Bacterial nanocellulose: A versatile biopolymer production using a cost-effective wooden disc based rotary reactor.

Bacterial nanocellulose (BNC) has various unique qualities, including high mechanical strength, crystallinity, and high water-holding capacity, which makes it appropriate for a wide range of industrial applications. But its lower yield coupled with its high production cost creates a barrier to its usage. In this study, we have demonstrated the better yield of BNC from an indigenous strain Komagataeibacter rhaeticus MCC-0157 using a rotary disc bioreactor (RDB) having a wooden disc. The RDB was optimized based on the type of disc material, distance between the disc, and rotation speed to get the highest yield of 13.0 g/L dry material using Hestrin-Schramm (H-S) medium. Further, the bioreactor was compared for the BNC production using reported medium, which is used for static condition; the RDB showed up to fivefold increase in comparison with the static condition reported. Komagataeibacter rhaeticus MCC-0157 was previously reported to be one of the highest BNC producing stains, with 8.37 g/L of dry yield in static condition in 15 days incubation. The designed RDB demonstrated 13.0 g/L dry yield of BNC in just 5 days. Other characteristics of BNC remain same as compared with static BNC production, although the difference in the crystallinity index was observed in RDB (84.44%) in comparison with static (89.74%). For the first time, wooden disc was used for rotary bioreactor approach, which demonstrated higher yield of BNC in lesser time and can be further used for sustainable production of BNC at the industrial level.

Fictibacillus fluitans sp. nov., isolated from freshwater pond.

A Gram-positive, aerobic, rod-shaped, spore-forming bacterium, designated NE201T, was isolated from a freshwater pond in Village Nerur, India. Growth was observed in the range of 15-45 °C temperature with optimum at 30 °C, pH range of 5-9 (optimum at 7.0), and at concentrations of NaCl ranging between 0 and 14% (optimum 0%, w/v). The 16S rRNA gene sequence showed the highest similarity with Fictibacillus enclensis NIO-1003T (JF893461) at 99.01% followed by F. rigui WPCB074T (EU939689) at 98.9% and F. solisalsi CGMCC 1.6854T (EU046268) at 98.66%. The digital DNA-DNA hybridization (dDDH) and orthoANI values for strain NE201T against F. enclensis NIO-1003T (GCA_900094955.1) were 33.7% and 87.68%, respectively. The phylogenetic analysis based on the 16S rRNA gene, 92 core genes derived from the genome, and 20 proteins involving over 20,236 amino acid positions revealed the distinct phylogenetic position of strain NE201T and the formation of a clearly defined monophyletic clade with F. enclensis. The strain NE201T showed a unique carbon utilization and assimilation pattern that differentiated it from F. enclensis NIO-1003T. The major fatty acids were anteiso -C15:0 (51.42%) and iso-C15:0 (18.88%). The major polar lipids were phosphatidylglycerol (PG), phosphatidylethanolamine (PE, and diphosphatidylglycerol (DPG). The antiSMASH analyzed genome of NE201T highlighted its diverse biosynthetic potential, unveiling regions associated with terpene, non-ribosomal peptide synthetases (NRPS), lassopeptides, NI-siderophores, lanthipeptides (LAP), and Type 3 Polyketide Synthases (T3PKS). The overall phenotypic, genotypic, and chemotaxonomic characters strongly suggested that the strain NE201T represents a novel species of genus Fictibacillus for which the name Fictibacillus fluitans sp. nov. is proposed. The type strain is NE201T (= MCC 5285 = JCM 36474).

Genome sequencing and protein modeling unraveled the 2AP biosynthesis in Bacillus cereus DB25.

