Aspergillus spp., a versatile cell factory for enzymes and metabolites: Interventions through genome editing

Aspergillus sp. is widely distributed in nature and plays significant roles in the degradation of lignocellulose biomass and extensively used in bioprocess and fermentation technology and many species are also a generally regarded safe. Many of the Aspergillus species are established cell factories due to their inherent capacity in secreting large number of hydrolytic enzymes. With the advent of next generation genomic technologies and metabolic engineering technologies, the production potential of Aspergillus cell factory has improved over the years. Various genome editing tools has been developed for Aspergillus like engineered nucleases, zinc finger nucleases, TALEN and CRISPR-Cas9 system. Currently, the CRISPR/Cas9-based technique is extensively used to enhance the effectiveness of gene manipulation in model system Aspergillus nidulans and other strains like Aspergillus oryzae, Aspergillus niger and Aspergillus fumigatus. This review describes the recent developments of genome editing technologies in Aspergillus the synthesis of heterologous proteins and secondary metabolites in the Aspergillus species.

Induction of systemic acquired resistance in Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg. by an endophytic bacterium antagonistic to Phytophthora meadii McRae

Abnormal leaf fall (ALF) disease caused by Phytophthora meadii McRae is one of the limiting factors of growth and latex yield in Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg. Endophytic bacteria residing within the host plant offer very high application potential as biocontrol agents for ALF disease management. Screening of bacterial endophytes from leaf, petiole and root tissues of Hevea brasiliensis was done for the selection of a potent antagonistic isolate against P. meadii. The most efficient antagonistic isolate was identified as Alcaligenes sp. The biochemical estimation of antagonist treated plants showed induction of peroxidase activity in leaves. Quantitative PCR analysis confirmed higher activity of lignin biosynthesis promoting class III peroxidase transcripts in antagonist treated plants of H. brasiliensis. Homology model of the peroxidase class III protein was built from the deduced amino acid sequence and was found to carry 36% helical and 5% beta sheets. Computer aided docking studies of the substrates (p-coumaryl, coniferyl and sinapyl alcohol) with the target proteins showed that the protein-substrate complexes were stabilized by various interactions and proposed that precursors of lignin biosynthesis were preferred by induced peroxidase in Hevea brasiliensis for lignifications. The present study revealed the potential of an antagonistic endophyte Alcaligenes sp. EIL-2 for inducing class III peroxidase involved in defence mechanism in Hevea brasiliensis.

Enzymatic hydrolysis of microwave assisted acid pretreated chili postharvest residue for production of value added products

Agricultural crop residues serve as a renewable source for production of bioethanol and other value added chemicals. Optimization of enzymatic saccharification may ensure cost-effective production of bioethanol and other industrially important products. Here, we attempted optimization of various process variables affecting enzymatic hydrolysis of microwave-assisted acid pretreated chili post-harvest residue was evaluated by adopting statistical design experiments. The optimum conditions of enzymatic hydrolysis were solid loading of 15% (w/w), cellulase loading of 20 FPU per gram of pretreated biomass and incubation time of 12 h. The high solid loading, low cellulase loading and low incubation time may lead to better process economics. Maximum reducing sugar yield of 0.205 g/g was observed. Fermentation inhibitors, such as furfural and 5-hydroxymethylfurfural were absent in the hydrolysate obtained after enzymatic saccharification of pretreated biomass and were found suitable for the production of various value added products like xylanases, bioethanol and biopolymer (poly-3-hydroxybutyrate).

Culture based approaches, dependent and independent, for microbial community fractions in petroleum oil reservoirs

Petroleum reservoir is an ecosystem having extreme environmental conditions of temperature, pressure and salinity. They possess highly anoxic conditions. Major microbial communities present in this environment include fermentative bacteria, sulphate reducing bacteria (SRB), syntrophic bacteria and methanogens. Phylogenetic diversity of microorganisms as well as their ecological role play an important role in the petroleum reservoir ecosystem. Past and present efforts to characterize microbial communities in oil field ecosystem by culture or cultivation-dependent and -independent approaches are discussed with highlights of microbial ecology of petroleum oil reservoir ecosystem(s). Novel strategies used to study culture independent diversity of microorganisms using metagenomic techniques have also been narrated.

