Complete genome sequence analysis of a plant growth-promoting phylloplane Bacillus altitudinis FD48 offers mechanistic insights into priming drought stress tolerance in rice.

Bacillus altitudinis FD48 is a multifunctional plant growth-promoting bacterium isolated from the phylloplane of rice and survives at --10 bars of osmotic potential (--1.0 MPa). It also serves as an ideal PGPM against drought stress by triggering antioxidant defense mechanisms in rice. To further unravel the genetic determinants of osmotic stress tolerance and plant growth-promoting traits, the whole genome sequence of FD48 was compared with its related strains. The whole genome analysis revealed a single chromosome with a total length of 3,752,533 bp (3.7 Mb) and an average G + C ratio of 41.19%. A total of 4029 genes were predicted, of which 3964 (98.4%) were protein-encoding genes (PEGs) and 65 (1.6%) were non-protein-coding genes. The interaction of FD48 with the host plants is associated with many chemotactic and motility-related genes. The ability of FD48 to colonize plants and maintain plant growth under adverse environmental conditions was evidenced by the presence of genes for plant nutrient acquisition, phytohormone synthesis, trehalose, choline, and glycine betaine biosynthesis, microbial volatile organic compounds (acetoin synthesis), heat and cold shock chaperones, translation elongation factor TU (Ef-Tu), siderophore production, DEAD/DEAH boxes, and non- ribosomal peptide synthase clusters (bacilysin, fengycin, and bacitracin). This study sheds light on the drought stress-resilient mechanism, metabolic pathways and potential activity, and plant growth-promoting traits of B. altitudinis FD48 at the genetic level.

Optimization and characterization of laccase (LccH) produced by Hexagonia hirta MSF2 in solid-state fermentation using coir pith wastes (CPW).

The accumulation of coir pith waste, a byproduct of coconut husk processing, poses environmental and logistical challenges. An innovative and sustainable solution involves using coir pith as a substrate for solid-state fermentation (SSF). In SSF, coir pith can be converted into valuable products, such as enzymes, organic acids, and bioactive compounds. The present study aimed to evaluate laccase production by Hexagonia hirta MSF2 through SSF using the coir pith waste as substrate. Physico-chemical parameters like moisture, pH, temperature, C source, N source, and CuSO4 concentrations were pre-optimized, and optimized through RSM. Laccase activity of 1585.24 U g-1 of dry substrate was recorded by H. hirta MSF2 on coir pith containing 1 % C source, 0.5 % N source, 0.25 mM of CuSO4 concentration, moisture content of 75 % at pH 4.6 and temperature 28 °C. Subsequently, the enzyme extraction parameters including, extraction buffer, mode of extraction, and temperature were optimized. The molecular weight of laccase was 66 kDa as observed by SDS-PAGE and native-PAGE. The optimum activity of partially purified laccase was achieved at 40 °C, and pH 4.0. Increasing salt concentration and use of different inhibitors affected the laccase activity. Organic solvents like dimethyl sulphoxide (DMSO) and methanol, and metal ions like BaCl2, CaCl2, CuSO4, and MnCl2 stimulated the laccase activity. Hence, coir pith used in SSF offers a dual benefit of waste management and enzyme synthesis through an eco-friendly and cost-effective approach.

Activity of volatiles induced by microbes and natural plants stifled the growth of Pythium aphanidermatum - the damping off in Tomato.