2-Acetyl-1-pyrroline (2AP) is an important and major flavor aroma compound responsible for the fragrance of basmati rice, cheese, wine, and several other food products. Biosynthesis of 2AP in aromatic rice and a few other plant species is associated with a recessive Betaine aldehyde dehydrogenase 2 (BADH2) gene. However, the literature is scant on the relationship between the functional BADH2 gene and 2AP biosynthesis in prokaryotic systems. Therefore, in the present study, we aimed to explore the functionality of the BADH2 gene for 2AP biosynthesis in 2AP synthesizing rice rhizobacterial isolate Bacillus cereus DB25 isolated from the rhizosphere of basmati rice (Oryza sativa L.). Full-length BcBADH2 sequence was obtained through whole genome sequencing (WGS) and further confirmed through traditional PCR and Sanger sequencing. Then the functionality of the BcBADH2 gene was evaluated in-silico through bioinformatics analysis and protein docking studies and further experimentally validated through enzyme assay. The sequencing and bioinformatics analysis results revealed a full-length 1485 bp BcBADH2 coding sequence without any deletion or premature stop codons. Full-length BcBADH2 was found to encode a fully functional protein of 54.08 kDa with pI of 5.22 and showed the presence of the conserved amino acids responsible for enzyme activity. The docking studies confirmed a good affinity between the protein and its substrate whereas the presence of BcBADH2 enzyme activity confirmed the functionality of BADH2 enzyme in B. cereus DB25. In conclusion, the findings of the present study suggest that B. cereus DB25 is able to synthesize 2AP despite a functional BADH2 gene and there may be a different molecular mechanism responsible for 2AP biosynthesis in bacterial systems, unlike that found in aromatic rice and other eukaryotic plant species.

In vitro anticancer evaluation of Enceleamycin A and its underlying mechanism.

It has become more crucial than ever to find novel anticancer compounds due to the rise in cancer mortality and resistance to the present chemotherapeutic drugs. Naphthoquinones are regarded as privileged structures for their ability to inhibit various cancers. The current study examined three novel furo-naphthoquinones (Enceleamycins A-C) previously isolated from Amycolatopsis sp. MCC 0218 for their anticancer potential. Enceleamycin A demonstrated considerable cytotoxicity for triple-negative breast cancer (TNBC) MDA-MB-231 cells with an IC50 value of 1.25 µg mL-1 (3.78 µM). It also showed the ability to inhibit MDA-MB-231 cell migration. Enceleamycin A raises intracellular ROS levels in TNBC cells, ultimately leading to apoptotic cell death, as demonstrated by Annexin V/PI staining. The molecular docking and simulation investigation revealed better binding affinity of Enceleamycin A with AKT2, which plays a vital role in breast cancer's invasiveness and chemo-resistance. Enceleamycin A inhibits the AKT2 enzyme in vitro with an IC50 value of 0.736 µg mL-1 (2.22 µM), further validating the docking study. The in silico physicochemical and pharmacokinetics characteristics of Enceleamycin A demonstrated its drug-likeness. Intriguingly, Enceleamycin A is non-hemolytic in nature. Taken together, Enceleamycin A could be a candidate molecule for treating TNBC cells by targeting the AKT2 signaling pathway.

Strain improvement for enhanced erythritol production by Moniliella pollinis Mutant-58 using jaggery as a cost-effective substrate.

Erythritol has been produced by various microorganisms including Yarrowia, Moniliella, Aureobasidium, and Candida strains. Due to its relatively high price, erythritol sweetener is used lesser than other polyols despite having many advantages. Therefore, in this study, Moniliella pollinis strain was improved for erythritol production by chemical mutagenesis and subsequently screening for cost-effective carbon sources for the enhanced erythritol yield. M. pollinis was subjected to N-methyl N-nitro N-nitroso guanidine (NTG), ethyl methyl sulfonate (EMS), and UV mutagenesis for improved erythritol production. The fmutant strains were evaluated for enhanced erythritol production medium optimization by using different carbon substrates at the shake flask level. To enhance the production of erythritol and statistical media, optimization was carried out using a central composite design (CCD). Among 198 isolated mutants, Mutant-58 strain generated by EMS mutagenesis was selected for further assessment. The Mutant-58 strain showed significant morphological changes as compared to the parent strain. Furthermore, statistically optimized media composition resulted in the higher production of erythritol (91.2 ± 3.4 g/L) with a yield of 40.7 ± 3.4 % in shake flask experiments. The optimized medium composition for erythritol production constitutes (g/L) 225 jaggery, 4.4 yeast extract (YE), 4.4 KH2PO4, 0.31 MgSO4, and pH 5.5. The present study demonstrated strain improvement, media, and process optimization resulting in a 30% increase in the erythritol production in the Mutant-58 as compared to the parent strain. This is also the first instance where jaggery has been used as a cost-effective carbon source alternative to glucose for industrial-scale erythritol production.

