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A radio-resistant bacterium labeled as strain TMC-6 was isolated from Thal desert, Pakistan and identified through 16S rRNA gene sequencing as Bacillus indicus strain TMC-6 (MN721293). The isolate was found to be resistant to UV radiation dose of 6.780 × 103 J/m2 and showed 50% survivability to mitomycin C (6 μg/ml) and H2O2 (30 mM). The bacterium showed yellowish orange coloration when grown on tryptone yeast glucose (TGY) medium. The cellular metabolite was extracted in methanol and purified through solid phase extraction with C18 column cartridge. The compound was characterized through UV/Visible spectrophotometry, Fourier Transform Infra-Red (FT-IR) spectroscopy and Liquid Chromatography Mass Spectrometry (LC–MS). The LC–MS analysis of the compound revealed a molar mass of 769 [m/z]− that matched the chemical formula C34H42O20 and identified as a glycosylated flavonoid xanthorhamnin. The compound showed significant antioxidant (77.05%) and metal chelation (79.80%) activities. Xanthorhamnin showed promising oxidative damage inhibitory actions in bovine serum albumin (65.32%) and mice liver lipids (71.61%) and prevented DNA strand breaks from oxidative stress. Cytotoxicity in brine shrimp larvae was observed when compared with mitomycin C indicating its effect toward cancerous cells. These findings concluded that xanthorhamnin from radio-resistant Bacillus indicus strain TMC-6 has high antioxidant, radioprotective, and antitumor properties against UV-mediated oxidative damages.

This study aims to determine UV-B resistance and to investigate computational analysis and antioxidant potential of methoxy-flavones of Micromonospora aurantiaca TMC-15 isolated from Thal Desert, Pakistan. The cellular extract was purified through solid phase extraction and UV-Vis spectrum analysis indicated absorption peaks at λmax 250 nm, 343 nm, and 380 nm that revealed the presence of methoxy-flavones named eupatilin and 5-hydroxyauranetin. The flavones were evaluated for their antioxidant as well as protein and lipid peroxidation inhibition potential using di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium (DPPH), 2,4-dinitrophenyl hydrazine (DNPH), and thiobarbituric acid reactive substances (TBARS) assays, respectively. The methoxy-flavones were further studied for their docking affinity and interaction dynamics to determine their structural and energetic properties at atomic level. The antioxidant potential, protein and lipid oxidation inhibition and DNA damage preventive abilities were correlated as predicted by computational analysis. The eupatilin and 5-hydroxyauranetin binding potential to their targeted proteins 1N8Q and 1OG5 is -4.1 and -7.5 kcal/mol, respectively. Moreover, the eupatiline and 5-hydroxyauranetin complexes illustrate van der Waals contacts and strong hydrogen bonds to their respective enzymes target. Both in-vitro studies and computational analysis results revealed that methoxy-flavones of Micromonospora aurantiaca TMC-15 can be used against radiation mediated oxidative damages due to its kosmotrophic nature. The demonstration of good antioxidant activities not only protect DNA but also protein and lipid oxidation and therefore could be a good candidate in radio-protective drugs and as sunscreen due to its kosmotropic nature.

Polysaccharides including resistant starch are categorized as dietary fiber and are used as an important prebiotic. Similar to soluble fibers, resistant starch also has a number of physiological effects that have been shown to be beneficial for health. Starch hydrolyzing enzymes, most importantly amylases, play essential roles in the production of resistant starch. This study aimed to develop α-amylase-treated maize flour with slow digestibility and unique physicochemical characteristics compared to native maize flour. In the current study, resistant starch type III from maize flour was prepared using α-amylase obtained from indigenously isolated Bacillus licheniformis. The α-amylase gene from B. licheniformis was amplified and cloned into the pET-24(a) vector, expressed in E. coli BL21 (DE3) cells and purified by metal ion affinity chromatography. The purified enzyme enhanced the yield of resistant starch 16-fold in maize flour. Scanning electron microscopy revealed that the granular structure of maize flour was disrupted into a dense network with irregular structure, and X-ray diffractograms confirmed the transformation from an amorphous to a crystalline structure upon α-amylase treatment. Thermogravimetric analysis revealed increased amylose content of α-amylase-treated maize flour. Moreover, α-amylase-treated maize flour resulted in a significant enhancement of the desired properties of maize flour, such as resistant starch content, amylose, milk absorption capacity, and iodine and fatty acid complexing ability, and a reduction in swelling power, water binding, oil absorption capacity, and in vitro digestibility compared to untreated maize flour. Resistant starch type III showed low digestibility and increased complexing ability with iodine and fatty acid and therefore could be a safe and beneficial alternative as a coating material for the delivery of active, sensitive ingredients to the colon.

