Valorization of rice straw and vascular aquatic weeds for sustainable prebiotic hemicellulosic autohydrolysate production: Extraction, characterization and fermentability.

This study describes the extraction and characterization of the hemicellulosic autohydrolysates (HAHs) derived from rice straw (RS) and vascular aquatic weeds like Typha angustifolia (TA) and Ceretophyllum demersum (CD). It further explores their capacity to sustain the proliferation of selected lactic acid bacteria (i.e., prebiotic activity) isolated from milk samples. To fractionate HAH from RS, TA and CD hot water extraction (HWE) method was used and RS, TA, and CD biomasses yielded 6.8, 4.99 and 2.98% of HAH corresponding to the hemicellulose extraction efficiencies of 26.15 ± 0.8%, 23.76 ± 0.6%, and 18.62 ± 0.4% respectively. The chemical characterization of HAH concentrates through HPLC showed that they comprised galactose, arabinose, xylose and glucose. The total phenol content of the RS, TA and CD-derived HAH concentrates were 37.53, 56.78 and 48.08 mg GAE/g. The five lactic acid bacteria (LAB) isolates Q1B, Q2A, Q3B, G1C and G2B selected for prebiotic activity assays generated mixed responses with the highest growth in RS-HAH for Q2A and the least in TA-HAH for Q3B. Further, the isolates Q2A, Q3B, G1C, and G2B, which showed the highest growth performance, were identified through MALDI-TOF and 16S rRNA sequencing as Lactobacillus brevis. All the tested LAB isolates showed diauxic growth in crude HAH preparations to maximize the utilization of carbon resources for their proliferation. This suggests that the selected LAB isolates are efficient degraders of hemicellulosic sugars. This paves the way for the valorization of lignocellulosic biomass to produce prebiotic hemicellulosic autohydrolysate and consequently enhances environmental sustainability by improving resource efficiency.

Evaluation of multi-heavy metal tolerance traits of soil-borne fungi for simultaneous removal of hazardous metals.

The demand for environment-friendly cleanup techniques has arisen due to an increase in environmental pollutants. Fungi is the most prevalent and effective class of heavy metal-resistant microorganisms with the ability to leach metals. The objective of the present study was to isolate the fungi from the agricultural soil of Kashmir valley, investigate their multi-metal tolerance to heavy metals and evaluate the metal uptake capacities of the resistant fungi. The fungi were isolated and identified on the basis of morphological and molecular approach (ITS1 and ITS4). The tolerance limits of the isolated fungal strains to various doses of lead (Pb), cadmium (Cd), zinc (Zn), chromium (Cr), copper (Cu), nickel (Ni), and cobalt (Co) was evaluated. Five fungal strains, Aspergillus niger, Fusarium oxysporum, Fusarium verticillioides, Aspergillus fischeri, Epicoccum mackenziei were isolated from the soil samples. To the best of our knowledge, this is the first report on the study of metal resistance of Aspergillus fischeri and Epicoccum mackenziei. Among the identified fungal species, Aspergillus niger and Fusarium oxysporum were found to be most tolerant with a minimum inhibitory concentration (MIC) of 600 ppm against Cu and Cr respectively. Results indicated removal of considerable amount of heavy metals by some of the fungi. The highest metal uptake of 8.31 mg/g was found in Fusarium verticillioides for Zn. Surprisingly, these fungal strains demonstrated resistance to metal concentrations above the levels that are universally acceptable for polluted soils, and hence prove to be appealing contenders for use as bioremediation agents for cleaning up heavy metal-polluted environments.

Biofilm formation and EPS production enhances the bioremediation potential of Pseudomonas species: a novel study from eutrophic waters of Dal lake, Kashmir, India.