Antibacterial and Anti-HIV Metabolites from Marine Streptomyces albus MAB56 Isolated from Andaman and Nicobar Islands, India.

Marine-derived actinobacteria have tremendous potential to produce novel metabolites with diverse biological activities. The Andaman coast of India has a lot of microbial diversity, but it is still a relatively unknown ecology for isolating novel actinobacteria with beneficial bioactive compounds. We have isolated 568 actinobacterial strains from mangrove rhizosphere sediments and sponge samples. Crude extracts from 75 distinct strains were produced by agar surface fermentation and extracted using ethyl acetate. In the disc diffusion method, 25 actinobacterial strains showed antimicrobial activity; notably, the strain MAB56 demonstrated promising broad-spectrum activity. Strain MAB56 was identified as Streptomyces albus by cultural, microscopic, and molecular methods. Conditions for bioactive metabolites from MAB56 were optimized and produced in a lab-scale fermenter. Three active metabolites (C1, C2, and C3) that showed promising broad-spectrum antimicrobial activity were isolated through HPLC-based purification. Based on the UV, FT-IR, NMR, and LC-MS analysis, the chemical nature of the active compounds was confirmed as 12-methyltetradecanoic acid (C1), palmitic acid (C2), and tridecanoic acid (C3) with molecular formulae C14H28O2, C16H32O2, and C13H26O2, respectively. Interestingly, palmitic acid (C2) also exhibited anti-HIV activity with an IC50 value of < 1 µg/ml. Our findings reveal that the actinobacteria from the Andaman marine ecosystems are promising for isolating anti-infective metabolites.

A Novel Finding: 2,4-Di-tert-butylphenol from Streptomyces bacillaris ANS2 Effective Against Mycobacterium tuberculosis and Cancer Cell Lines.

The aim of the present study is to identify actinobacteria Streptomyces bacillaris ANS2 as the source of the potentially beneficial compound 2,4-di-tert-butylphenol, describe its chemical components, and assess its anti-tubercular (TB) and anti-cancer properties. Ethyl acetate was used in the agar surface fermentation of S. bacillaris ANS2 to produce the bioactive metabolites. Using various chromatographic and spectroscopy analyses, the potential bioactive metabolite separated and identified as 2,4-di-tert-butylphenol (2,4-DTBP). The lead compound 2,4-DTBP inhibited 78% and 74% of relative light unit (RLU) decrease against MDR Mycobacterium tuberculosis at 100ug/ml and 50ug/ml concentrations, respectively. The Wayne model was used to assess the latent/dormant potential in M. tuberculosis H37RV at various doses, and the MIC for the isolated molecule was found to be 100ug/ml. Furthermore, the molecular docking of 2,4-DTBP was docked using Autodock Vinasuite onto the substrate binding site of the target Mycobacterium lysine aminotransferase (LAT) and the grid box was configured for the docking run to cover the whole LAT dimer interface. At a dosage of 1 mg/ml, the anti-cancer activity of the compound 2,4-DTBP was 88% and 89% inhibited against the HT 29 (colon cancer) and HeLa (cervical cancer) cell lines. According to our literature survey, this present finding may be the first report on anti-TB activity of 2,4-DTBP and has the potential to become an effective natural source and the promising pharmaceutical drug in the future.

An overview of erythritol production by yeast strains.

Erythritol is a 4-carbon polyol produced with the aid of microbes in presence of hyper-osmotic stress. It is the most effective sugar alcohol that is produced predominantly by fermentation. In comparison to various polyols, it has many precise functions and is used as a flavor enhancer, sequestrant, humectant, nutritive sweetener, stabilizer, formulation aid, thickener, and a texturizer. Erythritol production is a common trait in a number of the yeast genera viz., Trigonopsis, Candida, Pichia, Moniliella, Yarrowia, Pseudozyma, Trichosporonoides, Aureobasidium, and Trichoderma. Extensive work has been carried out on the biological production of erythritol through Yarrowia, Moniliella, Candida, and other yeast strains, and numerous strategies used to improve erythritol productivity through mutagenesis and genetic engineering are discussed in this review.