Hydrolytic bacteria are essential for the degradation of lignocellulose to produce biogas and organic fertilizers. In this study, sheep manure was used as substrate, and sheep manure slurry, yak rumen fluid and slurry from a biogas reactor (SBR) were used as inocula in single-stage anaerobic digestion. The SBR and rumen fluid inocula increased biogas production by 23% and 43%, respectively, when compared to solely sheep manure in the single-stage anaerobic digestion. The two-stage anaerobic digestion, with yak rumen fluid as inoculum in the hydrolytic reactor, increased the biogas production by 59, 86, and 58% compared with the control. Microbial analysis of the effluent revealed that yak rumen fluid contained hydrolytic bacteria such as Proteiniphilum, Jeotgalibaca, Fermentimonas, and Atopostipes to enhance the degradation of sheep manure and increase biogas production. It was concluded that yak rumen fluid, rich in hydrolytic bacteria, increases the degradability of sheep manure and improves production of volatile fatt acids and biogas.

Curli fimbriae, a virulent factor of the Avian Pathogenic Escherichia coli (APEC), is responsible for adhesion, biofilm formation, and colonization of pathogen. Major curli fimbriae protein is encoded by csgA gene. APEC is one of the leading causes of colibacillosis in poultry flocks and due to excessive use of antibiotics and vaccines in poultry, the emergence of various multi-drug resistant (MDR) bacterial strainsare is frequently reported. The growing concern of MDR bacterial strains necessitate novel antibacterial approaches to combat colibacillosis in poultry. RNA-based gene silencing is a very specific and robust strategy to target specific bacterial factors involved in pathogenicity and virulence. In this study, a phagemid-mediated sRNA expression system to target a vital gene, csgA, is employed. This comprises an M13 phagemid harboring a sRNA expression cassette and a pre-designed GUIDE sequences for the csgA target gene. To target the csgA gene at the mRNA level, a GUIDE sequence was computationally designed for pre-designed sRNA expression cassette. Online web tools were used to predict the binding energy, secondary structure, and off-target binding potential of the sRNA to optimize its expression. Results showed that the designed sRNA has a binding energy of - 29.60 kcal/mol with zero off-targets. After expression of the sRNA in the APEC cells, ̴ 45% reduction in the csgA level was observed via RT-PCR in the CS-APEC-O1 strains compared to the wt-APEC-O1. Similarly, the biofilm forming ability decreased by 40% in the CS-APEC-O1 strains. The swarming motility and hemagglutination efficiency were not affected by the sRNA expression. Future studies investigating the in vivo efficiency of M13 phagemid delivery are required to evaluate its candidacy in phage therapy.

This study was conducted to explore the frequency of positivity of cutaneous leishmaniasis (CL) in the tribal district Bajaur located near the Pak-Afghan border. The present study was conducted at the Leishmaniasis Center of Headquarter Hospital Khar District Bajaur, Pakistan. In total, 646 patients were recruited and included in the study after ethical approval and consent from the patients. CL was confirmed by taking blood samples from the sides of the lesion and observing them under a microscope using Giemsa staining. Information about demographic factors was collected from the study participants with a questionnaire and analyzed by SPSS. It was found that 73.8% of suspected patients were positive and 26.2% were negative for CL. There were 51.9% male and 48.1% female patients. The most frequently affected site was the face (42.6%), and most of the patients (85.8%) had only one lesion. The positivity of CL was higher among those under age 15 years. The area of most positivity, with 45.2% of the cases, was Tehsil Mamund. Most of the patients (46.6%) lived in stone houses, with 98.6% of patients having domestic animals in their houses. Approximately 198 patients were treated with intramuscular and intralesional injections of meglumine antimoniate, and their weekly follow-up revealed that 48% of patients recovered, while the remaining patients left the course of treatment at different stages of therapy. The positivity of CL is high in this area and is confirmed by the detection of Leishmania amastigotes in the blood collected from their lesions. Socioeconomic factors are the main underlying causes of the rapid spread of this disease and meglumine antimoniate is an effective drug.

Industrial applications require enzymes to be highly stable and economically viable in terms of reusability. Enzyme immobilization is an exciting alternative to improve the stability of enzymatic processes. Immobilization of β-1,4-xylanase produced by Bacillus licheniformis S3 is performed by using two polymer supports (agar-agar and calcium alginate). The maximum enzyme immobilization yield was achieved at a concentration of 3% agar, whereas a combination of sodium alginate, 4%, and calcium chloride, 0.3 M, was used for the formation of immobilized beads. The immobilization process increased the optimum reaction time from 10 min to 35 and 40 min for agar and calcium alginate, respectively, and the incubation temperature increased from 55°C to 60°C for agar, but it remained unchanged for calcium alginate. The pH profile of free and immobilized xylanase was quite similar in both cases. Both the techniques altered the kinetic parameters of immobilized β-1,4-xylanase as compared with the free enzyme. The diffusion limit of high molecular weight xylan caused a decline in Vmax of the immobilized enzyme, whereas there was an increase in the Km value. However, calcium alginate-immobilized enzyme displayed broad thermal stability as compared with agar-agar-immobilized enzyme and retained 57.1% of its initial activity at 80°C up to 150 min. Biotechnological characterization showed that the reusability of enzymes was the most striking finding, particularly of immobilized xylanase using agar-agar as immobilization carrier, which after six cycles retained 23% activity.

