In Vitro Biofilm-Mediated Biodegradation of Pesticides and Dye-Contaminated Effluents Using Bacterial Biofilms.

Overuse of pesticides in agricultural soil and dye-polluted effluents severely contaminates the environment and is toxic to animals and humans making their removal from the environment essential. The present study aimed to assess the biodegradation of pesticides (cypermethrin (CYP) and imidacloprid (IMI)), and dyes (malachite green (MG) and Congo red (CR)) using biofilms of bacteria isolated from pesticide-contaminated soil and dye effluents. Biofilms of indigenous bacteria, i.e., Bacillus thuringiensis 2A (OP554568), Enterobacter hormaechei 4A (OP723332), Bacillus sp. 5A (OP586601), and Bacillus cereus 6B (OP586602) individually and in mixed culture were tested against CYP and IMI. Biofilms of indigenous bacteria i.e., Lysinibacillus sphaericus AF1 (OP589134), Bacillus sp. CF3 (OP589135) and Bacillus sp. DF4 (OP589136) individually and in mixed culture were tested for their ability to degrade dyes. The biofilm of a mixed culture of B. thuringiensis + Bacillus sp. (P7) showed 46.2% degradation of CYP compared to the biofilm of a mixed culture of B. thuringiensis + E. hormaechei + Bacillus sp. + B. cereus (P11), which showed significantly high degradation (70.0%) of IMI. Regarding dye biodegradation, a mixed culture biofilm of Bacillus sp. + Bacillus sp. (D6) showed 86.76% degradation of MG, which was significantly high compared to a mixed culture biofilm of L. sphaericus + Bacillus sp. (D4) that degraded only 30.78% of CR. UV-VIS spectroscopy revealed major peaks at 224 nm, 263 nm, 581 nm and 436 nm for CYP, IMI, MG and CR, respectively, which completely disappeared after treatment with bacterial biofilms. Fourier transform infrared (FTIR) analysis showed the appearance of new peaks in degraded metabolites and disappearance of a peak in the control spectrum after biofilm treatment. Thin layer chromatography (TLC) analysis also confirmed the degradation of CYP, IMI, MG and CR into several metabolites compared to the control. The present study demonstrates the biodegradation potential of biofilm-forming bacteria isolated from pesticide-polluted soil and dye effluents against pesticides and dyes. This is the first report demonstrating biofilm-mediated bio-degradation of CYP, IMI, MG and CR utilizing soil and effluent bacterial flora from Multan and Sheikhupura, Punjab, Pakistan.

DNA Barcoding: Molecular Identification and Phylogenetic Analysis of Pheretimoid Earthworm (Metaphire sp. and Amynthas sp.) Based on Mitochondrial Partial COI Gene from Sialkot, Pakistan.

The extremely difficult and challenging process is identifying pheretimoid species, genus Metaphire and Amynthas involving increased homoplasy in various morphological characteristics. The molecular identification, phylogenetic relationships, and evolutionary divergence time of earthworms belonging to the pheretimoid complex were investigated in this study using partial mitochondrial COI (cytochrome C oxidase subunit I) gene sequences ranging from 550-680 bp. Results revealed that 86 pheretimoid earthworms were morphologically different from a total of 342 mature worms. Moreover, 11 pheretimoid species were molecularly identified, including Metaphire posthuma (02), M. anomala (01), M. houlleti (02), M. californica (01), M. birmanica (02), Amynthas minimus (01), A. morrisi (01), and M. bununa (01). A phylogenetic tree was constructed with bootstrap values of 95%, which supported a monophyletic lineage of two well-supported clades formed by 12 partial COI sequences and 48 GenBank sequences using Hirudo medicinalis as an outgroup. The monophyly of these obtained genera indicated overall similarity at species level. Today, species like Amynthas, Metaphire and Pheretima have worm diversity in the form of pheretimoid earthworms, which dates to the Late Miocene (11.2-5.3 Mya) and the Pliocene (5.3-2.4 Mya). Compared to all relevant pheretimoid species, genetic p-distance values ranged from 0.0% to 0.57% (less than 1%). These low range values demonstrated that both genera Metaphire and Amynthas, supported the theory, which states that there are shared similarities among the species, despite different morphology. The current study is the first attempt in Pakistan to identify earthworms through DNA barcoding thus providing a genomic stamp. The work explored the significance of COI gene sequences to construct molecular tools that will be useful to overcome the different obstacles in morphologically similar earthworm identification and their phylogenetic study.

Development of an Ultrasonic-Assisted Extraction Technique for the Extraction of Natural Coloring Substance Chlorophyll from Leaves of Carica papaya.

