirement for second-generation bio-based industries for economical yet efficient enzymatic cocktail to convert diverse cellulosic biomass into fermentable sugars. In our previous study, secretome of Penicillium funiculosum NCIM1228 showed high commercial potential by exhibiting high biomass hydrolyzing efficiency. To develop NCIM1228 further as an industrial workhorse, one of the major genetic interventions needed is global deregulation of cellulolytic genes to achieve higher enzyme production. Mig1 orthologs found in all yeast and filamentous fungi are transcriptional regulators that maintain carbon homeostasis by negatively regulating genes of secondary carbon source utilization. Their disruption has long been known to be beneficial for increasing the production of secreted enzymes for alternate carbon source utilization. Upon detailed genotypic and phenotypic analysis, we observed that NCIM1228 harbors a truncated yet functional allele of homolog of a well-known catabolite repressor, Mig1. Alleviation of carbon repression in NCIM1228 was attained by replacing functional Mig1134 allele with null allele Mig188. P. funiculosum having Mig188 null allele showed better growth characteristics and 1.75-fold better glucose utilization than parent strain. We also showed that visibly small colony size, one of the major characteristics of CCR disruptant strains in filamentous fungi, was not due to retarded growth, but altered hyphal morphology. CCR-disrupted strain PfMig188 showed profuse branching pattern in terminal hyphae resulting in small and compact colonies with compromised filamentous proliferation. We further observed that basal level expression of two major classes of cellulases, namely, cellobiohydrolase and endoglucanase, was regulated by Mig1134 in NCIM1228, whereas other two major classes, namely, xylanases and β-glucosidase, were only marginally regulated. Finally, CCR disruption in P. funiculosum NCIM1228 led to prolonged cellulase induction in production medium resulting in twofold increased cellulase activity than the parent strain with maximum secreted protein titer being > 14 g/l. CCR-disrupted P. funiculosum showed better growth, enhanced carbon source utilization, profuse branching pattern in terminal hyphae, and higher cellulase activity than parent strain. Our findings are particularly important in shedding light on important functions performed by Mig1 in addition to its role as negative regulator of alternate carbon source utilization in filamentous fungi.

Abstract Background Synechococcus sp. PCC 7002 is an attractive organism as a feedstock and for photoautotrophic production of biofuels and biochemicals due to its fast growth and ability to grow in marine/brackish medium. Previous studies suggest that the growth of this organism is limited by the HCO 3 − transport across the cytoplasmic membrane. Tools for genetic engineering are well established for this cyanobacterium, which makes it possible to overexpress genes of interest. Results In this work, we overexpressed two different native Na + -dependent carbon transporters viz., SbtA and BicA in Synechococcus sp. PCC 7002 cells under the influence of a strong light-inducible promoter and a strong RBS sequence. The overexpression of these transporters enhanced biomass by about 50%, increased intracellular glycogen about 50%, and increased extracellular carbohydrate up to threefold. Importantly, the biomass and glycogen productivity of the transformants with air bubbling was even higher than that of WT cells with 1% CO 2 bubbling. The overexpression of these transporters was associated with an increased carotenoid content without altering the chl a content. Conclusions Our work shows the utility of increased carbon transport in improving the growth as well as product formation in a marine cyanobacterium and will serve to increase the utility of this organism as a potential cell factory.

A prepared enzyme cocktail from different fungal enzyme preparations increases the hydrolysis of avicel/wheat straw by increasing synergism between the same or different types of cellulases.

Improved saccharification by exploiting the synergism between biomass degrading enzymes.

Butanol production from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of biobutanol production, both glucose and xylose should be utilized and converted into butanol. Here, we engineered a dual-operon-based synthetic pathway in the genome of E. coli MG1655 to produce n-butanol using CRISPR/Cas9 technology. Further deletion of competing pathway followed by fed-batch cultivation of the engineered strain in a bioreactor with glucose-containing complex medium yielded 5.4 g/L n-butanol along with pyruvate as major co-product, indicating a redox imbalance. To ferment xylose into butanol in redox-balanced manner, we selected SSK42, an ethanologenic E. coli strain engineered and evolved in our laboratory to produce ethanol from xylose, for integrating synthetic butanol cassette in its genome via CRISPR/Cas9 after deleting the gene responsible for endogenous ethanol production. The engineered plasmid- and marker-free strain, ASA02, produced 4.32 g/L butanol in fed-batch fermentation in completely defined AM1-xylose medium.

