Meta-Analysis of Cytotoxicity Studies Using Machine Learning Models on Physical Properties of Plant Extract-Derived Silver Nanoparticles.

Silver nanoparticles (Ag-NPs) demonstrate unique properties and their use is exponentially increasing in various applications. The potential impact of Ag-NPs on human health is debatable in terms of toxicity. The present study deals with MTT(3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium-bromide) assay on Ag-NPs. We measured the cell activity resulting from molecules' mitochondrial cleavage through a spectrophotometer. The machine learning models Decision Tree (DT) and Random Forest (RF) were utilized to comprehend the relationship between the physical parameters of NPs and their cytotoxicity. The input features used for the machine learning were reducing agent, types of cell lines, exposure time, particle size, hydrodynamic diameter, zeta potential, wavelength, concentration, and cell viability. These parameters were extracted from the literature, segregated, and developed into a dataset in terms of cell viability and concentration of NPs. DT helped in classifying the parameters by applying threshold conditions. The same conditions were applied to RF to extort the predictions. K-means clustering was used on the dataset for comparison. The performance of the models was evaluated through regression metrics, viz. root mean square error (RMSE) and R2. The obtained high value of R2 and low value of RMSE denote an accurate prediction that could best fit the dataset. DT performed better than RF in predicting the toxicity parameter. We suggest using algorithms for optimizing and designing the synthesis of Ag-NPs in extended applications such as drug delivery and cancer treatments.

An In Vitro and In Vivo Study of the Efficacy and Toxicity of Plant-Extract-Derived Silver Nanoparticles.

Silver nanoparticles (AgNPs) display unique plasmonic and antimicrobial properties, enabling them to be helpful in various industrial and consumer products. However, previous studies showed that the commercially acquired silver nanoparticles exhibit toxicity even in small doses. Hence, it was imperative to determine suitable synthesis techniques that are the most economical and least toxic to the environment and biological entities. Silver nanoparticles were synthesized using plant extracts and their physico-chemical properties were studied. A time-dependent in vitro study using HEK-293 cells and a dose-dependent in vivo study using a Drosophila model helped us to determine the correct synthesis routes. Through biological analyses, we found that silver nanoparticles' cytotoxicity and wound-healing capacity depended on size, shape, and colloidal stability. Interestingly, we observed that out of all the synthesized AgNPs, the ones derived from the turmeric extract displayed excellent wound-healing capacity in the in vitro study. Furthermore, the same NPs exhibited the least toxic effects in an in vivo study of ingestion of these NPs enriched food in Drosophila, which showed no climbing disability in flies, even at a very high dose (250 mg/L) for 10 days. We propose that stabilizing agents played a superior role in establishing the bio-interaction of nanoparticles. Our study reported here verified that turmeric-extract-derived AgNPs displayed biocompatibility while exhibiting the least cytotoxicity.

Characterization and Application of Biosurfactant Produced by Bacillus licheniformis R2.

The biosurfactant produced by Bacillus licheniformis R2 was characterized and studied for enhancing the heavy crude oil recovery at 80 °C in coreflood experiments. The strain was found to be nonpathogenic and produced biosurfactant, reducing the surface tension of medium from 70 to 28 mN/m with 1.1 g/l yield. The biosurfactant was quite stable during exposure to elevated temperatures (85 °C for 90 days), high salinity (10 % NaCl), and a wide range of pH (5-12) for 10 days. It was characterized as lipopeptide similar to lichenysin-A, with a critical micelle concentration of about 19.4 mg/l. The efficiency of crude biosurfactant for enhanced oil recovery by core flood studies revealed it to recovering additional 37.1 % oil from Berea sandstone cores at 80 °C. The results are indicative of the potential for the development of lipopeptide biosurfactant-based ex situ microbial enhanced heavy oil recovery from depleting oil fields with extreme temperatures.

Evaluation of hydroxylamine oxidoreductase as a functional and phylogenetic marker to differentiate Nitrosomonas spp.