Antifungal compound from marine Serratia marcescens BKACT and its potential activity against Fusarium sp / Compuesto antifúngico de Serratia marcescens marina BKACT y su potencial actividad contra Fusarium sp

Ecofriendly biocontrol agents to control pathogenic fungi are in demand globally. The present study evaluated the antifungal potentials of marine bacteria Serratia marcescens BKACT against eight different Fusarium species. A highest 75.5 ± 0.80% of mycelial inhibition was observed against Fusarium foetens NCIM 1330. Structural characterization of the purified compound was analyzed by GC–MS and NMR techniques; based on the analysis, it is confirmed as 2, 4-di-tert butyl phenol (2, 4-DTBP) with chemical structure C14H22O. At 0.53 mM concentration, purified compound inhibited complete spore germination of F. foetens NCIM 1330. In vitro assay showed complete inhibition of F. foetens NCIM 1330 on the wheat seeds. Tested concentration does not show any toxic effect on germination of the seeds. By this study, we conclude that, 2, 4-DTBP is a suitable candidate to be used as biocontrol agent against Fusarium infection.(AU)

Exploring the core microbiota in scented rice (Oryza sativa L.) rhizosphere through metagenomics approach.

Rice is a major food crop cultivated around the globe. Specially scented rice varieties are of commercial importance but they are low-yielding. The rhizospheric microflora plays a significant role in improving yield and aroma. However, the core microbiome of the scented rice rhizosphere is comparatively less explored. Here, we analyzed the core microbiome associated with the rhizosphere of the scented (Ambemohar-157 and Dehradun basmati) in comparison with non-scented rice (Kolam and Arize 6444 Gold) cultivated at two different geoclimatic zones of India (Maharashtra and Uttarakhand) using the metagenomics approach. The alpha and beta diversity analysis showed that the microbial communities associated with scented and non-scented varieties significantly changes with respect to richness, diversity, and evenness. The taxonomic profiling revealed the variation in composition, diversity, and abundance of the microbiome in terms of phyla and genera associated with scented rice varieties over non-scented. The cluster analysis distinguishes the microbial communities based on their geographical positions. The core microbiome analysis revealed that scented rice rhizosphere shelters distinct and unique microbiota. 28.6 % of genera were exclusively present only in the scented rice rhizosphere. The putative functional gene annotation revealed the high abundance of genes related to the biosynthesis of 2-acetyl-1-pyrroline (2AP) precursors in scented rice. The precursor feeding analysis revealed proline as a preferred substrate by 2AP synthesizing bacteria. The 2AP precursor proline and proline metabolism genes showed a positive correlation. The scented rice-specific rhizobacteria pointed out in this study can be used as bio-inoculants for enhancing aroma, yield, and sustainable rice cultivation.

Artificial intelligence in microbial natural product drug discovery: current and emerging role.

Covering: up to the end of 2022Microorganisms are exceptional sources of a wide array of unique natural products and play a significant role in drug discovery. During the golden era, several life-saving antibiotics and anticancer agents were isolated from microbes; moreover, they are still widely used. However, difficulties in the isolation methods and repeated discoveries of the same molecules have caused a setback in the past. Artificial intelligence (AI) has had a profound impact on various research fields, and its application allows the effective performance of data analyses and predictions. With the advances in omics, it is possible to obtain a wealth of information for the identification, isolation, and target prediction of secondary metabolites. In this review, we discuss drug discovery based on natural products from microorganisms with the help of AI and machine learning.

Antifungal compound from marine Serratia marcescens BKACT and its potential activity against Fusarium sp.

Ecofriendly biocontrol agents to control pathogenic fungi are in demand globally. The present study evaluated the antifungal potentials of marine bacteria Serratia marcescens BKACT against eight different Fusarium species. A highest 75.5 ± 0.80% of mycelial inhibition was observed against Fusarium foetens NCIM 1330. Structural characterization of the purified compound was analyzed by GC-MS and NMR techniques; based on the analysis, it is confirmed as 2, 4-di-tert butyl phenol (2, 4-DTBP) with chemical structure C14H22O. At 0.53 mM concentration, purified compound inhibited complete spore germination of F. foetens NCIM 1330. In vitro assay showed complete inhibition of F. foetens NCIM 1330 on the wheat seeds. Tested concentration does not show any toxic effect on germination of the seeds. By this study, we conclude that, 2, 4-DTBP is a suitable candidate to be used as biocontrol agent against Fusarium infection.

