Molecular authentication, metabolite profiling and in silico-in vitro cytotoxicity screening of endophytic Penicillium ramusculum from Withania somnifera for breast cancer therapeutics.

In the present study, we isolated a potent endophytic fungus from the roots of Withania somnifera. The endophytic fungal strain was authenticated as Penicillium ramusculum SVWS3 based on morphological and molecular sequencing using four gene data and phylogenetic analyses. In vitro cytotoxicity studies unveiled the remarkable cytotoxic potential of the crude extract derived from P. ramusculum, exhibiting dose-dependent effects on MDA-MB-468 and MCF-7 cells. At a concentration of 100 µg/mL, the crude extract resulted in cell viability of 29.78% for MDA-MB-468 cells and 14.61% for MCF-7 cells. The IC50 values were calculated as 62.83 ± 0.93 µg/mL and 17.23 ± 1.43 µg/mL, respectively for MDA-MB-468 and MCF-7 cells. Caspase activation assay established the underlying mechanism of the crude extract depicting the activation of caspases 3 and 7, indicating the induction of apoptosis in MCF-7 cells. Chemotaxonomic profiling elucidated the ability of P. ramusculum to synthesize a diverse array of bioactive compounds, including Fasoracetam, Tryprostatin B, Odorinol, Thyronine, Brevianamide F, Proglumide, Perlolyrine, Tyrphostin B48, Baptifoline, etc. Molecular docking studies inferred that Baptifoline, Brevianamide F, Odorinol, Perlolyrine, Thyronine, Tryphostin B48, and Tryprostatin B were the lead compounds that could effectively interact with the five selected target receptors of breast cancer, further surpassing the positive controls analyzed. Pharmacokinetic profiling revealed that Baptifoline, Odorinol, and Thyronine depicted an excellent therapeutic profile of druggability. These findings collectively substantiate the anticancer activity of bioactive metabolites synthesized by P. ramusculum SVWS3. Hence, the endophytic P. ramusculum SVWS3 can be an authentic source for developing novel chemotherapeutic drug formulations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03906-3.

Longitudinal dynamics and clinically available predictors of poor response to COVID-19 vaccination in multiple myeloma.

Not available.

Determinants of durable humoral and T cell immunity in myeloma patients following COVID-19 vaccination.

OBJECTIVE: To describe determinants of persisting humoral and cellular immune response to the second COVID-19 vaccination among patients with myeloma. METHODS: This is a prospective, observational study utilising the RUDYstudy.org platform. Participants reported their second and third COVID-19 vaccination dates. Myeloma patients had an Anti-S antibody level sample taken at least 21 days after their second vaccination and a repeat sample before their third vaccination. RESULTS: 60 patients provided samples at least 3 weeks (median 57.5 days) after their second vaccination and before their third vaccination (median 176.0 days after second vaccine dose). Low Anti-S antibody levels (<50 IU/mL) doubled during this interval (p = .023) and, in the 47 participants with T-spot data, there was a 25% increase negative T-spot tests (p = .008). Low anti-S antibody levels prior to the third vaccination were predicted by lower Anti-S antibody level and negative T-spot status after the second vaccine. Independent determinants of a negative T-spot included increasing age, previous COVID infection, high CD4 count and lower percentage change in Anti-S antibody levels. CONCLUSIONS: Negative T-spot results predict low Anti-S antibody levels (<50 IU/mL) following a second COVID-19 vaccination and a number of biomarkers predict T cell responses in myeloma patients.

Manifestation of Guillain-Barre Syndrome in a Case of Monkeypox Virus Infection: A Rare Case Report.

Monkeypox virus (MPXV) is one of the rare zoonotic infections caused by orthopoxvirus. MPXV has recently been an evolving threat to public health with its contagious human-to-human transmission. Various presentations of MPXV infection have been reported ranging from generalised symptoms such as fever, chills, body aches, and swollen lymph nodes to dermatological presentations. Neurological manifestations that have been reported include headaches, myalgia, seizures, and even mood disturbances. Postinfectious complications such as encephalitis, vision problems, and skin infections have also been noticed. Guillain-Barre Syndrome (GBS) is an acquired acute inflammatory polyradiculoneuropathy characterized by progressive, symmetrical, proximal, and distal tingling and weakness. Although various microorganisms are known to cause GBS and have been linked to the smallpox vaccine, they are rarely linked to MPXV disease. In this report, we describe a case of a confirmed monkeypox infection in a patient presenting with Guillain-Barre Syndrome.

