Shashwati Ghosh Sachan

Prospecting cold deserts of north western Himalayas for microbial diversity and plant growth promoting attributes

Microbial communities in different samples collected from cold deserts of north western Himalayas, India, were analyzed using 16S rRNA gene sequencing and phospholipid fatty acids (PLFA) analysis. A total of 232 bacterial isolates were characterized employing 16S rDNA-Amplified Ribosomal DNA Restriction Analysis with the three restriction endonucleases Alu I, Msp I and Hae III, which led to formation of 29-54 groups for the different sites, adding up to169 groups. 16S rRNA gene based phylogenetic analysis, revealed that 82 distinct species of 31 different genera, belonged to four phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. PLFA profiling was performed for concerned samples which gave an estimate of microbial communities without cultivating the microorganisms. PLFA analysis led to characterization of diverse group of microbes in different samples such as gram-negative, gram-positive bacteria, actinomycetes, cyanobacteria, anaerobic bacteria, sulphate reducing bacteria and fungi. The representative strains were screened for their plant growth promoting attributes, which included production of ammonia, HCN, gibberellic acid, IAA and siderophore; solubilization of phosphorus and activity of ACC deaminase. In vitro antifungal activity assay was performed against Rhizoctonia solani and Macrophomina phaseolina. Cold adapted microorganisms may serve as inoculants for crops growing under cold climatic conditions. To our knowledge, this is the first report for the presence of Arthrobacter nicotianae, Brevundimonas terrae, Paenibacillus tylopili and Pseudomonas cedrina in cold deserts and exhibit multifunctional PGP attributes at low temperatures.

Cold active hydrolytic enzymes production by psychrotrophic Bacilli isolated from three sub-glacial lakes of NW Indian Himalayas.

The diversity of culturable, cold‐active enzymes producing Bacilli was investigated from three sub‐glacial lakes of north western Indian Himalayas. Amplified ribosomal DNA restriction analysis (ARDRA) using three restriction enzymes Alu I, Msp I, and Hae III led to the clustering of 136 Bacilli into 26, 23, and 22 clusters at 75% similarity index from Chandratal Lake, Dashair Lake, and Pangong Lake, respectively. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 35 Bacilli that could be grouped in seven families viz.: Bacillaceae (48%), Staphylococcaceae (14%), Bacillales incertae sedis (13%), Planococcaceae (12%), Paenibacillaceae (9%), Sporolactobacillaceae (3%), and Carnobacteriaceae (1%), which included twelve different genera Bacillus, Desemzia, Exiguobacterium, Jeotgalicoccus, Lysinibacillus, Paenibacillus, Planococcus, Pontibacillus, Sinobaca, Sporosarcina, Staphylococcus, and Virgibacillus. Based on their optimal temperature for growth, 35 Bacilli were grouped as psychrophilic (11 strains), psychrotrophic (17 strains), or psychrotolerant (7 strains), respectively. The representative isolates from each cluster were screened for cold‐active enzyme activities. Amylase, β‐glucosidase, pectinase, and protease activities at 4 °C were detected in more than 80% of the strains while approximately 40, 31, 23, 14, 11, and 9% of strains possessed cellulase, xylanase, β‐galactosidase, laccase, chitinase, and lipase activity, respectively. Among 35 Bacilli, Bacillus amyloliquefaciens, Bacillus marisflavi, Exiguobacterium indicum, Paenibacillus terrae, Pontibacillus sp., Sporosarcina globispora, and Sporosarcina psychrophila were efficient producers of different cold‐active enzymes. These cold‐adapted Bacilli could play an important role in industrial and agricultural processes.

Production of natural value-added compounds: an insight into the eugenol biotransformation pathway

During the past few years, the production of natural value-added compounds from microbial sources has gained tremendous importance. Due to an increase in consumer demand for natural products, various food and pharmaceutical industries are continuously in search of novel metabolites obtained from microbial biotransformation. The exploitation of microbial biosynthetic pathways is both feasible and cost effective in the production of natural compounds. The environmentally compatible nature of these products is one major reason for their increasing demand. Novel approaches for natural product biogeneration will take advantage of the current studies on biotechnology, biochemical pathways and microbiology. The interest of the scientific community has shifted toward the use of microbial bioconversion for the production of valuable compounds from natural substrates. The present review focuses on eugenol biotransformation by microorganisms resulting in the formation of various value-added products such as ferulic acid, coniferyl alcohol, vanillin and vanillic acid.