During in situ biogas up-gradation by supplying hydrogen from an external source and enrichment of hydrogenotrophic methanogens, high pressure of H2 negatively affects hydrolytic and fermentative activities. To overcome this problem, the present study aimed to enrich the hydrogenotrophic methanogens by optimization of various parameters associated with gas recirculation along-with hydrogen supply from the external source. Due to recirculation of gases and supplied hydrogen, methane generation was two-fold higher in the optimal condition than in conventional anaerobic digestion, with the highest methane content of 99%. Additionally, the hydrogenotrophic methanogens were enriched, with a decrease in acetoclastic methanogens and an increase in Bathyarchaeia population, which utilizes H2 and CO2 to produce acetate and lactate as end products. The study concludes that recirculation increases methane production by converting H2 and CO2 into methane and enhances the degradation of organic matter left over undigested in the hydrolytic reactor.

The use of microbial technologies in agriculture is currently expanding quite rapidly with the identification of new bacterial strains, which are more effective in promoting plant growth. In the present study 18 strains of Pseudomonas were isolated from soil sample of Balochistan coastline. Among isolated Pseudomonas strains four designated as SP19, SP22, PS24, and SP25 exhibited biocontrol activities against phytopathogenic fungi, that is, Rhizopus microsporus, Fusarium oxysporum, Aspergillus niger, Alternaria alternata, and Penicillium digitatum; PS24 identified as Pseudomonas aeruginosa by 16srRNA gene bank accession number EU081518 was selected on the basis of its antifungal activity to explore its potential as plant growth promotion. PS24 showed multiple plant growth promoting attributes such as phosphate solubilization activity, indole acetic acid (IAA), siderophore, and HCN production. In order to determine the basis for antifungal properties, antibiotics were extracted from King B broth of PS24 and analyzed by TLC. Pyrrolnitrin antibiotic was detected in the culture of strain PS24. PS24 exhibited antifungal activities found to be positive for hydrogen cyanide synthase Hcn BC gene. Sequencing of gene of Hcn BC gene of strain PS24 revealed 99% homology with the Pseudomonas aeruginosa strain PA01. The sequence of PS24 had been submitted in gene bank accession number KR605499. Ps. aeruginosa PS24 with its multifunctional biocontrol possessions can be used to bioprotect the crop plants from phytopathogens.

Waste activated sludge generated from wastewater treatment plants makes an abundant source of biomass. Its effective utilization through anaerobic digestion (AD) requires pretreatment to disintegrate the sludge matrix and increase organic matter availability. In this study, dewatered waste activated sludge (DWAS) was subjected to alkaline, photocatalytic, and alkaline-photocatalytic pretreatment for its disintegration and subsequent methane production using different concentrations of sodium hydroxide and titania nanoparticles. Individual pretreatment resulted in maximum disintegration degree (DDsCOD) of 11.3 and 5.2% at 0.8% NaOH and 0.6 gTiO2/L, respectively. Alkaline-photocatalytic pretreatment yielded 37% DDsCOD at 0.8% NaOH–0.4 g/L TiO2. As compared to control, AD at 0.4% NaOH and 0.5 g/L TiO2 pretreatments yielded maximum methane, which was 50.4 and 32.6% higher. Similarly, alkaline-photocatalytic pretreatment at 0.4% NaOH-0.5 g/L TiO2 yielded methane as 462 N mL/g VS, which was 71.1% higher. Modified Gompertz model fitted the methane yield data well.

A cellulase-producing bacterium, designated as strain AK9, was isolated from a hot spring of Tatta Pani, Azad Kashmir, Pakistan. The bacterium was identified as Bacillus amyloliquefaciens through 16S rRNA sequencing. Cellulase from strain AK9 was able to liberate glucose from soluble cellulose and carboxymethyl cellulose (CMC). Enzyme was purified through size exclusion chromatography and a single band of ∼47 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified with recovery of 35.5%, 3.6-fold purity with specific activity of 31 U mg−1. The purified cellulase retained its activity over a wide range of temperature (50–70 °C) and pH (3–7) with maximum stability at 60 °C and pH 5.0. The activity inhibited by ethylenediaminetetraacetic acid (EDTA), suggested that it was metalloenzyme. Diethyl pyrocarbonate (DEPC) and β-mercaptoethanol significantly inhibited cellulase activity that revealed the essentiality of histidine residues and disulfide bonds for its catalytic function. It was stable in non-ionic surfactants, in the presence of various metal ions, and in water-insoluble organic solvents. Approximately 9.1% of reducing sugar was released after enzymatic saccharification of DAP-pretreated agro-residue, compared to a very low percentage by autohydrolysis treatment. Hence, it is concluded that cellulase from B. amyloliquefaciens AK9 can potentially be used in bioconversion of lignocellulosic biomass to fermentable sugars.