What do we know about the psychosocial issues associated with cancer during pregnancy? A scoping review and gap analysis.

OBJECTIVES: There is a global increase in the number of women diagnosed with cancer during their pregnancy and a nascent evidence base to guide their supportive care. The purposes of this study were to (1) map research on the psychosocial issues affecting women and their partners on diagnosis and treatment for cancer during pregnancy; (2) determine available supportive care or educational interventions; and (3) identify knowledge gaps for future research and development. DESIGN: Scoping review. SEARCH STRATEGY: Six databases were searched (Scopus, CINAHL, PsycINFO, Medline, Intermid, Maternal and Infant Health) to retrieve primary research (January 1995 to November 2021) investigating women and/or their partner's decision-making and their psychosocial outcomes during and after pregnancy. DATA EXTRACTION AND SYNTHESIS: Sociodemographic, gestational and disease characteristics of participants and psychosocial issues identified were extracted. Leventhal's self-regulatory model of illness provided a framework for mapping study findings enabling evidence synthesis and gap analysis. RESULTS: Twelve studies were included, conducted in eight countries in six continents. Most women (70% of 217) were diagnosed with breast cancer during pregnancy. Reporting of sociodemographic, psychiatric, obstetric and oncological characteristics that are important in assessing psychosocial outcomes was inconsistent. None of the studies had a longitudinal design and no supportive care or educational interventions were identified. The gap analysis highlighted the lack of evidence about pathways to diagnosis, impact of late effects and how internal/social resources may affect outcomes. CONCLUSIONS: Research has focused on women with gestational breast cancer. Little is known about those diagnosed with other cancers. We encourage future study designs to capture data on sociodemographic, obstetric, oncological and psychiatric characteristics and adopt a longitudinal approach to explore the longer term psychosocial impact on women and their families. Future research should include outcomes that are meaningful for women (and their partners) and draw on international collaboration to accelerate progress in this field.

Bacillus tequilensis PKDN31 and Bacillus licheniformis PKDL10 -As double headed swords to combat Fusarium oxysporum f. sp. lycopersici induced tomato wilt.

Wilt disease, caused by Fusarium oxysporum. f. sp. lycopersici, is a global threat to tomato production that needs to be addressed seriously. The current research envisages the use of two self-compatible Bacillus strains, Bacillus tequilensis PKDN31 and Bacillus licheniformis PKDL10, in a combinatorial approach. The spent supernatant of liquid cultures from strains PKDN31 and PKDL10 showed in vitro antifungal activity against Fusarium sp. attaining an inhibition percentage of 95.33% and 96.54%, respectively. The bacterial isolates lytic activity against Fusarium oxysporum was evaluated by scanning electron microscopic analysis and lytic enzyme production of amylase, lipase, protease and ß-1,3 glucanase. Furthermore, PKDN31 and PKDL10 produced siderophores and had root colonizing ability that enhanced the biocontrol efficiency. Combined in vivo inoculation of Bacillus tequilensis PKDN31 and Bacillus licheniformis PKDL10 on tomato seeds revealed that the strains could induce systemic resistance in tomato against Fusarium oxysporum. f. sp. lycopersici by increasing defence enzymes such as ß-1,3 glucanase, polyphenol oxidase, peroxidase, phenylalanine ammonia-lyase, chitinase, and total phenol accumulations. Pot culture experiments also proved the biocontrol efficacy of the above dual culture supplementation as this treatment displayed a better growth as well as defense against Fusarium challenge compared to the controls. The obtained results suggest that rhizobacterial isolates could be employed as systemic resistance inducers and biocontrol agents in tomato plants to protect against Fusarium wilt disease.

A Cross-Sectional Study of the Association of ABO Blood Group and Rh Type With Severity of COVID-19 Infection in a Tertiary Care Center of South India.