Transformation of ferulic acid to 4-vinyl guaiacol as a major metabolite: a microbial approach

The majority of the flavours and fragrances used worldwide are produced by chemical synthesis at low price. However, consumers prefer natural compounds because of increasing health and nutrition awareness in routine life. Hence, biotransformation is an alternative process to produce natural aroma compounds. Microorganisms have been gradually used more to produce natural aroma compounds with various applications in food, agriculture and pharmaceutical industries. This paper reviews the role of microorganisms in the transformation of ferulic acid to 4-vinyl guaiacol. The microbial processes based on biocatalytic method are discussed in terms of their advantages over chemical synthesis, plant cell cultures and enzyme catalyzed reactions. Thus, the transformation of ferulic acid by microorganisms could have possible use in food, pharmaceutical industry and become an increasingly important platform for the production of natural aroma compounds.

Analysis of gallstone composition and structure in Jharkhand region.

AimThe aim of this study was to analyze gallstones structurally and chemically as this may help to direct the measures for its treatment.MethodologyOn the basis of morphology, 459 gallstones were categorized into pigmented, cholesterol, and mixed gallstones and analyzed for their chemical structure and composition. Elementary analysis was done with the help of inductively coupled plasma, optical emission spectrophotometry. Fourier transform infrared spectroscopy was used for compound analysis. The effects of thermal stability were analyzed by thermal gravimetric analysis (TGA). Statistical analysis was done to correlate gallstone composition with their shape and number in gallbladder.ResultsPigmented gallstones were predominant and generally occurred in multiple forms, cholesterol as solitaire and black pigmented as slug (χ2 = 3.56; p < 0.001). Scanning electron microscopy showed the crystals of cholesterol in cholesterol gallstones while pigmented gallstones were more compact in structure. Both types of structure were seen in mixed stones while black pigmented stones were amorphous in nature. This difference in structures might be due to difference in chemical compositions. Cholesterol and mixed stones contained basically cholesterol, brown pigmented constituted bilirubin as a major component, and black stones differed from brown pigmented stones by the presence of Ca palmitate. Bilirubin and palmitate were thermally more stable than cholesterol; hence pigmented gallstones were thermally more stable than cholesterol gallstones, as seen in TGA study.ConclusionPigmented gallstones were most common in this study of gallstones from the Jharkhand region.

Biosurfactants: A Multifunctional Microbial Metabolite

During the 1960s, the science of biosurfactants was in its infancy and received attention mainly as hydrocarbon dissolving agents. Recently biosurfactants have emerged as the most versatile product of the modern microbial biotechnology. Owing to its biodegradable nature, it has been considered as “green chemical.” Recently, increasing awareness toward sustainable ecosystem and environmental protection has resulted in a concerted research in biosurfactant as a promising substitute of chemical surfactants. It can be produced from nonrenewable resources, with alternative synthesis from economical renewable feedstocks. Apart from the stability at relatively adverse environments, these compounds are readily biodegradable in the environment. Functional properties of these compounds are being conferred by their structural diversity which varies among the microbial community. These compounds may provide advantages in a particular ecological niche or may be responsible for the niche-specific behavior of the producer microorganism. Diverse properties encompassed by biosurfactants are wetting, dispersion, emulsification, foaming, cleansing, phase separation, reduction in viscosity, and surface activity. It serves a broad spectrum of industries like petroleum, paint, paper, textile, leather, agriculture, cosmetic, food processing, agriculture, and pharmaceutical industries, thereby plays a significant role in white biotechnology.

Microbial production of 4-vinylguaiacol from ferulic acid by Bacillus cereus SAS-3006

Abstract Ferulic acid is an abundant cinnamic acid derivative found in the plant kingdom. It is a commercially available substrate utilized to produce flavor compounds such as 4-vinylguaiacol (4-VG), vanillin, and vanillic acid. The isolate Bacillus cereus SAS-3006 was screened and selected based on its ability to produce 4-VG upon ferulic acid biotransformation. It was identified based on morphological and physiochemical characteristics and its 16S ribosomal DNA sequence (GenBank accession number: KF699134). A maximum amount (79.4 mg/L) of 4-VG accumulation was observed on the 5th day of incubation when the culture was grown on 2.5 mM ferulic acid as sole carbon source. Further conversion of 4-VG to other intermediates such as vanillin, vanillic acid, protocatechuic acid, acetovanillone, and vanillyl alcohol was not observed. In-vitro conversion of ferulic acid to 4-VG was also studied with cell extracts of B. cereus SAS-3006. The present study provides the first evidence for production of 4-VG as the sole product using B. cereus SAS-3006.