Enceleamycins A-C, Furo-Naphthoquinones from Amycolatopsis sp. MCC0218: Isolation, Structure Elucidation, and Antimicrobial Activity.

Three novel furo-naphthoquinones, enceleamycins A-C (1-3), and a new N-hydroxypyrazinone acid (4) were identified from the strain Amycolatopsis sp. MCC 0218, isolated from a soil sample collected from the Western Ghats of India. Their chemical structure and absolute and relative configurations were established by 1D and 2D NMR spectroscopy, single-crystal X-ray crystallography, and high-resolution mass spectrometry. Compounds 1 and 3 were active against methicillin-susceptible and -resistant Staphylococcus aureus with MIC values of 2-16 µg/mL.

Isolation and structural characterization of exopolysaccharide from marine Bacillus sp. and its optimization by Microbioreactor.

In the present study, a cost-effective, robust Microbioreactor based production optimization of levan like exopolysaccharide from marine Bacillus sp. SGD-03 was analysed. FE-SEM analysis has showed the significant fibrillar structure of EPS. Size exclusion chromatography and other analytical data revealed that, produced EPS has a molecular weight of 1.0 × 104 Da and is composed of fructose monosaccharide with hydroxyl, carbonyl, and ether groups. The backbone structure of EPS has a branching pattern of ß-(2,6) linkages which confirms the similarity with available levan like polymers. The cost-effective media composition for levan production was demonstrated. The maximum yield of crude levan obtained was 123.9 g/L by response surface methodology using robust BioLector Pro Microbioreactor, and same has been validated with shake flask, 1 L and 10 L pilot-scale fermentation.

High throughput sequencing based direct detection of SARS-CoV-2 fragments in wastewater of Pune, West India.

Given a large number of SARS-CoV-2 infected individuals, clinical detection has proved challenging. The wastewater-based epidemiological paradigm would cover the clinically escaped asymptomatic individuals owing to the faecal shedding of the virus. We hypothesised using wastewater as a valuable resource for analysing SARS-CoV-2 mutations circulating in the wastewater of Pune region (Maharashtra; India), one of the most affected during the covid-19 pandemic. We conducted study in open wastewater drains from December 2020-March 2021 to assess the presence of SARS-CoV-2 nucleic acid and further detect mutations using ARTIC protocol of MinION sequencing. The analysis revealed 108 mutations across six samples categorised into 39 types of mutations. We report the occurrence of mutations associated with Delta variant lineage in March-2021 samples, simultaneously also reported as a Variant of Concern (VoC) responsible for the rapid increase in infections. The study also revealed four mutations; S:N801, S:C480R, NSP14:C279F and NSP3:L550del not currently reported from wastewater or clinical data in India but reported worldwide. Further, a novel mutation NSP13:G206F mapping to NSP13 region was observed from wastewater. Notably, S:P1140del mutation was detected in December 2020 samples while it was reported in February 2021 from clinical data, indicating the instrumentality of wastewater data in early detection. This is the first study in India to demonstrate utility of sequencing in wastewater-based epidemiology to identify mutations associated with SARS-CoV-2 virus fragments from wastewater as an early warning indicator system.

Priestia veravalensis sp. nov., isolated from coastal sample.