BACKGROUND: Volatilomes from natural plants and microbes imparts diverse antifungal properties to suppress the growth of plant pathogens and therefore can be a suitable alternative of chemical fungicides. The present experiment was to study effect of volatiles produced by natural plants and microbes on the fungal growth of Pythium aphanidermatum, which is a tomato seedling pathogen. RESULTS: Isolate of P. aphanidermatum, causing damping off in tomato were isolated and incubated at 25 ± 2 °C. The isolate was tested for the anti-oomycetes activities of volatiles in vitro. The volatiles produced by the leaves of Mentha spicata and Cymbopogon citratus showed the maximum inhibitory effect of 45.56 and 24.70 percent, respectively on the mycelial growth of P. aphanidermatum, whereas, the pathogen was not inhibited on exposure to the volatiles of macro-basidiomycetes fungi. The volatiles of T. asperellum showed the maximum inhibitory effect of 69.26 percent against P. aphanidermatum. The study also included the identification of Volatile Organic Compounds (VOCs) involved in the suppression of pathogens by Headspace Gas Chromatography Mass Spectrometry (HS GCMS). The results revealed the production of carvone by the leaves of M. spicata; citronellol and geraniol by C. citratus; isopentyl alcohol and limonene by T. asperellum with increased peak area percentage and these compounds possessed antifungal properties. The vaporous action of isopentyl alcohol completely suppressed the mycelial growth of P. aphanidermatum, which is highly correlated to the T. asperellum extract on pathogenic growth. While the compounds, carvone, and citronellol showed the maximum inhibitory effect of 89.02 and 85.49 percent, respectively when used at 500 ppm and also altered the sporulation behavior of P. aphanidermatum. CONCLUSION: Results showed that volatiles of M. spicata and T. asperellum have anti-oomycetes action on pathogenic growth leading to a distortion of sporulation of P. aphanidermatum. High antifungal properties make VOCs suitable for incorporation as a new integrated plant disease management programs.

Flagellin, a plant-defense-activating protein identified from Xanthomonas axonopodis pv. Dieffenbachiae invokes defense response in tobacco.

BACKGROUND: Secretome analysis is a valuable tool to study host-pathogen protein interactions and to identify new proteins that are important for plant health. Microbial signatures elicit defense responses in plants, and by that, the plant immune system gets triggered prior to pathogen infection. Functional properties of secretory proteins from Xanthomonas axonopodis pv. dieffenbachiae (Xad1) involved in priming plant immunity was evaluated. RESULTS: In this study, the secretome of Xad1 was analyzed under host plant extract-induced conditions, and mass spectroscopic analysis of differentially expressed protein was identified as plant-defense-activating protein viz., flagellin C (FliC). The flagellin and Flg22 peptides both elicited hypersensitive reaction (HR) in non-host tobacco, activated reactive oxygen species (ROS) scavenging enzymes, and increased pathogenesis-related (PR) gene expression viz., NPR1, PR1, and down-regulation of PR2 (ß-1,3-glucanase). Protein docking studies revealed the Flg22 epitope of Xad1, a 22 amino acid peptide region in FliC that recognizes plant receptor FLS2 to initiate downstream defense signaling. CONCLUSION: The flagellin or the Flg22 peptide from Xad1 was efficient in eliciting an HR in tobacco via salicylic acid (SA)-mediated defense signaling that subsequently triggers systemic immune response epigenetically. The insights from this study can be used for the development of bio-based products (small PAMPs) for plant immunity and health.

Influence of Novel EnZolv Pretreatment on the Release of Reducing Sugar and Proximate Content of Banana Fiber.

Lignocellulosic biomass (LCB) from agriculture residues has gained a lot of attention in recent years for its conversion to useful by-products. The one drawback that the conversion of biomass faces is its recalcitrant nature which can be overcome by effective pretreatment technology. One such process is the EnZolv, a novel pretreatment technique used for delignification of biomass and it was recognized as an eco-friendly approach. The main objective of our present study is to optimize the novel EnZolv process parameters for enhanced release of reducing sugar from banana fiber. Banana fiber pre-optimization for EnZolv pretreated at 100% moisture content, incubated at 40 °C temperature, with an enzyme load of 50 U·g-1 of biomass for an incubation time of 5 h at a shaking speed of 100 rpm yielded enhanced sugar release of 1.7 mg·mL-1. The effect of pretreatment on proximate composition results in a decrease in the volatile matter (53%) and moisture percentage (1.07%) and an increase in the other parameters such as ash content (12%) and fixed carbon content (34%) under the optimized condition. A significantly higher release of phenol content 1264 µg·mL-1 equivalent to gallic acid suggests that EnZolv pretreatment confirms the degradation of lignin content in the biomass. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01130-4.

Lovastatin production by an oleaginous fungus, Aspergillus terreus KPR12 using sago processing wastewater (SWW).