Production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching.

Here, we described the production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching. Various process parameters affecting xylanase production by B. pumilus were optimized by adopting a Plackett-Burman design (PBD) as well as Response surface methodology (RSM). These statistical methods aid in improving the enzyme yield by analysing the individual crucial components of the medium. Maximum production was obtained with 4% yeast extract, 0.08% magnesium sulphate, 30 h of inoculum age, incubation temperature of 33.5 °C and pH 9.0. Under optimized conditions, the xylanase activity was 372 IU/ml. Media engineering improved a 5-fold increase in the enzyme production. Scanning electron microscopy (SEM) showed significant changes on the surface of xylanase treated pulps as a result of xylan hydrolysis. Increased roughness of paper carton fibres was apparent in scanning electron micrograph due to opening of the micro fibrils present on the surface by xylanase action. The untreated pulp did not show any such change. These results demonstrated that the B. pumilus MTCC 5015 xylanase was effective in bio-bleaching of paper carton.

Mixed Cultures Fermentation for the Production of Poly- ß-hydroxybutyrate

Poly-ß-hydroxybutyrate (PHB) is a biodegradable intracellular microbial product produced by many bacteria and it is comparable to some of the petrochemical derived thermoplastics such as polypropylene. One of the main barriers for the commercial exploitation is the high cost of the substrate for the production of biopolymer. The utilization of mixed microbial cultures facilitates the use of complex substrates thereby reducing the cost of PHB production. In the present study, mixed culture systems were evaluated for PHB production. Bacillus firmus NII 0830 was used for the production of PHB since it accumulates a large amount of PHB and a second organism Lactobacillus delbrueckii NII 0925 was used to provide lactic acid. FTIR and 1H NMR analyses revealed that the PHB extracted from pure culture and mixed culture showed exact match to that of standard PHB. Biodegradation studies of the PHB blends showed 87% degradation. It was also found that a consortium of organisms degraded the films faster than a single organism.

Evaluation of polymeric adsorbent resins for efficient detoxification of liquor generated during acid pretreatment of lignocellulosic biomass.

Production of fuel ethanol from lignocellulosic biomass conventionally includes biomass pretreatment, hydrolysis, and fermentation. The liquor generated during dilute acid pretreatment of biomass contains considerable quantities of pentose sugars as well as various degradation products of sugars and lignin, like furfural, hydroxymethyl furfural (HMF), organic acids, aldehydes and others, which are known to be inhibitory for microbial growth. This pentose rich liquor is a potent resource which can be used to produce alcohol or other value added metabolites by microbial fermentation. However, the presence of these inhibitory compounds is a major hindrance and their removal is essential for efficient utilization of this byproduct stream. In the present work, the polymeric adsorbent resins, XAD-4, XAD-7 and XAD-16 were evaluated for their ability to adsorb fermentation inhibitors like furfural and HMF from the acid pretreated liquor. These resins could remove 55-75% of furfural and 100% of HMF and more than 90% sugar remained un-adsorbed in the pretreated liquor. Desorption of furfural from stationary phase was evaluated by using ethanol and hot water. The results suggest that these polymeric resins may be used for detoxification of acid pretreatment liquor with selective removal of sugar degradation products without affecting the sugar content in the solution.

Production and characterization of poly-3-hydroxybutyrate from crude glycerol by Bacillus sphaericus NII 0838 and improving its thermal properties by blending with other polymers

The aim of this work was to study the production of poly-3-hydroxybutyrate (PHB) under nitrogen limited conditions by Bacillus sphaericus NII 0838 using crude glycerol from biodiesel industry as sole carbon source. Effect of various process parameters on PHB production such as glycerol concentration, inoculum size and pH of the medium were optimized. Characterization of extracted PHB was carried out by FT-IR, ¹H and 13C NMR. Results showed that the bacterial culture accumulated about 31 percent PHB in crude glycerol medium. The extracted PHB was blended with other polymers to improve its physical characteristics. The thermal properties of the polymer like melting temperature (Tm) and heat of fusion (ΔHf) were determined using DSC.