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.

Development & validation of scales to assess stigma related to COVID-19 in India

Background & objectives: COVID-19 pandemic has triggered social stigma towards individuals affected and their families. This study describes the process undertaken for the development and validation of scales to assess stigmatizing attitudes and experiences among COVID-19 and non-COVID-19 participants from the community. Methods: COVID-19 Stigma Scale and Community COVID-19 Stigma Scale constituting 13 and six items, respectively, were developed based on review of literature and news reports, expert committee evaluation and participants’ interviews through telephone for a multicentric study in India. For content validity, 61 (30 COVID-19-recovered and 31 non-COVID-19 participants from the community) were recruited. Test–retest reliability of the scales was assessed among 99 participants (41 COVID-19 recovered and 58 non-COVID-19). Participants were administered the scale at two-time points after a gap of 7-12 days. Cronbach’s alpha, overall percentage agreement and kappa statistics were used to assess internal consistency and test–retest reliability. Results: Items in the scales were relevant and comprehensible. Both the scales had Cronbach’s ? above 0.6 indicating moderate-to-good internal consistency. Test–retest reliability assessed using kappa statistics indicated that for the COVID-19 Stigma Scale, seven items had a moderate agreement (0.4-0.6). For the Community COVID-19 Stigma Scale, four items had a moderate agreement. Interpretation & conclusions: Validity and reliability of the two stigma scales indicated that the scales were comprehensible and had moderate internal consistency. These scales could be used to assess COVID-19 stigma and help in the development of appropriate stigma reduction interventions for COVID-19 infected, and mitigation of stigmatizing attitudes in the community.

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.

Whole cell based biocatalytic production of 2,5-furan dicarboxylic acid

Polyethylene furanoate (PEF), made of 2,5-furan dicarboxylic acid (FDCA), has immense application value and can play a role in reducing the dependence of non-renewable energy sources by replacing the petroleum based products such as polyethylene terephthalate (PET) and polybutylene terephthalate (PBT). The conventional chemical process for production of FDCA via oxidation of 5-hydroxymethyl furfuraldehyde (HMF) requires high temperature, high pressure, metal salts, organic solvents and toxic chemicals. Further, purification of FDCA makes the processes expensive as well as generates considerable pollutants and eco-friendly. The alternative bio-based approach, microbial conversion of substrates such as HMF, fructose and renewable biomass to FDCA sounds promising. In the present study, several soil isolates were evaluated for production of FDCA and one isolate, Enterobacter sp., showed potential for conversion of 5-(hydroxymethyl)furfural (HMF) to FDCA with an yield of 0.07g/L FDCA from 0.5g/L HMF in mineral salt media at 14 days of incubation. Changing the production media with 0.25% glucose and 0.25% glycerol showed an inhibition in the FDCA production by 7- and 2-folds, respectively.

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).

Bioprocess development for docosahexaenoic acid from novel fungal isolate of Fusarium solani.

Fungal cultures were isolated from soil samples collected from the Western Ghats regions of Kerala. Primary screening of isolated strains were done by Sudan black staining method and 15 lipid producing cultures were isolated. The fatty acid profiling of the positive strains were analyzed for docosahexaenoic acid (DHA) production. Selected oleaginous cultures were grown in submerged culture condition to study the biomass yield and poly unsaturated fatty acid, DHA production. The optimization of production process under submerged conditions was carried out using statistical experimental design and confirmation of DHA was done by GC analysis. Maximum DHA production of 150 mg/l was achieved on 4 days of incubation at submerged condition in the presence of glucose as carbon source.

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.

Anti-fungal potentials of extracellular metabolites of Western Ghats isolated Streptomyces sp. NII 1006 against moulds and yeasts.

Realization of hazardious effects of chemical fungicides has led to an interest in the usage of biocontrol agents. The present study, therefore, evaluates the biocontrol efficacy of Western Ghats (India) soil bacterial isolates. A potential strain NII 1006 was evaluated for its antagonistic property against a diverse range of moulds and yeasts. The strain was characterized morphologically, biochemically and molecularly, which revealed the isolate belonged to Streptomyces genus. Organic solvent extracts of NII 1006 culture filtrates inhibited the growth of the test pathogens indicating that growth suppression was due to extracellular anti-fungal metabolites present in the culture filtrates. The strain produced extracellular chitinase enzyme in addition to some stable partially purified anti-fungal compounds. Morphological changes such as hyphae degradation into debris and abnormal shapes were observed in test fungi and yeast grown on potato dextrose broth that contained the NII 1006 culture filtrate. The cell free supernatant has a tolerance to wide range of pH, temperature and enzymes such as lipase and protease. The biocontrol potential of NII 1006 strain may be correlated significantly with their ability to produce antibiotics as well as extracellular hydrolytic enzymes particularly chitinolytic enzyme.