BACKGROUND: Lovastatin is one of the first statins to be extensively used for its cholesterol-lowering ability. It is commercially produced by fermentation. Species belonging to the genus Aspergillus are well-studied fungi that have been widely used for lovastatin production. In the present study, we produced lovastatin from sago processing wastewater (SWW) under submerged fermentation using oleaginous fungal strains, A. terreus KPR12 and A. caespitosus ASEF14. RESULTS: The intra- and extracellular concentrations of lovastatin produced by A. terreus KPR12 and A. caespitosus ASEF14 were lactonized. Because A. caespitosus ASEF14 produced a negligible amount of lovastatin, further kinetics of lovastatin production in SWW was studied using the KPR12 strain for 9 days. Lovastatin concentrations in the intra- and extracellular fractions of the A. terreus KPR12 cultured in a synthetic medium (SM) were 117.93 and 883.28 mg L-1, respectively. However, these concentrations in SWW were 142.23 and 429.98 mg L-1, respectively. The yeast growth inhibition bioassay confirmed the antifungal property of fungal extracts. A. terreus KPR12 showed a higher inhibition zone of 14 mm than the ASEF14 strain. The two-way analysis of variance (ANOVA; p < 0.01) showed significant differences in the localization pattern, fungal strains, growth medium, and their respective interactions. The lovastatin yield coefficient values were 0.153 g g-1 on biomass (YLOV/X) and 0.043 g g-1 on the substrate, starch (YLOV/S). The pollutant level of treated SWW exhibited a reduction in total solids (TS, 59%), total dissolved solids (TDS, 68%), biological oxygen demand (BOD, 79.5%), chemical oxygen demand (COD, 57.1%), phosphate (88%), cyanide (65.4%), and void of nutrients such as nitrate (100%), and ammonia (100%). CONCLUSION: The starch-rich wastewater serves as a suitable medium for A. terreus KPR12 for the production of lovastatin. It simultaneously decontaminates the sago processing wastewater, enabling its reuse for irrigation/recreation.

Glycosyl hydrolase 11 (xynA) gene with xylanase activity from thermophilic bacteria isolated from thermal springs.

BACKGROUND: Hemicellulose is one of the copious polymer in lignocellulosic biomass (LCB). It is primarily composed of xylan linked by ß-1,4 glycosidic bonds. Xylanase preferentially cleaves the ß-1,4-glycosidic bonds in the xylan backbone resulting in complete hydrolysis of the biomass. Thermostable variants of glycoside hydrolases act as robust catalysts, not only in degradation but also during processing, to obtain specific carbohydrate-containing chemicals and materials (Ramasamy et al. in Madras Agric J 107(special):1. https://doi.org/10.29321/MAJ.2020.000382 , 2020). RESULTS: The xylanase production by two thermophilic bacteria isolated from thermal springs was evaluated. In addition, the gene encoding this industrially vital enzyme was isolated and characterized, and its protein structure was analyzed. The thermophilic bacteria producing xylanases were isolated from augmented sawdust and banana fiber biomass from hot springs of Himachal Pradesh and identified as Bacillus subtilis VSDB5 and Bacillus licheniformis KBFB4 using 16S rRNA gene sequencing. The persistent xylanase activity revealed that the enzyme is secreted extracellularly with the maximum activity of 0.76 IU mL-1 and 1.0 IU mL-1 at 6 h and 12 h of growth by KBFB4 and VSDB5, respectively, under submerged fermentation. Both the strains exhibited the maximum activity at pH 6 and a temperature of 50 °C. The xylanases of KBFB4 and VSDB5 were thermostable and retained 40% of their activity at 60 °C after incubation for 30 min. Xylanase of VSDB5 had wide thermotolerance and retained 20% of its activity from 60 to 80 °C, whereas xylanase of KBFB4 showed wide alkali tolerance and retained 80% of its activity until pH 10. The xylanase (xynA)-encoding gene (650 bp) cloned from both the strains using specific primers showed 98 to 99% homology to ß-1,4-endoxylanase gene. Further in silico analysis predicted that the xylanase protein, with a molecular weight of 23 kDa, had a high pI (9.44-9.65), which explained the alkaline nature of the enzyme and greater aliphatic index (56.29). This finding suggested that the protein is thermostable. Multiple sequence alignment and homology modeling of the protein sequence revealed that the gene product belonged to the GH11 family, indicating its possible application in bioconversion. CONCLUSION: The strains B. subtilis VSDB5 and B. licheniformis KBFB4 obtained from hot springs of Himachal Pradesh produced potent and alkali-tolerant thermostable xylanases, which belong to the GH11 family. The enzyme can be supplemented in industrial applications for biomass conversion at high temperatures and pH (or in processes involving alkali treatment).