A process for the production of high bioethanol titre was established through fed-batch and simultaneous saccharification and fermentation (FB-SSF) of wild, non-edible cassava Manihot glaziovii. FB-SSF allowed fermentation of up to 390g/L of starch-derived glucose achieving high bioethanol concentration of up to 190g/L (24% v/v) with yields of around 94% of the theoretical value. The wild cassava M. glaziovii starch is hydrolysable with a low dosage of amylolytic enzymes (0.1-0.15% v/w, Termamyl® and AMG®). The Automatic Gas Potential Test System (AMPTS) was adapted to yeast ethanol fermentation and demonstrated to be an accurate, reliable and flexible device for studying the kinetics of yeast in SSF and FB-SSF. The bioethanol derived stoichiometrically from the CO2 registered in the AMPTS software correlated positively with samples analysed by HPLC (R(2)=0.99).

The feasibility of anaerobic treatment of methanol condensate from pulp and paper mill in UASB reactor was investigated and compared with the anaerobic treatment of methanol. The UASB reactor treating methanol condensate was operated for 480 days with minimum problems of overload. COD removal from methanol condensate and methanol under normal operating conditions ranged from 84-86% to 86-98%, respectively. Under optimal conditions (OLR=5.0 g COD L(-1)day(-1), COD(influent)=11.40 g L(-1)) a methane yield of 0.29 NL CH(4) per g COD(removed) (at standard temperature and pressure) was achieved during the treatment of methanol condensate. The recovery time of the microorganisms after several overloads was decreasing each time probably due to the adaptation to methanol condensate. These findings indicate that methanol condensate can be efficiently treated in a UASB reactor with the benefit of biogas production. As a bonus effect of the treatment, much of the smell of the feed was eliminated.

The objective of this study was to characterise and evaluate a wild inedible cassava species, Manihot glaziovii as feedstock for bioenergy production. Tubers obtained from 3 different areas in Tanzania were characterised and evaluated for bioethanol and biogas production. These bioenergy carriers were produced both separately and sequentially and their energy values evaluated based on these two approaches. Composition analysis demonstrated that M. glaziovii is a suitable feedstock for both bioethanol and biogas production. Starch content ranged from 77% to 81%, structural carbohydrates 3-16%, total crude protein ranged from 2% to 8%. Yeast fermentation achieved ethanol concentration of up to 85g/L at a fermentation efficiency of 89%. The fuel energy of the bioethanol and methane from flour-peels mix ranged from 5 to 13 and 11 to 14MJ/kgVS, respectively. Co-production of bioethanol and biogas in which the peels were added to the fermentation residue prior to anaerobic digestion produced maximum fuel energy yield of (15-23MJ/kgVS).

A multi-channel analyzer was used to evaluate biogas potential of sugarcane bagasse (SCB). The Automatic Methane Potential Test System contained fifteen parallel reactors and the same number of gas flow meters attached to the acquisition system. The set of reactors - gas flow meters gave reproducible results during anaerobic digestion of chemically defined carbon source and the units were used to evaluate the biomethane potential of SCB after different pretreatments, such as treatment with water, acid, acid followed by enzymatic treatment and acid followed by treatment with inactive enzymes. Combined pretreatment with 2% sulphuric acid and enzymatic hydrolysis (3.5% enzymes) resulted in conversion of 79% to monomeric sugars present in SCB. SCB treated with acid followed by enzymatic hydrolysis achieved the methane yield of 200NL per kg VS(added). Enzymatic saccharification of acid pretreated SCB resulted in increase of methane yield by 16±5% compared to that from acid treated SCB. (Less)

The recently isolated anaerobic bacterium Caloramator boliviensis with an optimum growth temperature of 60 °C can efficiently convert hexoses and pentoses into ethanol. When fermentations of pure sugars and a pentose-rich sugarcane bagasse hydrolysate were carried out in a packed bed reactor with immobilized cells of C. boliviensis, more than 98% of substrates were converted. Ethanol yields of 0.40-0.46 g/g of sugar were obtained when sugarcane bagasse hydrolysate was fermented. These features reveal interesting properties of C. boliviensis in producing ethanol from a renewable feedstock.