Nitrosomonas genus belongs to beta-subclass of Proteobacteria and encompasses closely related species. Sequence independent techniques like single strand confirmation polymorphism (SSCP) was attempted in the present study to resolve AOB using ammonia monooxygenase (amoA) and hydroxylamine oxidoreductase (hao) gene fragments, unique to AOB. Variation in hydroxylamine oxidoreductase (HAO) enzyme zymogram of isolates observed in the study was also explored as an additional sequence independent method to substantiate the observations. Nitrosomonas europaea (standard strain) and 12 isolates, obtained by enriching environmental samples, were differentiated into six and four groups by SSCP analyses of amoA and hao gene fragments, respectively, whereas they could be resolved into six distinct groups through activity staining of HAO enzyme. amoA gene fragment was therefore found to be better than hao gene fragment in resolving the studied AOB based on richness and evenness with Simpson's index of diversity - 0.85. However, the ensembled use of these molecular methods (SSCP of amoA and hao gene fragments) and HAO enzyme zymogram in fingerprinting AOB provide better resolution and evenness, contributing significantly in AOB diversity studies. Grouping of AOB isolates by hao gene SSCP analysis followed almost the same pattern as that by 16S rRNA gene based sequence analysis, hence it is suitable as a phylogenetic marker.

Mutualism between autotrophic ammonia-oxidizing bacteria (AOB) and heterotrophs present in an ammonia-oxidizing colony.

Coexistence of an autotrophic ammonia-oxidizing bacterium (Nitrosomonas sp. RA) and heterotrophic bacteria was consistently observed when cultured in an inorganic medium without any external supply of organic carbon. The present study was undertaken to understand the association between autotrophs and the associated heterotrophs for which a system containing active autotrophs and heterotrophs controlled by Hg(2+) addition was developed. The study revealed interdependence of heterotrophs and Nitrosomonas sp. RA for growth under iron-limited condition. Growth of Nitrosomonas sp. RA was supported by siderophores produced by the associated heterotroph, Pusillimonas sp., thereby complementing its high iron requirement while the organics (such as pyruvate) excreted by Nitrosomonas sp. RA during its autotrophic growth supported the survival of heterotrophs in the inorganic medium. The study thus sheds light on the nature of the mutual interactions between heterotrophs and autotrophs that play a role in the ammonia-oxidizing system involved in wastewater treatment.

Development of a simultaneous partial nitrification, anaerobic ammonia oxidation and denitrification (SNAD) bench scale process for removal of ammonia from effluent of a fertilizer industry.

A simultaneous partial nitrification, anammox and denitrification (SNAD) process was developed for the treatment of ammonia laden effluent of a fertilizer industry. Autotrophic aerobic and anaerobic ammonia oxidizing biomass was enriched and their ammonia removal ability was confirmed in synthetic effluent system. Seed consortium developed from these was applied in the treatment of effluent in an oxygen limited bench scale SNAD type (1L) reactor run at ambient temperature (∼30°C). Around 98.9% ammonia removal was achieved with ammonia loading rate 0.35kgNH(4)(+)-N/m(3)day in the presence of 46.6mg/L COD at 2.31days hydraulic retention time. Qualitative and quantitative analysis of the biomass from upper and lower zone of the reactor revealed presence of autotrophic ammonia oxidizing bacteria (AOB), Planctomycetes and denitrifiers as the dominant bacteria carrying out anoxic oxidation of ammonia in the reactor. Physiological and molecular studies strongly indicate presence of anammox bacteria in the anoxic zone of the SNAD reactor.

Biosurfactant's role in bioremediation of NAPL and fermentative production.

Surfactants and biosurfactants are amphipathic molecules with both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases that have different degrees of polarity and hydrogen bonding which confers excellent detergency, emulsifying, foaming and dispersing traits, making them most versatile process chemicals. One of the major applications of (bio)surfactants is in environmental bioremediation field. Most synthetic organic compounds present in contaminated soils are only weakly soluble or completely insoluble in water, so they exist in the subsurface as separate liquid phase, often referred as a non-aqueous phase liquids (NAPL), which poses as threat to environment. Several studies have revealed the use of surfactants for remediation; however, several factors limit the use of surfactants in environmental remediation, mainly persistence of surfactants or their metabolites and thus potentially pose an environmental concern. Biosurfactants may provide a more cost-effective approach for subsurface remediation when used alone or in combination with synthetic surfactants. There are several advantages of biosurfactants when compared to chemical surfactants, mainly biodegradability, low toxicity, biocompatibility and ability to be synthesized from renewable feedstock. Despite having many commercially attractive properties and clear advantages compared with their synthetic counterparts, biosurfactants have not yet been employed extensively in industry because of their low yields and relatively high production and recovery costs. However, the use of mutants and recombinant hyperproducing microorganisms along with the use of cheaper raw materials and optimal growth and production conditions and more efficient recovery processes, the production of biosurfactant can be made economically feasible. Therefore, future research aiming for high-level production of biosurfactants must be focused towards the development of appropriate combinations of hyperproducing microbial strains, optimized cheaper production media and optimized process conditions, which will lead to economical commercial level biosurfactant production.