A 16S rRNA amplicon approach to the structural and functional diversity of bacterial communities associated with horse gram crop for drought mitigation and sustainable productivity.

AIM: In this study, 16S rRNA amplicon sequencing analyses were performed to determine the diversity of the bacterial community present in the soil, rhizosphere region, root nodules and seeds of the horse gram plant. METHODS AND RESULTS: We observed the dominance of Proteobacteria, Actinobacteria, Firmicutes, Acidobacteria, Bacteroidetes, Planctomycetes and Gemmatimonadetes across all four domains of the horse gram plant. For community analyses, the significance of the alpha diversity was estimated using the Shannon index, Simpson index and Chao1 index, which revealed no significant difference among the samples. However, the estimation of the beta diversity indicated a significant difference among the samples, with p < 0.001 and R2  = 1. A strong positive correlation was found between the rhizosphere and root nodule samples. Comparative genomics of the 16S rRNA gene showed that ammonium-oxidizing metabolism (amoA), nitrite-reducing metabolism (nirK) and nitrogen-fixing metabolism (nifH) were prominent mechanisms in all samples. The genes involved in the biosynthesis of amino acids, purine metabolism and nitrogen metabolism were identified as the key genes associated with the functional traits of microbial domains in horse gram. CONCLUSION: The culturable microbes associated with horse gram can be used as a substitute for synthetic fertilizers to maintain soil fertility and ecological health in agricultural practices. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the survival strategies of bacterial communities that positively respond to multiple gate selection helps in understanding the structural diversity and functional traits primarily focused on the development of beneficial microbial consortium for promoting plant growth.

Antifungal Volatilomes Mediated Defense Mechanism against Fusarium oxysporum f. sp. lycopersici, the Incitant of Tomato Wilt.

In this study, the volatilomes of naturally growing plant leaves were immobilized in a suitable substrate to enhance vapors' diffusion in the soil to eradicate the Fusarium wilt pathogens in Tomato. Volatilomes produced by Mentha spicata leaves immobilized in vermiculite ball was found to be effective and exhibit 92.35 percent inhibition on the mycelial growth of Fusarium oxysporum f. sp. lycopersici (FOL). Moreover, the volatilomes of M. spicata immobilized vermiculite balls were tested based on the distance traveled by the diffused volatilomes from the ball and revealed that the volatilomes of M. spicata traveled up to 20 cm distance from the center of PVC (Polyvinly chloride) chamber showed maximum reduction in colony growth of FOL at 12th day after inoculation. Tomato plants inoculated with FOL revealed increased expressions of defense gene, pathogenesis related protein (PR1) with 2.63-fold after 72 h and the gene, transcription factor (WRKY) increased with 2.5-fold after 48 h on exposure to the volatilomes of M. spicata vermiculite balls. To the best of our knowledge, this is the first report on development of volatilomes based vermiculite ball formulations. This result indicated that the volatilomes of M. spicata are promising phyto-fumigants for management of Tomato Fusarial wilt.

Optimization and scale-up of α-amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes.

BACKGROUND: Amylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box-Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied. RESULTS: The substrate optimization for α-amylase production by the Box-Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae, as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L. CONCLUSIONS: The process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect (p < 0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

Tyrosinase and laccase-producing Bacillus aryabhattai TFG5 and its role in the polymerization of phenols.