Enzymatic hydrolysis at high solids has potential to reduce both capital and operational expenditures. Here, dilute acid pretreatment of rice straw was carried out at on pilot scale (250 kg per day) at 162°C for 10 min and 0.35% acid concentration followed by enzymatic hydrolysis at different total solids loading. It showed that although total sugar concentration increased from 48 g/l to 132 g/l but glucan conversion reduced by 27% (84% to 66.2%) with increasing solids from 5% to 20% in batch mode. Therefore, two different fed-batch approaches were evaluated to improve the glucan conversion via sequential addition of substrate and/or enzyme. The maximum glucan conversion obtained in batch mode (Control) was only 61% and 66% after 24 h and 30h, respectively at 3 FPU enzyme/g solids while, 65% and 70% conversion was achieved at similar time and conditions by adding substrate in three batches of 50%, 25%, 25% at an interval of 0h, 4h, 8h respectively. Addition of surfactant (Ecosurf E6) further improved the conversion to 72% after 30h. The role of critical factors i.e. inhibitors, enzyme-lignin binding and viscosity was investigated during the course of hydrolysis in batch and fed-batch approaches. This study suggests a sustainable approach for improved hydrolysis at high solids by simple process fine-tunings.

The biological relevance of algae-derived marine oligosaccharides has potential significance in numerous applications of industrial biotechnology. Marine oligosaccharides are produced in algae naturally and/or by hydrolysis of derived polysaccharides. The potential of marine oligosaccharides is still unexplored, and these molecules, including their derivatives, are a versatile source of chemical diversity. In-depth knowledge on such molecules will provide novel insights, thus depicting their role in discovering mechanisms and the molecular functions within specific biological applications.

Exploring agricultural biomass for biofuel production necessitates pretreatment as a prerequisite step.

GH7 cellobiohydrolases (CBH1) are vital for the breakdown of cellulose. We had previously observed the enzyme as the most dominant protein in the active cellulose-hydrolyzing secretome of the hypercellulolytic ascomycete—Penicillium funiculosum (NCIM1228). To understand its contributions to cellulosic biomass saccharification in comparison with GH7 cellobiohydrolase from the industrial workhorse—Trichoderma reesei, we natively purified and functionally characterized the only GH7 cellobiohydrolase identified and present in the genome of the fungus. There were marginal differences observed in the stability of both enzymes, with P. funiculosum (PfCBH1) showing an optimal thermal midpoint (T m) of 68 °C at pH 4.4 as against an optimal T m of 65 °C at pH 4.7 for T. reesei (TrCBH1). Nevertheless, PfCBH1 had an approximate threefold lower binding affinity (K m), an 18-fold higher turnover rate (k cat), a sixfold higher catalytic efficiency as well as a 26-fold higher enzyme-inhibitor complex equilibrium dissociation constant (K i) than TrCBH1 on p-nitrophenyl-β-d-lactopyranoside (pNPL). Although both enzymes hydrolyzed cellooligomers (G2–G6) and microcrystalline cellulose, releasing cellobiose and glucose as the major products, the propensity was more with PfCBH1. We equally observed this trend during the hydrolysis of pretreated wheat straws in tandem with other core cellulases under the same conditions. Molecular dynamic simulations conducted on a homology model built using the TrCBH1 structure (PDB ID: 8CEL) as a template enabled us to directly examine the effects of substrate and products on the protein dynamics. While the catalytic triads—EXDXXE motifs—were conserved between the two enzymes, subtle variations in regions enclosing the catalytic path were observed, and relations to functionality highlighted. To the best of our knowledge, this is the first report about a comprehensive and comparative description of CBH1 from hypercellulolytic ascomycete—P. funiculosum NCIM1228, against the backdrop of the same enzyme from the industrial workhorse—T. reesei. Our study reveals PfCBH1 as a viable alternative for CBH1 from T. reesei in industrial cellulase cocktails.

The enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation biobased industries. While increasing efforts are being made to obtain indigenous cellulases for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge affecting its wide availability for the efficient utilization of cellulosic materials. This is because it is challenging to obtain an enzymatic cocktail with balanced activity from a single host. This report describes the annotation and structural analysis of an uncharacterized lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact on biomass deconstruction upon overexpression in a catabolite-derepressed strain of P. funiculosum . Cellobiohydrolase I (CBH1), which is the most important enzyme produced by many cellulolytic fungi for the saccharification of crystalline cellulose, was further overexpressed simultaneously with LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities and the corresponding improvement in saccharification performance (by ∼20%) under high-level substrate loading using a minimal amount of protein.