The present study was conducted with the aim of isolation and identification of the biofilm-forming denitrifying Pseudomonas bacterial strains from eutrophic waters of Dal lake, India, followed by the study of inter-relation of biofilm formation and denitrification potential of Pseudomonas strains. The bacterial strains were characterized by morphological observations and identified using 16S rDNA sequencing followed by the quantification of biofilm formation of these st by crystal violet (CV) assay using 96-well microtiter plate and extracellular polymeric substance (EPS) extraction. Lastly, the nitrate-reducing potential of all Pseudomonas species was studied. Our evaluation revealed that four different Pseudomonas species were observed to have the biofilm-forming potential and nitrate-reducing properties and the species which showed maximum biofilm-forming potential and maximum EPS production exhibited higher nitrate-removing capacity. Moreover, P. otitis was observed to have the highest denitrification capacity (89%) > P. cedrina (83%) > P. azotoform (79%) and the lowest for P. peli (70%). These results clearly signify a positive correlation of biofilm-forming capacity and nitrate-removing ability of Pseudomonas species. This study has for the first time successfully revealed the bioremediation potential of P. otitis, P. cedrina, P. azotoform, and P. peli species, thus contributing to the growing list of known nitrate-reducing Pseudomonas species. Based upon the results, these strains can be extrapolated to nitrate-polluted water systems for combating water pollution.

The hidden world of fish fungal pathogens: molecular identification and phylogenetic analysis in common carp, Cyprinus carpio.

Fungal infections pose a significant threat to aquaculture, causing substantial economic losses and ecological disruptions. The common carp (Cyprinus carpio), as a crucial farmed fish, requires in-depth research to uncover the underlying fungal pathogens affecting its health. In this study, we analyzed 150 samples of C. carpio to identify the fungal pathogens responsible for the infections based on clinical signs and symptoms. Further, we assessed fungal diversity and prevalence in the infected fish. The infected fish exhibited varying degrees of gross pathogenicity, with fins and skin heavily affected, intermediate infection observed in the head and gills, and the least infection found in the operculum. Morphological examination revealed distinct characteristics such as necrosis, lesions on the skin, fins, and gills, as well as loss of scales, hemorrhagic lesions, and red spots. Furthermore, the presence of gray and white cottony patches on the body confirmed ascomycete and zygomycete infections, while a dark white cottony mass indicated phycomycete infection. Some fish exhibited severe fungal infections, presenting prominently curved spines and necrosis with red spots on the skin. These isolates belonged to various fungal groups, including ascomycetes, zygomycetes, phycomycetes, deuteromycetes, and basidiomycetes. Among these, Fusarium oxysporum emerged as the most prevalent fungal pathogen, followed by Fusarium solani, Saprolegnia delica, and Saprolegnia parasitica. Molecular identification based on ITS and LSU rRNA sequences confirmed the presence of Saprolegnia delica, Mucor hiemalis, Coniothyrium telephii, Rhodotorula mucilaginosa, Penicillium cellarum, and Fusarium californicum in the fish samples. Phylogenetic analysis further supported the morphological and molecular data, providing insights into the relationship between the isolated fungal strains and known species from various geographical regions. Our study enhances our understanding of the diversity and prevalence of fish fungal pathogens in common carp, emphasizing the significance of employing molecular techniques for accurate identification. These comprehensive findings offer essential insights into the impact of fungal infections on common carp populations, laying the groundwork for targeted control measures to mitigate their effects on global aquaculture.

Isolation, molecular characterization and first report of Dothiorella gregaria associated with fruit rot of walnuts of Jammu and Kashmir, India.

Walnuts are known for their high levels of antioxidants, which are linked to various health benefits. However, challenges related to distribution and storage, as well as the risk of fungal infections, can affect the quality of walnut kernels. Fungal pathogens from the Botryosphaeriaceae family, including Dothiorella species and Diplodia species, can damage fruit and reduce its antioxidant content. To comprehend the cause of fruit rot in walnuts, Dothiorella gregaria isolates were studied using polyphasic methods, including multiple gene sequences and morphological identification, as well as analysis of polyphenol content and pathogenicity. The walnuts kernels purchased from market places of Jammu and Kashmir (J&K), India were observed to be affected by Dothiorella gregaria species causing the quality detoriation and decrease in polyphenol content thus undeniably with decreased antioxidant properties. D. gregaria Infected walnut kernels were having some brown and black spots and some were having white mycelial growth and however, most samples were asymptomatic. Pathogenicity testing revealed that the pathogen was able to develop all the symptoms under experimental conditions and the reisolated pathogen was morphologically similar to D. gregaria. The samples infected with this pathogen showed considerable decrease in polyphenol content, 10.9 ± 2.66 mgGAE/g (mean ± standard deviation) thus decreased antioxidant quality as compared to the samples which showed zero incidence of this pathogen, 52.50 ± 4.27 mgGAE/g (mean ± standard deviation). Furthermore, the pathogen was studied using polyphasic approach involving morphological, molecular and phylogenetic analysis. Combined nucleotide dataset of nuclear ribosomal ITS and tef1-α revealed that Dothiorella gregaria (NY6) formed a clade with Dothiorlla iberica (MAEC33), Dothiorella sarmentorium (MAEC28) and Dothiorella iberica (CAA905) strains with 83% bootstrap support. Besides, we observed six nucleotide changes, four were insertions or deletions and two were substitutions in the 502-bp region of the ITS rRNA gene when we compared our isolate to the most equivalent sequences submitted to NCBI GenBank. This is the first report of Dothiorella gregaria affecting walnuts purchased from various markets in J&K, India, causing fruit rot in walnuts after harvest. Given that local farmers store and export walnuts, it could pose an emerging threat to their livelihood. Thus, creating post-harvesting interventions for D. gregaria and knowing more about the fruit rot in walnuts can be benefited from morphological and molecular identification using several gene loci, genetic variability in the ITS rRNA gene, and total phenol analysis.