Dilute acid pretreatment and enzymatic hydrolysis of sorghum biomass for sugar recovery—A statistical approach.

Sorghum is one of the commercially feasible lignocellulosic biomass and has a great potential of being sustainable feedstock for renewable energy. As with any lignocellulosic biomass, sorghum also requires pretreatment which increases its susceptibility to hydrolysis by enzymes for generating sugars which can be further fermented to alcohol. In the present study, sorghum biomass was evaluated for deriving maximum fermentable sugars by optimizing various pretreatment parameters using statistical optimization methods. Pretreatment studies were done with H2SO4, followed by enzymatic saccharification. The efficiency of the process was evaluated on the basis of production of the total reducing sugars released during the process. Compositional analysis was done for native as well as pretreated biomass and compared. The biomass pretreated with the optimized conditions could yield 0.408 g of reducing sugars /g of pretreated biomass upon enzymatic hydrolysis. The cellulose content in the solid portion obtained after pretreatment using optimised conditions was found to be increased by 43.37% with lesser production of inhibitors in acid pretreated liquor.

Production, purification and properties of fungal chitinases—A review.

After cellulose, chitin is the second most abundant organic and renewable polysaccharide in nature. This polymer is degraded by enzymes called chitinases which are a part of the glycoside hydrolase family. Chitinases have many important biophysiological functions and immense potential applications especially in control of phytopathogens, production of chito-oligosaccharides with numerous uses and in treatment and degradation of chitinous biowaste. At present many microbial sources are being explored and tapped for chitinase production which includes potential fungal cultures. With advancement in molecular biology and gene cloning techniques, research on fungal chitinases have made fast progress. The present review focuses on recent advances in fungal chitinases, containing a short introduction to types of chitinases, their fermentative production, purification and characterization and molecular cloning and expression.

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.

Causes of Male Re-canalisation (Vasovasostomy (Reversal Surgery)) in Vasectomy Client in Nepal.

Re-canalisation is the process of restoring flow to or reuniting an interrupted channel of a bodily tube (as a blood vessel or vas deferens) In Nepal in 2012–2013 surgeons performed 20,588 vasectomies with an incidence of 1.64 new acceptors per 100 women of reproductive age. Divorce rate is getting high as women are empowered (education, job), they can depend on themselves. The purpose of the study was two-fold. Firstly it was to assess the suitability for male recanalization and factors predicting for recanalization. Secondly, it was to analyze the reasons following vasectomies recanalization.Both qualitative and quantitative methods was used with 18 Key in-depth Interview, 111 men at a FPAN central clinic lalitpur and Valley branch Koteshwor seeking vasectomy sterilization reversal between May 2011 and May 2014.Results showed that individual excised the Re-canalisation ranged from 27 to 54 years with median age of 38. On the address of the respondents for the recanalization 66.7 percent were from outside valley followed by 33.3 percent from inside valley. On the cause of recanalization, 51.7% of the respondents were second marriage followed by 27.6%. The association between Children and cause of recanalization is statistically significant (P=0.046) with Cramer’s strength of association of 56.4%. Labor migrant are main restorant due to divorce,change of life style and the income.It was concluded that the commonest cause of the recanalization is second marriage. The risk of recanalization and its implications should be explained to the patient.

Extracellular methionine amino peptidase (MAP) production by Streptomyces gedanensis in solid-state fermentation

A bioprocess was developed for extracellular MAP production from Streptomyces gedanensis by solid-state fermentation. Response surface methodology of Box Behken Design was performed to evaluate the interaction effects of most significant variables {inoculum size, (NH4)2SO4 concentration, MgSO4.7H2O and tryptone) on MAP production after the single parameter optimization and it resulted a maximum MAP production of 55.26 IU/g PUF after 120 h of fermentation. The concentrated crude MAP displayed a pH and temperature optimum of 8.5 and 50°C. By analyzing the thermal stability, the MAP was found to be stable in a temperature range of 50 to 55°C but lost about 50% of its activity at 65°C after 30 min. This is a first report of this kind of study for MAP.