Background The novel coronavirus disease 2019 (COVID-19) was declared a pandemic that had affected 224 countries, causing >2.1 million deaths worldwide. The association of the different ABO blood groups with the risk and severity of COVID-19 infections has been speculated in many studies. This study aims to determine the incidence of COVID-19 infections among various blood groups and the association of ABO blood groups and Rh type with the severity of COVID-19 infection as well as with other outcome predictors of COVID-19 infection including neutrophil-to-lymphocyte ratio, D-dimer, ferritin, lactate dehydrogenase, and C-reactive protein. Methodology This was a retrospective study conducted among 150 serologically positive patients >18 years of age who underwent treatment in a district government hospital over two months. Patients were categorized into severity groups, and laboratory data were divided into those corresponding to severe disease and otherwise, in accordance with national guidelines. Appropriate statistical analysis was performed. Results The frequency of blood groups A, B, AB, and O was 30.7%, 29.4%, 13.7%, and 39%, respectively. There was a statistically significant number of patients belonging to non-O blood groups who developed a severe COVID-19 infection (group C) (p = 0.005). There was an increased risk of multiorgan failure (p = 0.035), non-invasive ventilation (p = 0.005), intubation, and mortality among non-O blood groups, and was the maximum for A blood group even after adjusting for age and pre-existing comorbidities. Increased D-dimer levels were noted in non-O blood groups (p = 0.037). No statistically significant association was found between Rh typing and the severity of COVID-19 infection. Conclusions Our findings provide evidence that individuals with non-O blood groups are susceptible to developing more severe COVID-19 infections and should take active preventive measures. Moreover, they should be cautiously monitored and treated once infected.

Leads and hurdles to sustainable microbial bioplastic production.

Indiscriminate usage, disposal and recalcitrance of petroleum-based plastics have led to its accumulation leaving a negative impact on the environment. Bioplastics, particularly microbial bioplastics serve as an ecologically sustainable solution to nullify the negative impacts of plastics. Microbial production of biopolymers like Polyhydroxyalkanoates, Polyhydroxybutyrates and Polylactic acid using renewable feedstocks as well as industrial wastes have gained momentum in the recent years. The current study outlays types of bioplastics, their microbial sources and applications in various fields. Scientific evidence on bioplastics has suggested a unique range of applications such as industrial, agricultural and medical applications. Though diverse microorganisms such as Alcaligenes latus, Burkholderia sacchari, Micrococcus species, Lactobacillus pentosus, Bacillus sp., Pseudomonas sp., Klebsiella sp., Rhizobium sp., Enterobacter sp., Escherichia sp., Azototobacter sp., Protomonas sp., Cupriavidus sp., Halomonas sp., Saccharomyces sp., Kluyveromyces sp., and Ralstonia sp. are known to produce bioplastics, the industrial production of bioplastics is still challenging. Thus this paper also provides deep insights on the advancements made to maximise production of bioplastics using different approaches such as metabolic engineering, rDNA technologies and multitude of cultivation strategies. Finally, the constraints to microbial bioplastic production and the future directions of research are briefed. Hence the present review emphasizes on the importance of using bioplastics as a sustainable alternative to petroleum based plastic products to diminish environmental pollution.

Immune response to COVID-19 vaccination is attenuated by poor disease control and antimyeloma therapy with vaccine driven divergent T-cell response.

Myeloma patients frequently respond poorly to bacterial and viral vaccination. A few studies have reported poor humoral immune responses in myeloma patients to COVID-19 vaccination. Using a prospective study of myeloma patients in the UK Rudy study cohort, we assessed humoral and interferon gamma release assay (IGRA) cellular immune responses to COVID-19 vaccination post second COVID-19 vaccine administration. We report data from 214 adults with myeloma (n = 204) or smouldering myeloma (n = 10) who provided blood samples at least three weeks after second vaccine dose. Positive Anti-spike antibody levels (> 50 iu/ml) were detected in 189/203 (92.7%), positive IGRA responses were seen in 97/158 (61.4%) myeloma patients. Only 10/158 (6.3%) patients were identified to have both a negative IGRA and negative anti-spike protein antibody response. In all, 95/158 (60.1%) patients produced positive results for both anti-spike protein serology and IGRA. After adjusting for disease severity and myeloma therapy, poor humoral immune response was predicted by male gender. Predictors of poor IGRA included anti-CD38/anti-BCMA (B-cell maturation antigen) therapy and Pfizer-BioNTech vaccination. Further work is required to understand the clinical significance of divergent cellular response to vaccination.