Catalytic and thermodynamic characterization of protease from Halobacterium sp. SP1(1).

Osmolytes KCl, glycerol, mannitol, trehalose, sucrose, betaine, proline and Na-glutamate at different concentrations (5-30%) were investigated as effective solutes for retaining the activity of Halobacterium sp. SP1(1) protease in the absence of NaCl. Maximum activity was observed in the presence of 30% Na-glutamate. Kinetic and thermodynamic parameters for casein hydrolysis revealed that the protease was equally efficient in the presence of Na-glutamate as in NaCl. The enzyme was active over a broader range of temperature (20-80 degrees C) and was highly stable even at 80 degrees C with Na-glutamate. Thermodynamic parameters (DeltaH*, DeltaS*, G*) for irreversible inactivation of protease at different temperatures (20-80 degrees C) were determined in the presence of Na-glutamate and NaCl. The efficiency of these osmolytes for thermal stability of protease was 30% (1.6 M) Na-glutamate > 4 M ( approximately 25%) NaCl > 2 M (approximately 10%), suggesting that the effect exerted by the osmolyte depends not only on its chemical nature but also on its concentration. Na-glutamate was thus found to play an important role in thermal stabilization of enzyme substituting for NaCl. Moreover, substitution of NaCl by Na-glutamate may increase the applicability of halophilic enzymes in biotechnology and industry, which is otherwise limited to high NaCl concentrations.

Selective plugging strategy-based microbial-enhanced oil recovery using Bacillus licheniformis TT33.

The selective plugging strategy of microbial enhanced oil recovery involves the use of microbes that grow and produce exopolymeric substances, which block the high permeability zones of an oil reservoir, thus allowing the water to flow through the low permeability zones leading to increase in oil recovery. Bacillus licheniformis TT33, a hot water spring isolate, is facultatively anaerobic, halotolerant, and thermotolerant. It produces EPS as well as biosurfactant and has a biofilm-forming ability. The viscosity of its cell-free supernatant is 120 mPas at 28 degrees C. Its purified EPS contained 26% carbohydrate and 3% protein. Its biosurfactant reduced the surface tension of water from 72 to 34 mN/m. This strain gave 27.7+/-3.5% oil recovery in a sand pack column. Environmental scanning electron microscopy analysis showed bacterial growth and biofilm formation in the sand pack. Biochemical tests and amplified ribosomal DNA restriction analysis confirmed that the oil recovery obtained in the sand pack column was due to Bacillus licheniformis TT33.

Catalase and superoxide dismutase double staining zymogram technique for Deinococcus and Kocuria species exposed to multiple stresses.

Superoxide dismutase (SOD) and catalase expression is associated with oxidative stress. Existing techniques for the individual staining of SOD and catalase have been described in the past. The objective of this study was to achieve a simple and rapid technique for the double staining of bacterial SOD and catalase on the same polyacrylamide gel. SOD detection was carried out using nitro-blue tetrazolium (NBT) dye reduction followed by ferricyanide precipitation for negative staining of the catalase enzyme on the same gel. The staining procedure resulted in pale blue SOD bands while catalase appeared as yellow bands against a greenish blue background on the same gel. This technique was used to detect changes in the polymorphic forms of these enzymes in Deinococcus radiodurans R1 and Kocuria sp. C2 subjected to stresses like UV and gamma radiation and desiccation.

Organic solvent tolerance of Halobacterium sp. SP1(1) and its extracellular protease.

Halophilic archaea belonging to three different genera- Halobacterium, Haloarcula and Haloferax, were isolated from Kandla salt pans. The isolates had an optimum requirement of 25% NaCl for growth. Increase in organic solvent tolerance of isolates was observed at higher NaCl concentrations. Among the three isolates Halobacterium sp. SP1(1) was found to be more tolerant than Haloarcula sp. SP2(2) and Haloferax sp. SP1(2a). The extracellular protease of Halobacterium sp. SP1(1) showed higher solvent tolerance compared to the organism itself. The enzyme was highly tolerant to toluene, xylene, n-decane, n-dodecane and n-undecane, majority of which are frequently used in paints. These findings may help in understanding the mechanism of organic solvent tolerance in halophilic archaea and their application in antifouling coatings. Also, best to our knowledge the present study is the first report on organic solvent tolerance of haloarchaeal extracellular protease.

Evaluation of bioemulsifier mediated Microbial Enhanced Oil Recovery using sand pack column.