BACKGROUND: Tyrosinases and laccases are oxidoreductase enzymes that are used widely in the food, feed, textile, and biofuel industries. The rapidly growing industrial demand for bacterial oxido-reductases has encouraged research on this enzyme worldwide. These enzymes also play a key role in the formation of humic substances (HS) that are involved in controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants besides increasing carbon sequestration and mitigating greenhouse gas emission in the environment. The present study aimed to screen and characterize extracellular tyrosinase and laccase-producing soil bacteria that could be utilized in the polymerization of phenols. RESULTS: Twenty isolates from different soil samples collected from forest ecosystems were characterized through ARDRA using restriction digestion with AluI, HpaII, and HaeIII restriction enzymes. The results of Hierarchical Cluster Analysis (HCA) revealed a 60 % similarity coefficient among 13 out of 20 isolates, of which, the isolate TFG5 exhibited only 10 % similarity when compared to all the other isolates. The isolate TFG5 exhibited both tyrosinase (1.34 U.mL- 1) and laccase (2.01 U.mL- 1) activity and was identified as Bacillus aryabhattai. The increased polymerization activity was observed when B. aryabhattai TFG5 was treated with phenols. The monomers such as catechol, p-Hydroxy benzoic acid, ferulic acid, and salicylic acid were polymerized efficiently, as evidenced by their FT-IR spectra depicting increased functional groups compared to the standard mushroom tyrosinase. CONCLUSIONS: The polymerization ability of B. aryabhattai TFG5 could be applied to phenol-rich wastewater treatment for efficient precipitation of phenols. Furthermore, tyrosinases can be used for enhancing the synthesis of HS in soil.

Bioconversion of sago processing wastewater into biodiesel: Optimization of lipid production by an oleaginous yeast, Candida tropicalis ASY2 and its transesterification process using response surface methodology.

BACKGROUND: Biodiesel is an eco-friendly and renewable energy source and a valuable substitute for petro-diesel. Sago processing wastewater (SWW), a by-product of the cassava processing industry, has starch content ranging from 4 to 7 g L-1 and serves as an outstanding source for producing microbial lipids by the oleaginous microorganisms. In the present study, Candida tropicalis ASY2 was employed to optimize single-cell oil (SCO) production using SWW and subsequent transesterification by response surface methodology. Variables such as starch content, yeast extract, airflow rate, pH, and temperature significantly influenced lipid production in a preliminary study. The lipid production was scaled up to 5 L capacity airlift bioreactor and its optimization was done by response surface methodology. The dried yeast biomass obtained under optimized conditions from 5 L bioreactor was subjected to a direct transesterification process. Biomass: methanol ratio, catalyst concentration, and time were the variables used to attain higher FAME yield in the transesterification optimization process. RESULTS: Under optimized conditions, the highest lipid yield of 2.68 g L-1 was obtained with 15.33 g L-1 of starch content, 0.5 g L-1 of yeast extract, and 5.992 L min-1 of airflow rate in a bioreactor. The optimized direct transesterification process yielded a higher FAME yield of 86.56% at 1:20 biomass: methanol ratio, 0.4 M catalyst concentration, and a time of 6.85 h. CONCLUSIONS: Thus, this optimized process rendered the microbial lipids derived from C. tropicalis ASY2 as potentially alternative oil substitutes for sustainable biodiesel production to meet the rising energy demands.

Bacillus aryabhattai TFG5-mediated synthesis of humic substances from coir pith wastes.

BACKGROUND: Humic substances (HS) form the largest proportion among all the constituents of soil organic matter and are a key component of the terrestrial ecosystem. HS plays a multifunctional role in the environment by controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants. The rate of formation of HS in soils determines its productivity and carbon sequestration capacity. Enhancement of HS synthesis in the soil through the microbial route not only increases CO2 sequestration but also mitigates the greenhouse gas emissions in the environment. RESULT: In this study, we attempted to understand the mechanism of formation and enhancement of HS from coir pith wastes using the tyrosinase produced by Bacillus aryabhattai TFG5. The bacterium TFG5 isolated from the termite garden produced the tyrosinase (1.34 U mL-1) and laccase (2.1 U mL-1) at 48 h and 60 h of fermentation, respectively. The extracellular tyrosinase from B. aryabhattai TFG5 was designated as TyrB. Homology modeling of TyrB revealed a structure with a predicted molecular mass of 35.23 kDa and two copper ions in the active center with its conserved residues required for the tyrosinase activity. TyrB efficiently transformed and polymerized standard phenols, such as p-cresol, p-hydroxyl benzoic acid, Levo DOPA, and 2,6 DMP, besides transforming free phenols in coir pith wash water (CWW). Additionally, UV-Vis and FT-IR spectra of the degradation products of the coir pith treated with TyrB revealed the formation of HS within 3 days of incubation. Furthermore, the E472/664 ratio of the degradation products revealed a higher degree of condensation of the aromatic carbons and the presence of more aliphatic structures in the HS. CONCLUSION: The results confirmed the influence of TyrB for the effective synthesis of HS from coir pith wastes. The results of the present study also confirm the recently accepted theory of humification proposed by the International Humic Substances Society.

