Browsing by Subject "Bacteria"

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Open Access
DNA and its bending: a glimpse of mechanism and implication in bacteriology
(University of North Bengal, 2010-03) Mukherjee, Sayani; Dam, Bomba; Sen, Sukannta K
The doub le hdkol DN A s lrurturc vorlcs from one s pecie., to oth er. Donel DNA Is nl so ovn llnblc In the cell. Bending is c.xprcs$c
Open Access
Studies on microbial diversity of some fish products of North Bengal
(University of North Bengal, 2022) Yonle, Rujas; Pal, Joydeb
Fish is an important source of nutrition in many people's diets around the world. Fish and fish products are regularly consumed by people of various ethnicities in North Bengal. The current study examines the various types of fish consumed in North Bengal. During the survey, three types of locally prepared fish products were documented: Loah ko Dalla, Sidol, and Jhinghe Maacha. Among the three fish products, Loah ko Dalla was primarily consumed in parts of the Darjeeling and Kalimpong districts of North Bengal, particularly in villages along the river Rangeet, Balasan and Relli. Only the Rajbanshi tribes of North Bengal plains were found to consume the product Sidol. Jhinghe Maacha, on the other hand, was a popular fish product consumed widely throughout North Bengal. The fish products Loah ko Dalla and Sidol were rarely sold in the open market and were only produced for domestic consumption, whereas Jhinghe Maacha was readily available in all of North Bengal's fish markets. Four Loah ko Dalla sample batches, three Sidol sample batches, and four Jhinghe Maacha sample batches were collected from various parts of North Bengal. A total of 219 isolates of microorganisms were isolated from the eleven samples. All of the samples collected were found to have a 100% prevalence of LAB (Lactic acid Bacteria). All of the fish products had a microbial load of LAB ranging from 103 to 105 cfu/g. From the 148 LAB strains isolated from all of the fish products, 113 were cocci and were identified as Lactococcus plantarum, Leuconostoc mesenteroides, Pediococcus pentosaceus, Enterococcus faecium, and Enterococcus faecalis, while the remaining 35 were non-spore forming rods and were identified as Lactobacillus fructosus and Lactobacillus plantarum. A total of 62 spore former strains were isolated from the 11 samples of fish products collected, out of which 42 strains were endospore forming rods and 20 strains were aerobic cocci. The prevalence of endospore forming rods in all the fish samples were 91 % and aerobic cocci 64%. The microbial load of the spore formers was found to be< 103 cfu/gm with values ranging from 101 to 104 cfu/g. The isolated strains of endospore forming rods were identified as Bacillus subtilis and Bacillus pumilus. The isolated 20 strains of aerobic cocci were all identified as Micrococcus sp. The Micrococcus had a prevalence of 64% in all the samples analysed. The microbial load of yeast in all the samples were found to be <1 cfu/g and no mould could be ascertained from all the products analysed. All 9 strains of yeast were isolated from only Loah ko Dalla and were identified as Candida sp. The prevalence of the yeast in all the samples was found to be 27%. The total viable count of the 11 samples was found between 103 to 105 cfu/g. The major food pathogens Bacillus cereus, Staphylococcus aureus and Enterobacteriaceae were isolated from all the fish products. Bacillus cereus has a prevalence of 73 % in all the fish products analysed with microbial load ranging between 102 to 103 cfu/ g. Staphylococcus aureus was also isolated from all the fish samples with a prevalence of 64% and microbial load ranging between 102 to 103 cfu/g. Enterobacteriaceae was also isolated from all the fish samples with a prevalence of 82% and microbial load of 103 cfu/g in all the fish samples. The proximate analysis of all the fish samples revealed the pH of 6 to7 in all the samples with no detectable acidity. Sida! had the highest moisture content of 50%, and Jhinghe maacha had the highest ash content of 82.44 %. The protein content was highest in Jhinghe Maacha and lowest inLoah ko Dalla with 54.55% and 9.27% respectively. The fat content was highest with 50% in Loah ko Dalla and lowest in Sida! with 21. 7 5%. All fish samples were tested for mineral content, specifically calcium, iron, magnesium, manganese, and zinc. The calcium content in Loah ko dalla, Sida!, and Jhinghe maacha was 38.42 mg/lO00g, 50.24 mg/lO00g, and 129.65 mg/lO00g, respectively. The iron content in Loah ko dalla, Sida!, and Jhinghe maacha was 56.51 mg/lO00g, 41.28 mg/lO00g, and 35.50 mg/1 000g, respectively. The magnesium content in Loah ko dalla, Sida!, and Jhinghe maacha was 77.25 mg/l000g, 129.63 mg/l000g, and 137.95 mg/l000g, respectively. Manganese content in Loah ko dalla was 2.41 mg/lO00g, Sidol 3.99 mg/lO00g, and Jhinghe maacha 1.2 mg/1 000g. The zinc content in Loah ko dalla was 4.13 mg/1 000g, Sidol 8.25 mg/lO00g, and Jhinghe Maacha 3.60 mg/l000g. The findings of the research indicate the presence of LAB, spore formers, and yeast in all of the fish products. Although none of the pathogens exceeded the hazard limit, the presence of pathogenic bacteria Bacillus cereus, Staphylococcus aureus, and Enterobacteriaceae in the entire fish sample indicates poor handling and preservation practices. The research finding also indicates the fish products contain substantial amount of food value in terms of protein, fat and nutrient content.