Beautiful green leaves of Papaya are the rich source of Chlorophyll. Green color of chlorophyll has been used for a very long time as a natural colorant. Carica papaya has been considered as a good example and reasonable source of natural phytochemicals, which makes it suitable to color the food items and beverages. The aim of the present investigation is to develop the process of ultrasonic extraction in combination with solid phase extraction (SPE) to extract out chlorophyll with high yield as well as high degree of clarity. Newly customized ultrasonic-assisted extraction technique for the extraction of chlorophyll from Carica papaya leaves is optimized by taking different parameters like time, temperature, solvents concentrations, and raw material under consideration. Furthermore, the extract was purified by means of SPE and examined by using UV-Vis spectrophotometer. The highest yield of chlorophyll (dye) extract was found as 40% in solvent solution having 80 mL of ethanol and 20 mL of water with 5 minutes of extraction time, 35°C of temperature, and 1 grams of raw material in the sonication bath. Furthermore, the SPE purified sample was characterized by means of the UV-Vis spectrophotometer and here the total chlorophyll content was 34 mg/g, including chlorophyll a with a concentration of 14.1246 mg/g and chlorophyll b with concentration of 19.845 mg/g respectively. Consequently, sonication method can be suggested as a good method to get better concentration of chlorophyll.

Impact of Interplay between Obese Gut Microbiota and Diet in Developing Obesity in Synthetic Community Mice.

This study aims at investigating the effects of cultured gut microbiota (GM) of obese human coupled high fat diet (HFD) or chow diet (CD) in development of obesity in mice. 20 stool samples were collected from obese patients and isolated bacteria were identified morphologically and biochemically. Identified isolates were mixed in equal proportions to synthesize obese GM. In vivo study was performed using obese GM combined with HFD/CD using mouse model for three months. Albino mice were treated with ampicillin from one week prior to birth until weaning of the pups at seven weeks of age and then inoculated with obese GM. Sixteen mice were divided into four groups: i.e. group 1 (G1) mice fed with CD, group 2 (G2) mice with HFD, group 3 (G3) mice with GM + HFD and group 4 (G4) mice with GM + CD. Mice from groups 3-4 were considered synthetic community (SC) mice due to transfer of synthesize human GM. 16S rRNA sequencing identified five abundant bacteria as Pseudomonas aeruginosa, Staphylococcus sp., Escherichia coli, Morganella morganii, and Klebsiella oxytoca (accession numbers: MZ150742-MZ150746). In vivo study indicated that GM combination with either HFD/CD caused significantly increased body weight in SC mice (BMI; Kg/m2) compared to HFD or CD fed mice groups. One way ANOVA revealed highly significant increase (p ≤ 0.001) in levels of total cholesterol (TC), triglycerides and low density lipoprotein (LDL) in GM coupled diet groups (G3-G4; SC mice) compared to significant increase in HFD group (G2) versus CD group (G1). Our study is first of its kind to report significant effects of using purified strains as obese GM plus diet (HFD/CD) in inducing obesity in SC mice and elevated serum liver parameters as metabolic indicators, hence providing strong evidence about significance of modified GM combination with HFD in developing obesity in SC mice.

Role of modified diet and gut microbiota in metabolic endotoxemia in mice.

This study was aimed at investigating the effect of cultured gut microbiota (GM) from obese humans coupled HFD in inducing metabolic endotoxemia in humanized mice. In total, 30 strains were isolated from 10 stool samples of obese patients. Following morphological and biochemical characterization, 16S rRNA gene sequencing of six abundant isolates identified these Klebsiella aerogenes, Levilactobacillus brevis, Escherichia coli, Staphylococcus aureus, Bacillus cereus and Bacillus subtilis (MZ052089-MZ052094). In vivo trial using above isolates, known as human gut microbiota (HGM), was performed for six months. Sixteen mice were distributed into four groups, i.e., G1 (control) mice fed with chow diet, group 2 (G2) with HFD, group 3 (G3) with HFD + HGM and group 4 (G4) with chow diet + HGM. Body mass index (BMI) and plasma endotoxins were measured pre- and post-experiment. In vivo study revealed that HFD + HGM caused significant increase (3.9 g/cm at 20 weeks) in the body weight and BMI (0.4 g/cm post-experiment) of G3 mice compared to the other groups. One-way ANOVA showed significantly higher level of endotoxins (2.41, 4.08 and 3.7 mmol/L) in mice groups G2, G3 and G4, respectively, indicating onset of metabolic endotoxemia. Cecal contents of experimental mice groups showed a shift in microbial diversity as observed by all isolates belonging to either Firmicutes or Bacteroidetes phyla, respectively. In conclusion, current study reported that minor alteration in GM composition through HFD feeding and cultured GM transfer has significant impact in development of metabolic endotoxemia, possibly via modified intestinal permeability.

Formulation and Characterization of Calcium-Fortified Jelly and Its Proximate Composition and Sensory Analysis.