In vitro Sequestration of Molecular and Mass Spectra Characterized Metallophilic Cadmium Tolerant Bacteria for Sustainable Agriculture.

Due to industrialization, the contamination of toxic metals in soils is currently one of the major concerns to scientists worldwide. The presence of high concentrations of heavy metals including cadmium in the environment is mainly attributed to human activities. Being a highly toxic metal, cadmium can enter plant cell transporters usually used for the uptake of essential cations, such as iron, calcium, and zinc. This study deals with the appraisement of response and tolerance shown by various bacteria in varied cadmium concentrations (100-1,000 ppm). The optical density (OD) of the isolates was measured to determine the minimum inhibitory concentration (MIC) of cadmium. Isolated bacteria have been identified using 16S rRNA gene sequence and Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the 72 isolates, 07 (Bacillus pumilus, Enterobacter kobei, Klebsiella pneumonia, Pseudomonas mandelii, Pseudomonas putida, Pseudomonas avellanae, and Staphylococcus equorum), isolates had efficacy for cadmium tolerance and showed sequestration potential at varying MIC. Furthermore, K. pneumonia was observed to have the highest (900 ppm) tolerance for cadmium and the lowest (600 ppm) was shown by E. kobei. Besides, K. pneumonia showed the highest (75.2%) sequestration potential while the least (52.4%) potential was observed for P. putida. These cadmium tolerant species can be implemented in contaminated environments for detoxification and elimination of cadmium from these agricultural fields. Graphical Abstract.

Molecular characterization of lipase from a psychrotrophic bacterium Pseudomonas sp. CRBC14.

Lipases from Pseudomonas species are particularly useful due to their broader biocatalytic applications and temperature activity. In this study, we amplified the gene encoding wild-type cold-active lipase from the genome of psychrotrophic bacterium isolated from the Himalayan glacier. The isolated CRBC14 strain was identified as Pseudomonas sp. based on the 16S rRNA gene sequence. Lipase activity was determined by observing the hydrolysis zone on nutrient agar containing tributyrin (1%, v/v). The sequence analysis of cold-active lipase revealed a protein of 611 amino acids with a calculated molecular mass of 63.71 kDa. The three-dimensional structure of this lipase was generated through template-supported modeling. Distinct techniques stamped the model quality, following which the binding free energies of tributyrin and oleic acid in the complex state with this enzymatic protein were predicted through molecular mechanics generalized born surface area (MMGBSA). A relative comparison of binding free energy values of these substrates indicated tributyrin's comparatively higher binding propensity towards the lipase. Using molecular docking, we evaluated the binding activity of cold-active lipase against tributyrin and oleic acid. Our docking analysis revealed that the lipase had a higher affinity for tributyrin than oleic acid, as evidenced by our measurement of the hydrolysis zone on two media plates. This study will help to understand the bacterial diversity of unexplored Himalayan glaciers and the possible application of their cold-adapted enzymes.

Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil.