Open Access
Studies on siderophore producing and arsenic resistant bacteria isolated from agricultural soil and their role in biocontrol and bioremediation
(University of North Bengal, 2024) Pradhan, Smriti; Saha, Dipanwita
Arsenic is a toxic metalloid which cause serious health effects to human populationworldwide. Considered as a Group I carcinogen, it exists naturally in soil and ground water,however, human activities lead to high amount of arsenic pollution in these environments.Arsenic finds its entry into human food chain through different agricultural crops grown inarsenic polluted agricultural fields or fields where arsenic contaminated ground water is used for irrigation. In India, West Bengal tops the list of most contaminated states with several reports of arsenic toxicity in human. Therefore, there is an immense necessity for the implementation of efficient arsenic remediation strategies without harming the natural environment as done by the conventional methods for arsenic removal. In this consideration, arsenic remediation using microorganisms can be a cost effective and safer approach. Additionally, since several years, microorganisms have been used for controlling the agricultural crop loss caused due to the attack by plant pathogens. Rhizosphere microorganisms as biocontrol agents are being extremely used as an alternative to the use of synthetic chemicals for the better management of crop production. Moreover, nowadays, rhizobacteria are also receiving much attention in terms of their application in bioremediation of toxic compounds from soil. Therefore, through this study we aimed to focus on the rhizosphere inhabiting bacteria which can be applied as a potent multifunctional agent for arsenic bioremediation as well as in the biocontrol of plant pathogens. The work was conducted with a primary aim of isolating some highly arsenic resistant siderophoregenic strains with strong antagonism against plant pathogens. We then focused on the arsenic removal efficiency of potent isolates considering their field trials for arsenic bioremediation. Another aspect of this study was to prepare a stable and effective bioformulation for management of plant disease. We also isolated and characterized siderophore, suggesting its role as an antifungal metabolite as well as arsenic chelator. Overall, the work was conducted with the following objectives: (i) Isolation of siderophoregenic bacterial strains from agricultural soils and their screening for antifungal activity and arsenic resistance; (ii) Identification and phylogenetic analysis of the selective isolates based on 16S rRNA gene sequence; (iii) Evaluation of antagonistic potential of selected bacterial strains against plant pathogenic fungi; (iv) Characterisation of siderophore and metal complexation studies between siderophore and arsenic; (v) Analysing the presence of genetic determinants of arsenic resistance and antifungal property in the bacterial isolates; (vi) Batch study for arsenic removal from the soil by potent isolate; (vii) Studying the biocontrol potential of selected bacterial strain under greenhouse conditions. For bacterial isolation, rhizosphere soils were collected from agricultural fields of different regions of Darjeeling, Jalpaiguri, Cooch Behar, Alipurduar, Malda and Purulia districts of West Bengal. A total of 821 isolates were obtained from which 603 isolates were screened as siderophore producers. 500 siderophorogenic isolates with prominent haloes were tested for their resistance towards both forms of arsenic i.e., arsenate (AsV) and arsenite (AsIII) from which 230 isolates were able to grow in presence of both AsV and AsIII. Further, in vitro antifungal activity of these isolates was tested by dual culture method against six plant pathogens viz. Lasiodiplodia theobromae, Colletotrichum gloeosporioides, Curvularia eragrostidis, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani. Here, thirty isolates were found to show strong antagonism against all test pathogens among which, three isolates BM3, CDG7 and GST18 were the most efficient antagonists. All thirty isolates were tested for the production of antifungal metabolites including extracellular lytic enzymes and HCN followed by detection of PGPR traits such as phosphatase and IAA production. Maximum isolates showed amylase production followed by lipase and amylase whereas a smaller number of isolates produced chitinase, cellulase and pectinase. Only two isolates showed HCN production. Four isolates were detected with both phosphatase and IAA production where highest amount of IAA was produced by BM3. Biofilm formation, which is also an important mechanism of biocontrol agents, was observed in two different media i.e., Luria Bertani (LB) and M9 yeast extract (M9YE) by all thirty isolates. Comparatively, maximum biofilm production occurred in LB medium where seven isolates appeared as strong biofilm formers. The identity of thirty potential isolates was revealed through morphological, biochemical and phylogenetic characterization. Through 16S rRNA gene sequencing and BLAST similarity search, isolates were distinguished into eight different genera where majority of the isolates (60%) belonged to genus Bacillus followed by Pseudomonas, Serratia, Microbacterium, Lysinibacillus, Proteus, Ensifer and Micrococcus. The sequences of all thirty isolates were deposited in NCBI GenBank and accession numbers were assigned as follows: MN133951, MN133999, MN865204, MN133963, MT032417, MN923204, MN148541, MN120803, MN865978, MN809373, MN809382, MN122130, MN809526, MN809529, MN108490, MN148539, MN809348, MN809357, MN809577, MN912102, MN120791, MN814034, MN865987, MN915155, MN814036, MN918097, MN809367, MN865968 and MN809363. The isolates were also subjected to hemolysis test considering the safety during field application where eighteen isolates were found to be non-hemolytic. Further, the level of arsenic resistance was checked by determining the minimum inhibitory concentration (MIC) of AsV and AsIII against thirty isolates in two different media i.e., LB and minimal salt (MS) medium. Highest MIC value of AsV was observed against M. luteus BPA2 irrespective of the growth medium. This strain also showed maximum resistance towards AsIII in MS medium while in case of LB medium, highest MIC value of AsIII was observed for B. amyloliquefaciens BM3. Arsenic transformation has been one of the mechanisms adopted by microorganisms to cope with arsenic toxicity. In this study, we tested arsenic reducing as well as arsenic oxidizing ability of all thirty isolates where nine isolates reported arsenate reduction and eight isolates showed arsenite oxidation in silver nitrate test. M. luteus BPA2 showed both the abilities of arsenic transformation. Detection for the genetic determinants of arsenate reduction (arsC gene for arsenate reductase) and arsenite oxidation (aoxB gene for arsenite oxidase) was also done inorder to correlate with the arsenic resistant phenotype of four highly resistant isolates. Successful PCR amplification of arsC gene was observed in all four isolates M. luteus BPA2, B. amyloliquefaciens BM3, P. putida BPA1 and L. macroides SUT34. However, aoxB gene was amplified in three isolates except L. macroides SUT34. The amplicons were cloned in pGEMT Easy vector and sequenced. The obtained gene sequences were submitted to GenBank through BankIt tool and the provided accession numbers are: arsC (OR875842, OL405606, OR195443 and OR228422) and aoxB (OR875841, OL405605 and OR875843). The growth study performed for M. luteus BPA2 and B. amyloliquefaciens BM3 (with highest MIC values) under arsenic stress in LB and MS media revealed higher impact of AsIII on the growth of both bacteria than AsV. Moreover, arsenic stress lowered the growth rate more in MS medium than LB medium. In comparison, M. luteus BPA2 showed better growth under arsenic stress in both media than B. amyloliquefaciens BM3. Scanning electron microscopy showed slightly irregular surface morphology of M. luteus BPA2 cells whereas an increase in cell size was observed in B. amyloliquefaciens BM3 under arsenic stress. M. luteus BPA2 and B. amyloliquefaciens BM3 showed efficient arsenic removal ability in both in vitro and in vivo studies. The quantitative study for siderophore production done through CAS shuttle assay revealed varied level of siderophore production by thirty isolates with B. amyloliquefaciens BM3 producing highest amount of siderophore (94.15 p.s.u). The chemical nature of siderophore was also detected where eighteen isolates produced hydroxamate type, thirteen isolates showed carboxylate type and only seven isolates were detected with catecholate type of siderophore. Siderophore from B. amyloliquefaciens BM3 was extracted and purified though Amberlite XAD2 and Sephadex LH20 column chromatography. Following purification, thin layer chromatography was done where a single spot was observed with Rf 0.84 under UV light (365 nm) and also by spraying with FeCl3. The compound was identified as bacillibactin through spectroscopic analyses including FT-IR, NMR and LCMS. Bacillibactin was studied for its metal chelating ability through qualitative and quantitative CAS assay which revealed that this siderophore could chelate both forms of arsenic apart from iron and the binding affinity was in the order Fe> AsV>AsIII. This observation was further confirmed by fluorescence spectroscopic analysis. SEM study of the interaction between our most potent biocontrol agent B. amyloliquefaciens BM3 and F. oxysporum shows several morphological abnormalities in the mycelia of fungal pathogen. The genes of three antifungal metabolites i.e, chitinase (chiA), bacilysin (bacAB) and surfactin (srfA) were successfully amplified through PCR in B. amyloliquefaciens and subsequently cloned and sequenced. The obtained gene sequences were submitted to GenBank and the following accession numbers were provided: chiA (OL335882), bacAB (MT740320) and srfA (OR228422). Soil inoculation of talc based formulation of B. amyloliquefaciens BM3 was considerably effective in controlling the Fusarium wilt disease in brinjal. The disease control efficiency of this strain was found to be more or less similar to that of the fungicide (thiophanate methyl). Moreover, this strain remained viable in talc formulation upto seven months at room temperature. In conclusion, the current study reports the recovery of useful strains from soil sample with multipurpose potential. The major findings of this study suggest the application of rhizospheric strains for cost effective removal of arsenic from soil. Moreover, we can also consider the development of formulation with the recovered strain as biocontrol products in eco-friendly agriculture. This study also witnesses the first report of arsenic chelation by bacillibactin thereby leading a new insight towards the application of this compound in combating arsenic toxicity in soils.