Calcium is a dynamic mineral. Recent discoveries designate that low intake of calcium generates deficiencies and path to other diseases. Food fortification could play a key role to overcome this problem. To cope with this deficiency problem, jellies were formulated with food-grade calcium salts and chicken eggshell powder. In the present study, three different concentrations of calcium salts, as well as eggshell powder were used to formulate jellies. The results of the sensory evaluation indicated that the two jelly products (A&D) in the current study were suitable for consumers. Results of Atomic Absorption Spectrophotometer revealed Jelly A and jelly D had 151±0.05 ppm and 133±0.06 ppm calcium concentration, respectively. Proximate analysis of Jelly A showed that it has 6.0±0.01% ash, 9.2±0.1% moisture, 0.4±0.01 g crude protein, 82.79±0.001 g crude fiber, and 0.61±0.001 g crude fat, while the jelly D that was made with chicken eggshell powder exhibited 6.0±0.01% ash, 10.1±0.1% moisture, 0.5±0.01 g protein, 84.54±0.01 g crude fiber and 1.61±0.01 g crude fat. Therefore, these two jelly A & D were greatly appreciated among other attributes. In spite of naturally available calcium-rich sources, calcium-fortified jellies can be consumed by individuals who are incapable to take sufficient calcium from their diet.

Biogenic Fabrication, Characterization and Drug Loaded Antimicrobial Assay of Silver Nanoparticles Using Centratherum anthalminticum (L.) Kuntze.

Bionanotechnology is considered a safe and ecofriendly route for the biosynthesis of metal nanoparticles from plant extracts, microorganisms, and biomaterials. The present study was focused on the fabrication of silver nanoparticles (<50 nm) biogenically from the novel Centratherum anthelmminticum's aqueous seed extract. The obtained nanoproduct was evaluated by X-ray diffraction analysis (XRD), Scanning electron microscopy (SEM), UV-Visible spectroscopy, FTIR and Raman spectroscopy. The particle size and surface charge were estimated by Dynamic light scattering (DLS) and Zeta potential measurements. The nanoparticles showed cubic close packed (ccp) morphology with miller indices (111), (200), (220), (311) and (222). The λmax for synthesized silver nanoparticles was measured in the range of 436 nm, 464 nm and 467 nm for 1 mM, 5 mM and 10 mM samples, respectively. The bioreduction of silver ions exhibited a gradual color change which confirms the formation of silver nanoparticles under UV-visible spectrum. Ag-O and Ag-N stretching vibrations corresponding to the bond formation between silver and oxygen of the carboxylate group and nitrogen of amine was corroborated by the presence of a sharp peak in Raman spectra at 245 cm-1. Antimicrobial activity was assessed against eight bacterial and three fungal strains. The silver nanoparticles fabricated from 10 mM AgNO3 solution showed significant results against all Gram-negative bacteria, with the further restriction in growth of C. albicans and A. niger. From in-vitro antimicrobial assay, it was observed that drug-loaded silver nanoparticles (Ciprofloxacin +10 mM) displayed a stronger potential than the synthesized silver nanoparticles and ciprofloxacin alone to restrain the development of E. coli, and E. aerogenes.

Neuropharmacological potential of various morphological parts of Camellia sinensis L.

Camellia sinensis L. has long been used as a therapeutic agent for the Central nervous system (CNS) due to the presence of flavonoids. The present study aimed to evaluate the dose-dependent Neuropharmacological behavioral potential of Camellia sinensis seed and leaf extracts on mice. To evaluate the differential potential of leaf and seed extract various doses were prepared and examined in open field, head dip, rearing, cage cross, swimming and traction tests. One-way ANOVA set at P* < 0.05 followed by POST HOC LSD (P* < 0.01) was applied to evaluate the significant difference among the treatments. Herein both seed and leaf extract showed significant results at high doses. Interestingly leaf extract at high dose showed significant effect on mice CNS in open field and head dip test, while seed at high dose revealed significant stimulus on mice CNS in rearing, cage cross, swimming and traction tests. Overall results showed that seed produced more stimulant effect and less calmness as compared to leaf extract was. Tea leaves had already known as potential CNS stimulant drugs; current investigation suggests that tea seed can be used as an alternative CNS stimulant agent with more effective stimulant action.

Taxonomic study of one generic and two new species record to the flora of Pakistan using multiple microscopic techniques.

Recent field exploration and collections has led to the findings of several new species in Pakistan. Here we reported two new species Ajuga reptance L and Sphagneticola trilobata (L.) Prusk for the first time in Pakistan flora as these species were neither listed in any other literature nor identified before in Pakistan. These species were found as a result of taxonomic studies performed in the year 2019 in District Rawalpindi and Islamabad, Pakistan. Microscopic techniques were used for the confirmation of foliar epidermal and pollen micromorphological features. Detailed study (morphological, palynological, and foliar epidermal) was provided for the correct identification and delimitation of the species using both light and scanning electron microscopy. Morphological results were compared with the flora of Taiwan and China.