Understanding health anxiety in the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic continues to be one of the greatest public health challenges faced by the UK. Reported rates of psychiatric difficulties have increased and the mechanisms by which the pandemic has affected mental health requires investigation. AIMS: The aim of the study was to understand the psychological impact of the COVID-19 pandemic on health anxiety in the general population, with a focus on Oxford Health NHS Foundation Trust, which provides specialist mental health care within Oxfordshire and Buckinghamshire. METHODS: We invited participants aged 18+ across Oxfordshire and Buckinghamshire to complete an online questionnaire consisting of standardised questionnaires measuring psychological wellbeing, non-standardised questions to provide demographic information, and information about social circumstances and behaviour during the COVID-19 pandemic. Participants were then invited via email to complete a 3-month follow-up questionnaire. RESULTS: About 324 participants completed the baseline survey while 199 completed the follow-up. Our analysis demonstrated that higher scores on a health anxiety inventory were related to reports of depression, anxiety, loneliness, poor quality of life and obsessive-compulsive symptoms and panic. We also identified several predictors of health anxiety such as being female, having a pre-existing physical or mental health condition, and poor tolerance of uncertainty. Lifestyle and demographic factors such as changes in financial situation, changes in employment status, having a close relative or friend with COVID-19, being able to do grocery shopping, being a key worker, and ethnicity were not found to predict health anxiety. CONCLUSIONS: Awareness of the impact of health anxiety on people needs to be consistently high among healthcare professionals, especially in the continuing fallout from the COVID-19 pandemic. The relationship between health anxiety and fatigue, sleep quality, gender and co-morbid mental and physical health difficulties needs to be investigated further to determine areas of intervention.

Prospective Assessment of Tumour Burden and Bone Disease in Plasma Cell Dyscrasias Using DW-MRI and Exploratory Bone Biomarkers.

Novel biomarkers for tumour burden and bone disease are required to guide clinical management of plasma cell dyscrasias. Recently, bone turnover markers (BTMs) and Diffusion-Weighted Magnetic Resonance Imaging (DW-MRI) have been explored, although their role in the prospective assessment of multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) is unclear. Here, we conducted a pilot observational cohort feasibility study combining serum BTMs and DW-MRI in addition to standard clinical assessment. Fifty-five patients were recruited (14 MGUS, 15 smouldering MM, 14 new MM and 12 relapsed MM) and had DW-MRI and serum biomarkers (P1NP, CTX-1, ALP, DKK1, sclerostin, RANKL:OPG and BCMA) measured at baseline and 6-month follow-up. Serum sclerostin positively correlated with bone mineral density (r = 0.40-0.54). At baseline, serum BCMA correlated with serum paraprotein (r = 0.42) and serum DKK1 correlated with serum free light chains (r = 0.67); the longitudinal change in both biomarkers differed between International Myeloma Working Group (IMWG)-defined responders and non-responders. Myeloma Response Assessment and Diagnosis System (MY-RADS) scoring of serial DW-MRI correlated with conventional IMWG response criteria for measuring longitudinal changes in tumour burden. Overall, our pilot study suggests candidate radiological and serum biomarkers of tumour burden and bone loss in MM/MGUS, which warrant further exploration in larger cohorts to validate the findings and to better understand their clinical utility.

Characterization of the major antifungal extrolite from rice endophyte Lysinibacillus sphaericus against Rhizoctonia solani.

Sheath blight of rice caused by Rhizoctonia solani is regarded as one of the most widely distributed diseases of rice, and is one of the major production constraints for rice in India and most rice-growing countries of Asia. Biological control of plant diseases using antagonistic bacteria is now considered as a promising alternative to the use of hazardous chemical fungicides or bactericides. Several bacterial endophytes have been reported to support growth and improve the health of the plants and therefore, may be important as biocontrol agents. In the present study, putative antifungal metabolites were extracted from rice foliage endophyte Lysinibacillus sphaericus KJ872548 by solvent extraction methods and purified using HPTLC techniques. Separated bands were subjected to assess the in vitro antagonistic activity toward rice sheath blight pathogen Rhizoctonia solani using a dual culture method. Partially purified active fraction B2 obtained from HPTLC analysis showed the highest percentage of inhibition (76.9%). GC MS and FTIR analyses of B2 revealed the compound as1, 2-Benzenedicarboxylic acid butyl 2-Ethylhexyl ester, a strong antifungal volatile organic compound. Light microscopic analysis of the fungal mycelium from the dual culture plate of both culture filtrate and 1, 2-Benzenedicarboxylic acid butyl 2-Ethylhexyl ester disclosed strong mycolytic activity as evident by mycelial distractions and shrinkage. This is the first report on antifungal production by endophytic Lysinibacillus sphaericus against R. solani, the rice sheath blight pathogen. The findings of this study biologically prospect the endophyte L. sphaericus as an inexpensive broad spectrum bioagent for eco-friendly, economic and sustainable agriculture.