Aspergillus caespitosus ASEF14, an oleaginous fungus as a potential candidate for biodiesel production using sago processing wastewater (SWW).

BACKGROUND: Oleaginous microorganisms are sustainable alternatives for the production of biodiesel. Among them, oleaginous fungi are known for their rapid growth, short life cycles, no light requirement, easy scalability, and the ability to grow in cheap organic resources. Among all the sources used for biodiesel production, industrial wastewater streams have been least explored. We used oleaginous fungi to decontaminate sago processing wastewater and produce biodiesel. RESULTS: Among the 15 isolates screened for lipid production and starch utilization using the Nile red staining assay and amylase plate screening, three isolates accumulated > 20% (w/w) of their dry cell mass as lipids. The isolate ASEF14 exhibited the highest lipid accumulation (> 40%) and was identified as Aspergillus caespitosus based on the 28S rRNA gene sequencing. The maximum lipid content of 54.4% in synthetic medium (SM) and 37.2% in sago processing wastewater (SWW) was produced by the strain. The Fourier-transform infrared (FTIR) spectroscopy of the fungal oil revealed the presence of functional peaks corresponding to major lipids. Principal component analysis (PCA) of the FTIR data revealed major changes in the fatty acid composition during the transition from the growth phase (Days 1-3) to the lipid accumulation phase (Days 4-7). The fatty acid methyl esters (FAME) analysis of fungal oil from SWW contained 43.82% and 9.62% of saturated and monounsaturated fatty acids, respectively. The composition and percentage of individual FAME derived from SWW were different from SM, indicating the effect of nutrient and fermentation time. The fuel attributes of the SM- and SWW-grown fungal biodiesel (kinematic viscosity, iodine value, cetane number, cloud and pour point, linolenic acid content, FA > 4 double bonds) met international (ASTM D6751, EN 14214) and national (IS 15607) biodiesel standards. In addition to biodiesel production, the strain removed various contaminants such as total solids (TS), total suspended solids (TSS), total dissolved solids (TDS), dissolved oxygen (DO), chemical oxygen demand (COD), biological oxygen demand (BOD), total nitrogen (TN), total phosphorus (TP), and cyanide up to 58.6%, 53.0%, 35.2%, 94.5%, 89.3%, 91.3%, 74.0%, 47.0%, and 53.84%, respectively, from SWW. CONCLUSION: These findings suggested that A. caespitosus ASEF14 is a potential candidate with high lipid accumulating ability (37.27%), capable of using SWW as the primary growth medium. The medium and incubation time alter the FAME profile of this fungus. The physical properties of fungal oil were in accordance with the biodiesel standards. Moreover, it decontaminated SWW by reducing several polluting nutrients and toxicants. The fungal biodiesel produced by this cost-effective method could serve as an alternate path to meet global energy demand.

Optimized culture conditions for bacterial cellulose production by Acetobacter senegalensis MA1.