Psychrotrophic Microbiomes: Molecular Diversity and Beneficial Role in Plant Growth Promotion and Soil Health

Prospecting the cold habitats has led to the isolation of a great diversity of psychrotrophic microbes belonging to different groups. The cold-adapted microbes have potential biotechnological applications in agriculture, medicine, and industry as they can produce cold-adapted enzymes (amylase, cellulase, chitinase, laccase, lipase, pectinase, protease, xylanase, β-galactosidase, and β-glucosidase), antifreezing compounds, and antibiotics and possess diverse multifunctional plant growth-promoting attributes (production of ammonia, hydrogen cyanide, indole-3-acetic acid, and siderophores; solubilization of phosphorus, potassium, and zinc; 1-aminocyclopropane-1-carboxylate deaminase activity and biocontrol activity against plant pathogenic microbes). Cold-adapted microbes are ubiquitous in nature and have been reported from Antarctica, permanently ice-covered lakes, cloud droplets, ice cap cores from considerable depth, snow, glaciers, and those associated with plants growing in cold habitats. Cold-adapted microbial communities can be studied using culture-dependent and culture-independent techniques. Microbes recovered using both techniques revealed the occurrence of different and diverse major groups, viz., Actinobacteria, Ascomycota, Bacteroidetes, Basidiomycota, Chlamydiae, Chloroflexi, Cyanobacteria, Euryarchaeota, Firmicutes, Gemmatimonadetes, Mucoromycota, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Thaumarchaeota, and Verrucomicrobia. On the review of isolated cold-adapted microbes, it was found that Proteobacteria was the most dominant phylum followed by Firmicutes and Actinobacteria. This book chapter deals with the isolation, characterization, and biodiversity of cold-tolerant microbes from Antarctica; Himalayan cold desert; glaciers; ice-capped rivers; plant-associated, subalpine region of Uttarakhand; and different sub-glacial lakes. The biotechnological applications of cold-adapted microbes have been discussed. The benificial and potential cold-adapted microbes may have applications in diverse processes in agriculture, industry, and allied sectors.

Mutational analysis of phenolic acid decarboxylase from Enterobacter sp. Px6-4. towards enhancement of binding affinity: A computational approach

Microbial Ferulic Acid Decarboxylase (FADase) catalyses the conversion of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol) via non-oxidative decarboxylation. In this article, we present a computational, three-dimensional structural and functional analysis of FADase from Enterobacter sp. P × 6-4 (3NX1) which can be used to generate enhanced bindings of substrates. The enzymatic catalytic site and binding sites have been critically evaluated. Sequential site directed mutations on enzyme have also been introduced for formation of a greater number of hydrogen bonds. Four mutants were generated based on our hypothesis. Active sites of mutated FADases have been analyzed with dynamic cross-correlation maps and principle components analysis. All structures were validated and optimized through energy minimization. Docking studies were also carried out between ferulic acid and different mutated enzymes. The protein (wild and mutants) complexes were further validated with molecular simulation. Mutant3 was found to have better affinity towards ferulic acid. Mutant3 also forms a higher number of hydrogen bonds with the substrate to facilitate greater interaction. This current work will help industry to create new and novel mutants to produce vanillin.

Ferulic Acid Decarboxylase from Bacillus cereus SAS-3006: Purification and Properties

The cell-free extract of Bacillus cereus SAS-3006 strain grown on the medium containing ferulic acid was used as a source of the enzyme ferulic acid decarboxylase. This key enzyme, playing a vital role in the conversion of ferulic acid, was purified to homogeneity by anion-exchange chromatography from Bacillus cereus SAS-3006. The optimal pH and temperature for enzyme activity were found to be 6.5 and 37 °C, respectively. Kinetic studies indicated the K m and V max values of the purified ferulic acid decarboxylase were 0.0118 mM and 0.333 U, respectively. The enzyme activity was not inhibited by any of the studied metal ions viz. Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, and Zn2+ at 2.0 mM of concentration. The enzyme was found to be activated and expressed inside the cells adequately by the substrate ferulic acid.