Induction of defence response in Oryza sativa L. against Rhizoctonia solani (Kuhn) by chitosan nanoparticles.

Rice is a major food source for more than half of the world population. It is the only crop whose cultivation utilizes large area of land for food production. Recent surveys on rice production revealed that its potential yield has drastically reduced and its demand has exceeded over its production. In this context, the present study focuses on the biocontrol ability of chitosan nanoparticles (ChNP) against rice sheath blight pathogen (ShB) Rhizoctonia solani. To determine the percent disease suppression, detached leaf assay followed by greenhouse experiments were performed. The defence enzyme levels as well as the total phenol content were analysed to understand the mechanism of action of ChNP against ShB. This study recommends ChNP as a cost-effective alternative for chemical fungicides with potential biocontrol efficacy. ChNP was found to suppress 90% disease in detached leaf assay and 75% under greenhouse conditions. The enzyme specific activity of all the defence enzymes were significantly higher than the chemical control. The peroxidase, phenylalanine ammonia-lyase and chitinase enzymes were found to be the most active defence enzymes with 0.19,7.28 and 118.16 U/min/ml/mg protein compared to 0.01, 4.99 and 62.22 U/min/ml/mg protein for control. Hence, the current study suggest that ChNP, a non-toxic biodegradable biopolymer, can be an effective biocontrol agent against ShB caused by R.solani. It is a potent plant immunity booster that can be used as a suitable alternative to commercially available chemical fungicides. Further field trials in this respect are needed to determine the dosage for their application in rice fields.

Sequence analysis and docking performance of extracellular chitinase from Bacillus pumilus MCB-7, a novel mangrove isolate.

Bacterial chitinases has a major role in chitinaceous waste management, biological control of pests and phytopathogens. In the present study, exochitinase gene ChitA encoding extracellular chitinase from the mangrove bacteria Bacillus pumilus MCB-07 was genetically characterized. Oligonucleotide primers specific to chitinase gene of Bacillus pumilus were designed and amplified by PCR. The purified PCR product was successfully cloned in pGEM-T vector and transformed into Escherichia coli DH5-α competent cells. Nucleotide sequence alignment of the chitinase gene revealed 96 % similarity whereas 94 % of the catalytic domain of 598 amino acids is conserved with protein family GH18 chitinases, which is a novel report for Bacillus pumilus. The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which demonstrated that chitinase of Bacillus pumilus MCB-07 is a novel gene. Multiple sequence alignment of chitinase gene sequences and its predicted amino acid sequences were also evaluated and the sequence was deposited in GenBank with accession number KT966736.1. Homology modeling of the chitinase depicted the typical (α/ß) 8 TIM barrel structure. Molecular docking of the protein was performed by Autodock 4.2.6 and the docked pocket contained Val 113, Met 114, Gln 99, Ala 75 and Cys 98 as the key binding residues. The molecular docking of Bacillus pumilus chitinase, revealed the involvement of a phenylalanine of the catalytic domain in the catalytic process of chitin to mono and oligomers of NAG. The amino acid exhibited both hydrophobic and hydrogen bond interactions of chitin molecules with phenylalanine.

Exploring the efficacy of antagonistic rhizobacteria as native biocontrol agents against tomato plant diseases.

As the environmental and health concerns alert the necessity to move towards a sustainable agriculture system, biological approach using indigenous plant growth-promoting rhizobacteria (PGPR) gains a strong impetus in the field of plant disease control. In this context, the present review article addresses the usage of rhizospheric antagonistic bacteria as a suitable alternative to control tomato fungal diseases namely Fusarium wilt and early blight disease. Biological control has been considered to be an eco-friendly, safe and effective method for disease management. The inherent traits of PGPR to antagonize a pathogen through various mechanisms has been investigated extensively to utilize them as potent biocontrol agents (BCA). Hence, the article provides a detailed account on different biocontrol mechanisms displayed by BCA. It is also suggested that the use of bacterial consortium ensures consistent performance by BCA in field conditions. Likewise, this review also deals with the opportunities and obstacles faced during commercialization of these antagonistic bacteria as biocontrol agents in the market.