BACKGROUND: Cellulose, the most versatile biomolecule on earth, is available in large quantities from plants. However, cellulose in plants is accompanied by other polymers like hemicellulose, lignin, and pectin. On the other hand, pure cellulose can be produced by some microorganisms, with the most active producer being Acetobacter xylinum. A. senengalensis is a gram-negative, obligate aerobic, motile coccus, isolated from Mango fruits in Senegal, capable of utilizing a variety of sugars and produce cellulose. Besides, the production is also influenced by other culture conditions. Previously, we isolated and identified A. senengalensis MA1, and characterized the bacterial cellulose (BC) produced. RESULTS: The maximum cellulose production by A. senengalensis MA1 was pre-optimized for different parameters like carbon, nitrogen, precursor, polymer additive, pH, temperature, inoculum concentration, and incubation time. Further, the pre-optimized parameters were pooled, and the best combination was analyzed by using Central Composite Design (CCD) of Response Surface Methodology (RSM). Maximum BC production was achieved with glycerol, yeast extract, and PEG 6000 as the best carbon and nitrogen sources, and polymer additive, respectively, at 4.5 pH and an incubation temperature of 33.5 °C. Around 20% of inoculum concentration gave a high yield after 30 days of inoculation. The interactions between culture conditions optimized by CCD included alterations in the composition of the HS medium with 50 mL L- 1 of glycerol, 7.50 g L- 1 of yeast extract at pH 6.0 by incubating at a temperature of 33.5 °C along with 7.76 g L- 1 of PEG 6000. This gave a BC yield of wet weight as 469.83 g L- 1. CONCLUSION: The optimized conditions of growth medium resulted in enhanced production of bacterial cellulose by A. senegalensis MA1, which is around 20 times higher than that produced using an unoptimized HS medium. Further, the cellulose produced can be used in food and pharmaceuticals, for producing high-quality paper, wound dressing material, and nanocomposite films for food packaging.

Non-rhizobial endophytic (NRE) yeasts assist nodulation of Rhizobium in root nodules of blackgram (Vigna mungo L.).

The signal orchestration between legumes and the rhizobia attribute to symbiotic nitrogen fixation through nodule formation. Root nodules serve as a nutrient-rich reservoir and harbor diverse microbial communities. However, the existence of non-rhizobial endophytes (NRE) and their role inside the root nodules are being explored; there is no evidence on yeast microflora inhabiting nodule niche. This study focused on unraveling the presence of yeast in the root nodules and their possible function in either nodulation or signal exchange. From the root nodules of blackgram, two yeast strains were isolated and identified as Candida glabrata VYP1 and Candida tropicalis VYW1 based on 18S rRNA gene sequencing and phylogeny. These strains possessed plant growth-promoting traits viz., IAA, ACC deaminase, siderophore, ammonia, and polyamine production. The functional capacity of endophytic yeast strains, and their interaction with Rhizobium sp. was further unveiled via profiling volatile organic compounds (VOC). Among the VOCs, α-glucopyranoside and pyrroloquinoline pitches a pivotal role in activating lectin pathways and phosphorous metabolism. Further, lectin pathways are crucial for nodulating bacterium, and our study showed that these endophytic yeasts assist nodulation by Rhizobium sp. via activating the nod factors. The plant growth-promoting traits of NRE yeast strains coupled with their metabolite production, could recruit them as potential drivers in the plant-microbe interaction.

Antimicrobial activity and spectroscopic characterization of surfactin class of lipopeptides from Bacillus amyloliquefaciens SR1.

A bacterial isolate screened from wet land soil sample, found to posses antimicrobial activity against an array of fungal plant pathogens viz., Rhizoctonia solani, Sclerotium rolfsii, Alternaria solani, Fusarium oxysporum under in vitro dual culture plate assay. Further the isolate was identified into Bacillus amyloliquefaciens based on 16S rRNA sequencing. The antimicrobial fraction from the extracellular supernatant of the isolate comprises chiefly of surfactin molecules and also iturin and fengycin group of compounds. The surfactins were partially purified by tangential flow ultra-filtration and quantified with liquid chromatography yielding 316.1 mg L-1. Further the surfactin molecules were characterized by HPLC separation, FT-IR, LC-MS spectroscopy and PCR amplification of antibiotic genes. The surfactin molecule with m/z 1022 performed for MS-MS fragmentation and produced two different patterns of ion dissociation.

EnZolv delignification of cotton spinning mill waste and optimization of process parameters using response surface methodology (RSM).