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, purification, characterization and over-expression of xylanases from actinomycetes.

Xylanases are a group of depolymerizing enzymes often used for the hydrolysis of xylan (present in hemicellulose) to monomeric sugars and comprise endo-xylanases (EC 3.2.1.8) and β-xylosidases (EC 3.2.1.37). They often act in synergy with other enzymes for complete hydrolysis of hemicellulose. Xylanases find several industrial applications, for example in food and feed industries, paper and pulp industries and more recently have acquired a great role in biomass to biofuels program. Bacteria and fungi can best produce xylanases. Recent developments in rDNA technology have resulted in molecular cloning and expression of xylanases in heterologous and homologous hosts. In view of significance of the actinomycetes for the production of biotechnological products, attempts have been made in recent years to explore them for the production of industrial enzymes, including xylanses, aiming to find the enzyme with novel features. This review provides the state-of-art information and developments on the xylanases from actinomycetes, presenting the production, purification, characterization and over-expression from various actinomycetes cultures.

Recent developments in microbial oils production: a possible alternative to vegetable oils for biodiesel without competition with human food?

Since centuries vegetable oils are consumed as human food but it also finds applications in biodiesel production which is attracting more attention. But due to being in competition with food it could not be sustainable and leads the need to search for alternative. Nowdays microbes-derived oils (single cell oils) seem to be alternatives for biodiesel production due to their similar composition to that of vegetable oils. However, the cold flow properties of the biodiesel produced from microbial oils are unacceptable and have to be modified by an efficient transesterification. Glycerol which is by product of transesterification can be valorised into some more useful products so that it can also be utilised along with biodiesel to simplify the downstream processing. The review paper discusses about various potent microorganisms for biodiesel production, enzymes involved in the lipid accumulation, lipid quantification methods, catalysts used in transesterification (including enzymatic catalyst) and valorisation of glycerol.

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.

Arginine specific aminopeptidase from Lactobacillus brevis

The proteolytic system of lactic acid bacteria contribute to the development of flavor during the ripening of cheese through the generation of short peptides and free amino acids, which directly or indirectly act as flavor precursors. Newly isolated lactic acid bacteria (LAB) as well as those procured from culture collection centers were screened for the production of various substrate specific aminopeptidases. Among all the strains screened, L. brevis (NRRL B-1836) was found to produce quantifiable amount of intracellular arginine specific aminopeptidase (EC 3.4.11.6). The productivity of arginine aminopeptidase in 5 L fermentor was 36 IU/L/h. The Luedeking and Piret model was tested for intracellular production of aminopeptidase and the data seemed to fit well, as the correlation coefficient was 0.9964 for MRS. The αAP and βAP was 0.4865 and 0.0046, respectively in MRS medium indicating that the yield was predominantly depended on growth. The culture produced lactic acid and also tolerated pH 2.0-3.0 and 0.3-0.5 percent bile salts, the most important probiotic features.

Arginine specific aminopeptidase from Lactobacillus brevis

The proteolytic system of lactic acid bacteria contribute to the development of flavor during the ripening of cheese through the generation of short peptides and free amino acids, which directly or indirectly act as flavor precursors. Newly isolated lactic acid bacteria (LAB) as well as those procured from culture collection centers were screened for the production of various substrate specific aminopeptidases. Among all the strains screened, L. brevis (NRRL B-1836) was found to produce quantifiable amount of intracellular arginine specific aminopeptidase (EC 3.4.11.6). The productivity of arginine aminopeptidase in 5 L fermentor was 36 IU/L/h. The Luedeking and Piret model was tested for intracellular production of aminopeptidase and the data seemed to fit well, as the correlation coefficient was 0.9964 for MRS. The αAP and βAP was 0.4865 and 0.0046, respectively in MRS medium indicating that the yield was predominantly depended on growth. The culture produced lactic acid and also tolerated pH 2.0-3.0 and 0.3-0.5 percent bile salts, the most important probiotic features.