As an approach to the exploration of cold-active enzymes, in this study, we isolated a cold-active protease produced by psychrotrophic bacteria from glacial soils of Thajwas Glacier, Himalayas. The isolated strain BO1, identified as Bacillus pumilus, grew well within a temperature range of 4-30 °C. After its qualitative and quantitative screening, the cold-active protease (Apr-BO1) was purified. The Apr-BO1 had a molecular mass of 38 kDa and showed maximum (37.02 U/mg) specific activity at 20 °C, with casein as substrate. It was stable and active between the temperature range of 5-35 °C and pH 6.0-12.0, with an optimum temperature of 20 °C at pH 9.0. The Apr-BO1 had low Km value of 1.0 mg/ml and Vmax 10.0 µmol/ml/min. Moreover, it displayed better tolerance to organic solvents, surfactants, metal ions and reducing agents than most alkaline proteases. The results exhibited that it effectively removed the stains even in a cold wash and could be considered a decent detergent additive. Furthermore, through protein modelling, the structure of this protease was generated from template, subtilisin E of Bacillus subtilis (PDB ID: 3WHI), and different methods checked its quality. For the first time, this study reported the protein sequence for psychrotrophic Apr-BO1 and brought forth its novelty among other cold-active proteases.

Biochemical Characterization and Functional Analysis of Heat Stable High Potential Protease of Bacillus amyloliquefaciens Strain HM48 from Soils of Dachigam National Park in Kashmir Himalaya.

A novel temperature stable alkaline protease yielding bacteria was isolated from the soils of Dachigam National Park, which is known to be inhabited by a wide variety of endemic plant and animal species of Western Himalaya. This high-potential protease producing isolate was characterized and identified as Bacillus amyloliquefaciens strain HM48 by morphological, Gram's staining and biochemical techniques followed by molecular characterization using 16S rRNA approach. The extracellular protease of B. amyloliquefaciens HM48 was purified by precipitating with ammonium sulfate (80%), followed by dialysis and Gel filtration chromatography increasing its purity by 5.8-fold. The SDS-PAGE analysis of the purified enzyme confirmed a molecular weight of about ≈25 kDa. The enzyme displayed exceptional activity in a broad temperature range (10-90 °C) at pH 8.0, retaining its maximum at 70 °C, being the highest reported for this proteolytic Bacillus sp., with KM and Vmax of 11.71 mg/mL and 357.14 µmol/mL/min, respectively. The enzyme exhibited remarkable activity and stability against various metal ions, surfactants, oxidizing agent (H2O2), organic solvents and displayed outstanding compatibility with widely used detergents. This protease showed effective wash performance by exemplifying complete blood and egg-yolk stains removal at 70 °C and efficiently disintegrated chicken feathers making it of vital importance for laundry purpose and waste management. For functional analysis, protease gene amplification of strain HM48 yielded a nucleotide sequence of about 700 bp, which, when checked against the available sequences in NCBI, displayed similarity with subtilisin-like serine protease of B. amyloliquefaciens. The structure of this protease and its highest-priority substrate ß-casein was generated through protein modeling. These protein models were validated through futuristic algorithms following which protein-protein (protease from HM48 and ß-casein) docking was performed. The interaction profile of these proteins in the docked state with each other was also generated, shedding light on their finer details. Such attributes make this thermally stable protease novel and suitable for high-temperature industrial and environmental applications.

MALDI-TOF-MS and 16S rRNA characterization of lead tolerant metallophile bacteria isolated from saffron soils of Kashmir for their sequestration potential.

Toxic metal contamination in soils due industrialization is nowadays a concern to the scientists worldwide. The current study deals with the evaluation of response and tolerance by isolated metallophilic bacteria in different lead concentrations (100 ppm to 1000 ppm). By taking optical densities of the isolates, the minimum inhibitory concentration (MIC) of Pb2+ were determined.16S rRNA and MALDI-TOF MS were used for the identification of the bacteria. Total of 37 isolates were observed, among them 04 (Staphylococcus equorum, Staphylococcus warneri, Bacillus safensis and Bacillus thuringiensis), isolated were detected having efficacy of Pb2+tolerance and sequestration at varying MIC. Furthermore, B. thuringiensis was observed to have highest (900 ppm) tolerance for lead and lowest (500 ppm) for Staphylococcus warneri. Moreover, the highest (65.3%) sequestration potential has been observed for B. thuringiensis and least (52.8%) for S. warneri. The tolerance and sequestration potential properties of these isolated species can be utilised to exterminate heavy metals and reduce their toxicity from the contaminated environment.

Comparative evaluation of extraction methods for total proteins from Crocus sativus L. (Saffron).