BACKGROUND: EnZolv is a novel enzyme-based, eco-friendly biomass pretreatment process that has shown great potential in the field of textile engineering and biotechnology. It employs laccase from Hexagonia hirta MSF2 and 2% ethanol in the process of delignification. The process is designed to evaluate optimal conditions to remove lignin and other impurities from cotton spinning mill waste (CSMW), without compromising the quality and strength of the fibers. CSMW is a low-cost and readily available source of cellulose, making it an ideal candidate for delignification using EnZolv. By optimizing the pretreatment conditions and harnessing the potential of enzymatic delignification, this research aims to contribute to more sustainable and efficient ways of utilizing lignocellulosic biomass in various industries for the production of biochemical and bioproducts. RESULTS: The present study emphasizes the EnZolv pretreatment in the delignification of cotton spinning mill wastes irrespective of the cellulose content. EnZolv process parameters such as, moisture content, enzyme load, incubation time, incubation temperature, and shaking speed were optimized. Under pre-optimized conditions, the percent lignin reduction was 61.34%, 61.64%, 41.85%, 35.34%, and 35.83% in blowroom droppings (BD), flat strips (FS), lickerin fly (LF), microdust (MD) and comber noils (CN), respectively. Using response surface methodology (RSM), the statistically optimized EnZolv pretreatment conditions showed lignin reduction of 59.16%, 62.88%, 48.26%, 34.64%, and 45.99% in BD, FS, LF, MD, and CN, respectively. CONCLUSION: Traditional chemical-based pretreatment methods often involve harsh chemicals and high energy consumption, which can have detrimental effects on the environment. In contrast, EnZolv offers a greener approach by utilizing enzymes that are biodegradable and more environmentally friendly. The resulting fibers from EnZolv treatment exhibit improved properties that make them suitable for various applications. Some of the key properties include enhanced cellulose recovery, reduced lignin content, and improved biophysical and structural characteristics. These improvements can contribute to the fiber's performance and processability in different industries and future thrust for the production of cellulose-derived and lignin-derived bioproducts.

Archaeal JAB1/MPN/MOV34 metalloenzyme (HvJAMM1) cleaves ubiquitin-like small archaeal modifier proteins (SAMPs) from protein-conjugates.

Proteins with JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains are widespread among all domains of life, yet poorly understood. Here we report the purification and characterization of an archaeal JAMM/MPN+ domain protein (HvJAMM1) from Haloferax volcanii that cleaves ubiquitin-like small archaeal modifier proteins (SAMP1/2) from protein conjugates. HvJAMM1 cleaved SAMP1/2 conjugates generated in H. volcanii as well as isopeptide- and linear-linked SAMP1-MoaE in purified form. Cleavage of linear linked SAMP1-MoaE was dependent on the presence of the SAMP domain and the C-terminal VSGG motif of this domain. While HvJAMM1 was inhibited by size exclusion chromatography and metal chelators, its activity could be restored by addition of excess ZnCl2 . HvJAMM1 residues (Glu31, His88, His90, Ser98 and Asp101) that were conserved with the JAMM/MPN+ active-site motif were required for enzyme activity. Together, these results provide the first example of a JAMM/MPN+ zinc metalloprotease that independently catalyses the cleavage of ubiquitin-like (isopeptide and linear) bonds from target proteins. In archaea, HvJAMM1 likely regulates sampylation and the pools of 'free' SAMP available for protein modification. HvJAMM1-type proteins are thought to release the SAMPs from proteins modified post-translationally as well as those synthesized as domain fusions.

Delignification of the cotton stalk and ginning mill waste via EnZolv pretreatment and optimization of process parameters using response surface methodology (RSM).

The present study aimed to add value to cotton waste biomass using a more eco-friendly process, EnZolv which delignifies cotton stalk and cotton ginning mill waste. A maximum delignification of 68.68% and 65.51% was obtained using pre-optimized EnZolv parameters in cotton stalk (CS) and ginning mill waste (GMW), respectively. Optimized EnZolv process removed 78.68% of lignin in CS using Response Surface Methodology (RSM) in Box-Behnken design at 0% moisture content, 50 U laccase g-1 of biomass, 5 h incubation time, 50 °C incubation temperature, and 150 rpm shaking speed. Similarly, RSM-based delignification of 70.53% in GMW was achieved under the optimized EnZolv conditions of 98.75 % moisture content, 41.59 U laccase g-1 of biomass, 9.3 h incubation time, 46.15 °C incubation temperature, and 150 rpm shaking speed.