Bacillus licheniformis K125, isolated from an oil reservoir, produces an effective bioemulsifier. The crude bioemulsifier showed 66% emulsification activity (E(24)) and reduced the surface tension of water from 72 to 34 mN/m. It contains substantial amount of polysaccharide, protein and lipid. This bioemulsifier is pseudoplastic non-Newtonian in nature. It forms oil in water emulsion which remains stable at wide range of pH, temperature and salinity. It gave 43+/-3.3% additional oil recovery upon application to a sand pack column designed to simulate an oil reservoir. This is 13.7% higher than that obtained from crude lipopeptide biosurfactants produced by the standard strain, Bacillus mojavensis JF2 and 8.5% higher than hot water spring isolate, Bacillus licheniformis TT42. The increased oil recovery obtained by using the crude bioemulsifier can be attributed to its combined surface and emulsification activity. Its mechanism of oil recovery must be similar to the mechanism exhibited by surfactant-polymer flooding process of chemical enhanced oil recovery.

Biosurfactant production using molasses and whey under thermophilic conditions.

Biosurfactant production was studied by Bacillus licheniformis K51, B. subtilis 20B, B. subtilis R1 and Bacillus strain HS3 using molasses or cheese whey as a sole source of nutrition at 45 degrees C. The isolates were able to grow and produce biosurfactant under shaking as well as static conditions. Maximum biosurfactant production was achieved with molasses at 5.0-7.0% (w/v). The biosurfactant retained its surface-active properties after incubation at 80 degrees C at a wide range of pH values and salt concentrations for nine days. Oil displacement experiments in sand pack columns with crude oil showed 25-33% recovery of residual oil.

Enhanced growth and nodulation of pigeon pea by co-inoculation of Bacillus strains with Rhizobium spp.

Endophytic bacteria which are known to reside in plant tissues have often been shown to promote plant growth. Present study deals with the isolation of putative endophytes from the surface sterilized root nodules of pigeon pea (Cajanus cajan) designated as non-rhizobial (NR) isolates. Three of these non-rhizobial isolates called NR2, NR4 and NR6 showed plant growth promotion with respect to increase in plant fresh weight, chlorophyll content, nodule number and nodule fresh weight when co-inoculated with the rhizobial bioinoculant strain IC3123. The three isolates were neither able to nodulate C. cajan nor did they show significant plant growth promotion when inoculated alone without Rhizobium spp. IC3123. All the three isolates were gram positive rods with NR2 and NR4 showing endospore formation and formed one single cluster in Amplified Ribosomal DNA Restriction Analysis (ARDRA). Partial sequences of 16S rRNA genes of NR4 and NR6 showed 97% similarity to Bacillus megaterium. The Bacillus strains NR4 and NR6 were able to produce siderophores which the rhizobial bioinoculant IC3123 was able to cross-utilize. Under iron starved conditions IC3123 showed enhanced growth in the presence of the Bacillus isolates indicating that siderophore mediated interactions may be underlying mechanism of beneficial effect of the NR isolates on nodulation by IC3123.

Production of biosurfactant and antifungal compound by fermented food isolate Bacillus subtilis 20B.

A biosurfactant producing strain, Bacillus subtilis 20B, was isolated from fermented food in India. The strain also showed inhibition of various fungi in in-vitro experiments on Potato Dextrose Agar medium. It was capable of growth at temperature 55 degrees C and salts up to 7%. It utilized different sugars, alcohols, hydrocarbons and oil as a carbon source, with preference for sugars. In glucose based minimal medium it produced biosurfactant which reduced surface tension to 29.5 mN/m, interfacial tension to 4.5 mN/m and gave stable emulsion with crude oil and n-hexadecane. The biosurfactant activity was stable at high temperature, a wide range of pH and salt concentrations for five days. Oil displacement experiments using biosurfactant containing broth in sand pack columns with crude oil showed 30.22% recovery. The possible application of organism as biocontrol agent and use of biosurfactant in microbial enhanced oil recovery (MEOR) is discussed.

Siderophore cross-utilization amongst nodule isolates of the cowpea miscellany group and its effect on plant growth in the presence of antagonistic organisms.