Broadly speaking proteomic studies are one of the various techniques of utmost importance for understanding complex biological processes that occur under inductive conditions and revealing the multidimensional aspects of Crocus sativus in biological systems. In order to get an insight into the molecular changes and to characterize the variations in protein expression of C. sativus, a detailed proteomic analysis on one-dimensional gel electrophoresis is one of the basic steps to accomplish. We have compared total protein profiles of C. sativus extracted by three different recipes and analyzed on 10% sodium dodecyl sulfate polyacrylamide gels. Gels were subjected to densitometric analysis for further characterization. Among three different protocols NP-40 extraction buffer recipe resulted in the extraction of proteins most efficiently with minimum background and streaking. There was maximum solubilization of proteins with high efficiency. Such a profile can be used for high precision analysis of differential protein expression. This work is an attempt to assist researchers in effective extraction of proteins from C. sativus. As a researcher faces a perplexing array of choices as where to start we describe a method based on our collective analysis of the different protein protocols. This paper presents a method that could be applied at the outset of any proteomic study.

Fish antimicrobial peptides (AMP's) as essential and promising molecular therapeutic agents: A review.

Antimicrobial peptides (AMPs) are generally considered as an essential component of innate immunity, thereby providing the first line of defense against wide range of pathogens. In addition, they can also kill the pathogens which are generally resistant to number of antibiotics, thereby providing the avenues for the development of future therapeutic agents. Fishes are constantly challenged by variety of pathogens which not only shows detrimental effect on their health but also increases risk of becoming resistant to conventional antibiotics. As fishes rely more on innate immunity, AMPs can serve as a potential defensive weapons in fishes for combating emerging devastating diseases. Generally, AMPs show multidimensional properties like rapid diffusion to the site of infection, recruitment of other immune cells to infected tissues and vigorous potential to rapidly neutralize broad range of pathogens (bacterial, fungal and viral). AMPs also exhibit diverse biological effect like endotoxin neutralization, immunomodulation and induction of angiogenesis in mammals. Due to these properties AMPs have become one of the most promising therapeutic agents to be studied. Till date, many AMPs have been isolated from the fishes but not fully characterized at molecular level. This review provides an overview of the structures, functions, and putative mechanisms of major families of fish AMPs. Further, we also highlighted how fish AMPs can be used as a novel therapeutic tool which is the theme of future research in drug development.

A report on the incidence of Trichodina heterodentata from fishes of Jammu, J&K India.

The present study was aimed at screening for the presence of protozoan's among Cyprinid fishes collected from various fish ponds and farms in Jammu division of Jammu and Kashmir (J and K) state. Out of 75 fishes collected from local water bodies of Jammu division, only 35, (49.6 %) were infested with Trichodina. Trichodina infestations were studied in the period from November to February. In light infestation Trichodina was usually present on gills, fins and skin of apparently healthy fish. Clinical signs of Trichodiniasis appeared on fish with heavy infections and in presence of one or more stress factors including, rough handling during transportation from ponds, over crowdedness, malnutrition, high concentration of free ammonia and low oxygen concentration. Clinical signs of Trichodiniasis in fish such as sluggish movement, loss of appetite, black colouration, necrosis and ulcer on different parts of the body, detached scales and excessive accumulation of mucous in gill pouches were also observed.

Enhanced production of α-amylase by Penicillium chrysogenum in liquid culture by modifying the process parameters.

In this paper, we have assessed the role of changing physicochemical parameters and substrate types on the production of α-amylase enzyme from Penicillium chrysogenum, with a view to determining the optimal conditions required for its maximum production. The findings of this research revealed that, at pH 6 using linseed oil cake as substratum, α-amylase enzyme production was maximum (550.0 U/mL), when the fungi was incubated for 6 days at 30 °C in 0.1 M acetate buffer. Further, reasonably good production of the α-amylase enzyme was also observed at pH 9 with all the experimented carbon sources as substrates. Moreover, statistical analysis, using analysis of variance (ANOVA) carried out to study the impact of different studied parameters on the α-amylase enzyme production revealed that incubation period of 6-18 days is highly significant (p = 0.01) factor in amylotic activity of the P. chrysogenum. Under the researched out optimal conditions, P. chrysogenum is an economically viable option for the industrial and biotechnological production of α-amylase enzyme.