Enhancing Activity by Supercritical CO2 Mediated Immobilization of Lipase on Mesocellular Foam in Preparation of Hexyl Laurate.

Hexyl laurate is employed in several cosmetics having great demand. It could be synthesized catalytically like a "natural" perfume using a lipase. The use of mesocellular foam silica (MCF) for immobilization of lipases could be made using supercritical CO2 as a medium to enhance its activity in comparison with the normal techniques. Three different catalysts were supported on MCF such as Candida antractica B (CALB), Amano AYS, and Porcine pancreas (PPL), and their activity was evaluated in the preparation of hexyl laurate from lauric acid and hexyl alcohol. CALB@ MCF was the best among all. A systematic study was conducted to assess the effects of different operating parameters. It was ternary complex mechanism with inhibition by hexyl alcohol. The enzyme was reusable and the process is green.

Removal of Selected Pharmaceuticals and Personal Care Products from Wastewater using Soybean Peroxidase.

Personal care products and pharmaceuticals have been reported in various concentrations in the effluent of municipal sewage treatment plants (STP). Although they are generally found in the nanogram to microgram per liter range, many of them might have adverse health effects on humans at these concentrations. Conventional treatments applied at the STP are unable to effectively remove most of these recalcitrant compounds, thus there is a necessity for development of alternative treatment techniques. In this article, the efficiency of enzymatic treatment using soybean peroxidase in treating some commonly found micropollutants is discussed. The target compounds were, two phenolic surfactant breakdown products, nonylphenol and octylphenol, two antimicrobial agents, Triclosan and sulfamethoxazole and three phenolic steroids. The effects of the most important parameters pH, enzyme concentration and peroxide concentration have been evaluated for each compound. The treatment of synthetic wastewater was shown to be effective (≥95% removal), except for sulfamethoxazole, in concentration ranges of 10 s of µM at neutral pH with 2-5 mU/L of catalytic activity and 2-3 molar equivalents of hydrogen peroxide. The effectiveness of the treatment has also been determined for lower concentrations (6-9 nM) which approximate those in real wastewater. A matrix effect was found in the treatment of Triclosan in spiked real wastewater indicating that re-optimization of important parameters for STP treatment would be required to achieve high removal efficiency. A reverse-phase, solid-phase extraction technique was used to concentrate target analytes in real wastewater, enabling chromatographic detection by UV absorbance.

Nitrogen fixing bacterial diversity in a tropical estuarine sediments.

Microorganisms play a significant role in biogeochemical cycles, especially in the benthic and pelagic ecosystems. Role of environmental parameters in regulating the diversity, distribution and physiology of these microorganisms in tropical marine environment is not well understood. In this study, we have identified dinitrogen (N2) fixing bacterial communities in the sediments by constructing clone libraries of nitrogenase (nifH) gene from four different stations in the Cochin estuary, along the southeastern Arabian Sea. N2 fixing bacterial clones revealed that over 20 putative diazotrophs belong to alpha-, beta-, gamma-, delta- and epsilon- proteobacteria and firmicutes. Predominant genera among these were Bradyrhizobium sp. (α-proteobacteria), Dechloromonas sp. (ß-proteobacteria); Azotobactor sp., Teredinibacter sp., Methylobacter sp., Rheinheimera sp. and Marinobacterium sp. (γ-proteobacteria); Desulfobacter sp., Desulfobulbus sp. and Desulfovibrio sp. (δ -proteobacteria); Arcobacter sp. and Sulfurospirillum sp. (ε-proteobacteria). Nostoc sp. was solely identified among the cyanobacterial phylotype. Nitrogen fixing Sulfate reducing bacteria (SRBs) such as Desulfobulbus sp., Desulfovibrio sp., Desulfuromonas sp., Desulfosporosinus sp., Desulfobacter sp., were also observed in the study. Most of the bacterial nifH sequences revealed that the identities of N2 fixing bacteria were less than 95% similar to that available in the GenBank database, which suggested that the sequences were of novel N2 fixing microorganisms. Shannon-Weiner diversity index of nifH gene ranged from 2.95 to 3.61, indicating an inflated diversity of N2 fixing bacteria. Canonical correspondence analysis (CCA) implied positive correlation among nifH diversity, N2 fixation rate and other environmental variables.