Nodule isolates from the cowpea miscellany group of legumes produced varying concentrations of catecholate and hydroxamate types of siderophores under iron-limiting conditions. The nodule isolates differed with respect to siderophore cross-utilizing abilities; some were proficient at using siderophores of other nodule isolates (homologous siderophores) while others could utilize siderophores produced by other rhizospheric bacteria (heterologous siderophores). Utilization of siderophore of rhizospheric bacterium PsB, a plant pathogen, benefited the nodule isolate G11 in terms of growth under iron-limiting laboratory conditions, while PsB was clearly inhibited in the presence of G11. Plate assays showed that siderophore of G11 could withhold iron from PsB and hence PsB was inhibited in the presence of G11. Isolates G11 and PsB when applied simultaneously to peanut seedlings under sterile soil conditions, provided a clear advantage to the plant in terms of reduction in the inhibitory effect of PsB. The count of the nodule isolate G11 increased in the soil when co-inoculated with PsB, as compared to when inoculated alone. Thus, the increased growth of the plant can be attributed to the iron sequestration and plant growth promoting properties of G11. The isolate G11 could utilize the siderophores produced by many other rhizospheric isolates while the siderophore of G11 was not being utilized by these rhizospheric isolates.

Statistical optimization of medium components for the production of biosurfactant by Bacillus licheniformis K51.

The nutritional medium requirement for biosurfactant production by Bacillus licheniformis K51 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were H3PO4, CaCl2, H3BO3, and Na-EDTA. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were (g/l): glucose, 1.1; NaNO3, 4.4; MgSO4 x 7H2O, 0.8; KCl, 0.4; CaCl2, 0.27; H3PO4, 1.0 ml/l; and trace elements (mg/l): H3BO3, 0.25; CuSO4, 0.6; MnSO4, 2.2; Na2MoO4, 0.5; ZnSO4, 6.0; FeSO4, 8.0; CoCl2, 1.0; and Na-EDTA, 30.0. Using this statistical optimization method, the relative biosurfactant yield as critical micelle dilution (CMD) was increased from 10x to 105x, which is ten times higher than the non-optimized rich medium.

Functional expression of Escherichia coli fhuA gene in Rhizobium spp. of Cajanus cajan provides growth advantage in presence of Fe3+: ferrichrome as iron source.

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 DeltafhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.

Multiple-stress tolerance of ionizing radiation-resistant bacterial isolates obtained from various habitats: correlation between stresses.

Isolation of five ionizing radiation (IR)-resistant bacteria by screening of isolates from various habitats classified as common and stressed is reported. IR-resistant isolates exhibited varying degrees of resistance to gamma-radiation and were classified as highly and moderately radiation resistant. Resistance to ultraviolet (UV) radiation correlated well with gamma-radiation resistance, whereas a comparable desiccation resistance for all the highly and moderately radiation-resistant isolates was observed. However, salt tolerance failed to correlate with IR resistance, indicating a divergent evolution of the salt tolerance and radiation resistance. Characterization of isolates by the amplified rDNA restriction analysis profiling attested to the clustering of these isolates with their stress phenotype. 16S rRNA gene-based analysis of the isolates showed that the bacteria with similar-resistance physiologies clustered together and belonged to related genera. Hydrogen peroxide resistance and mitomycin survival patterns of the isolates indicated the roles of oxidative-stress tolerance in desiccation survival and recombination repair in higher radiation resistance, respectively.

Siderophore cross-utilization amongst rhizospheric bacteria and the role of their differential affinities for Fe3+ on growth stimulation under iron-limited conditions.

The majority of bacteria isolated from rhizospheres of Arachis hypogea (Groundnut) and Vigna radiata (Mung bean) predominantly produced catechol-type siderophores except for a few fluorescent pseudomonads that produced hydroxamates in addition to catecholates. The rhizospheric isolates differed in their ability to cross-utilize siderophores produced by other rhizospheric isolates (heterologous); some were highly proficient at utilizing heterologous siderophores, while others were poor cross-utilizers. Isolate G9, which utilized hydroxamate as well as catecholate siderophores, was found to be an efficient siderophore cross-utilizer, while isolates G2 and G6 were poor-utilizers of catecholate and non-utilizers of hydroxamate siderophores. Growth stimulation of two isolates G9 and G6 was seen when grown in the presence of externally supplied heterologous siderophores, which they cross-utilized. The iron-regulated outer membrane protein (IROMP) profiles differed for the most cross-utilizer and the least cross-utilizer strains, but in both the cases no new outer membrane proteins (OMP) were induced in response to the exogenous siderophores supplied. The growth of the organisms in the presence of heterologous siderophores that they failed to cross-utilize led to growth inhibition in the case of isolate G9. This appears to be due to a lower affinity of the siderophore of G9 as compared to the exogenously supplied G6 siderophore. A simple method was devised to measure relative affinities of respective siderophores for iron based on CAS solution decolorization by the siderophore preparations. The effect on the growth of the differential affinities of the siderophores for iron and the interactions of the organisms through cross-utilization is also discussed.