The taxonomic position of two isolates, SGD-V-76T and SGD-M-37, isolated from sediment sample of Veraval coast, India, was examined using the polyphasic taxonomic approach. The morphological and chemotaxonomic characteristics of these two organisms are typical of the genus Priestia. The phylogenetic analyses performed using almost complete 16S rRNA gene sequences demonstrated that the isolate belongs to the Bacillaceae family, and forms a clade within the cluster containing Priestia flexus MTCC 2909T, Priestia aryabhattai B8W22T and Priestia megaterium KCTC 3007T and both strains showed highest similarity of > 98% with 3-29 nucleotide differences. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant isoprenoid quinone was MK-7 and the G + C content of strains was 37.5-37.7 mol%. However, the DNA-DNA hybridization and the phenotypic characteristics revealed that, the strain SGD-V-76T and strain SGD-M-37 are similar species but different from any known Priestia species with ANI values of 79.2, 79.3 and 79.2 and the dDDH values of 17.7, 17.8 and 18.0% respectively. On the basis of phenotypic characteristics, phylogenetic analysis and the results of biochemical and physiological tests, and genomic data strain SGD-V-76T was clearly distinguished from closely related members of the Priestia genus. Based on the above data analysis strain SGD-V-76T (= DSM28242T = KCTC33802T = CIP111056T = NCIM5510T) represents a novel species of the genus Priestia, and we propose the name Priestia veravalensis sp. nov.

Rhizobacterial consortium mediated aroma and yield enhancement in basmati and non-basmati rice (Oryza sativa L.).

Basmati and non-basmati rice varieties are commercially important. Aromatic rice varieties are low yielding and recently depletion in aroma is observed due to the shift towards modern agriculture. Therefore, it is necessary to restore the aroma and increase the yield through sustainable agriculture. The use of microbial bioinoculants is one of the promising ways to achieve these targets. With these objectives, rhizospheric bacterial strains Enterobacter hormaechei (AM122) and Lysinibacillus xylanilyticus (DB25) having the property of synthesizing 2-acetyl-1-pyrroline (2AP) were isolated from the rhizosphere of two aromatic rice varieties, Ambemohar-157 and Dehradun Basmati respectively and their effect on plant growth, aroma and yield enhancement under mono-inoculation and consortium conditions was analyzed. The bacterial inoculum in consortium resulted in significant improvement in vegetative growth, yield and 2AP content over mono inoculation and control. The study highlights the potential of E. hormaechei and L. xylanilyticus in plant growth, yield and aroma enhancement in basmati and non-basmati rice varieties. These strains can be taken up further for developing a commercial bioformulation.

Molecular insights of fungal endophyte co-inoculation with Trichoderma viride for the augmentation of forskolin biosynthesis in Coleus forskohlii.

To understand the compatibility of three native endophytic fungi Phialemoniopsis cornearis (SF1), Macrophomina pseudophaseolina (SF2) and Fusarium redolens (RF1) with Trichoderma viride (TV1) on Coleus forskohlii in enhancing plant growth and forskolin content, field experiments were conducted. Co-inoculation of RF1+TV1 showed significant improvement in plant growth (52%), root biomass (67%), and in-planta forskolin content (94%), followed by treatment with SF2+TV1 and SF1+TV1. qRT-PCR was carried out to quantify expression of five key forskolin biosynthetic pathway genes (CfTPS2, CfTPS3, CfTPS4, CfCYP76AH15, and CfACT1-8) in RF1+TV1 treated C. forskohlii plants. Elevated expression of CfTPS2, CfTPS4, CfCYP76AH15 and CfACT1-8 genes was observed with RF1+TV1 combination as compared to uninoculated C. forskohlii plants. Besides, RF1+TV1 treatment considerably reduced the severity of nematode infection of C. forskohlii plants under field conditions. Thus, congruent properties of F. redolens (RF1) were witnessed with co-inoculation of T. viride (TV1) under field conditions which resulted in enhanced forskolin content, root biomass, and reduced nematode infections in C. forskohlii. Overall, this approach could be an economical and sustainable step towards cultivation of commercially important medicinal plants.

Total synthesis of (-)-2-methoxy-2-butenolide-3-cinnamate and its antimicrobial potentials.

The first total synthesis of (-)-2-methoxy-2-butenolide-3-cinnamate (butenolide cinnamate) was achieved using commercially available starting material. The synthesized compound was found to have promising antibacterial activity against Gram-negative strains Escherichia coli (ATCC 8739), Salmonella typhimurium (ATCC 23564) and Pseudomonas aeruginosa (ATCC 19154) with a minimum inhibitory concentration of 2.0 µg/mL, 1.0 µg/mL and 2.0 µg/mL, respectively. Notably, the compound was more potent against Gram-negative test strains than the Gram-positive test strains.[Figure: see text].