Phenotypic, genomic and in planta characterization of Bacillus sensu lato for their phosphorus biofertilization and plant growth promotion features in soybean.

Bacillus sensu lato were screened for their capacity to mineralize organic phosphorus (P) and promote plant growth, improving nitrogen (N) and P nutrition of soybean. Isolates were identified through Type Strain Genome Server (TYGS) and Average Nucleotide Identity (ANI). ILBB95, ILBB510 and ILBB592 were identified as Priestia megaterium, ILBB139 as Bacillus wiedmannii, ILBB44 as a member of a sister clade of B. pumilus, ILBB15 as Peribacillus butanolivorans and ILBB64 as Lysinibacillus sp. These strains were evaluated for their capacity to mineralize sodium phytate as organic P and solubilize inorganic P in liquid medium. These assays ranked ILBB15 and ILBB64 with the highest orthophosphate production from phytate. Rhizocompetence and plant growth promotion traits were evaluated in vitro and in silico. Finally, plant bioassays were conducted to assess the effect of the co-inoculation with rhizobial inoculants on nodulation, N and P nutrition. These bioassays showed that B. pumilus, ILBB44 and P. megaterium ILBB95 increased P-uptake in plants on the poor substrate of sand:vermiculite and also on a more fertile mix. Priestia megaterium ILBB592 increased nodulation and N content in plants on the sand:vermiculite:peat mixture. Peribacillus butanolivorans ILBB15 reduced plant growth and nutrition on both substrates. Genomes of ILBB95 and ILBB592 were characterized by genes related with plant growth and biofertilization, whereas ILBB15 was differentiated by genes related to bioremediation. Priestia megaterium ILBB592 is considered as nodule-enhancing rhizobacteria and together with ILBB95, can be envisaged as prospective PGPR with the capacity to exert positive effects on N and P nutrition of soybean plants.

Antimicrobial activity and comparative metabolomic analysis of Priestia megaterium strains derived from potato and dendrobium.

The growth of endophytic bacteria is influenced by the host plants and their secondary metabolites and activities. In this study, P. megaterium P-NA14 and P. megaterium D-HT207 were isolated from potato tuber and dendrobium stem respectively. They were both identified as Priestia megaterium. The antimicrobial activities and metabolites of both strains were explored. For antimicrobial activities, results showed that P. megaterium P-NA14 exhibited a stronger inhibition effect on the pathogen of dendrobium, while P. megaterium D-HT207 exhibited a stronger inhibition effect on the pathogen of potato. The supernatant of P. megaterium P-NA14 showed an inhibition effect only on Staphylococcus aureus, while the sediment of P. megaterium D-HT207 showed an inhibition effect only on Escherichia coli. For metabolomic analysis, the content of L-phenylalanine in P. megaterium P-NA14 was higher than that of P. megaterium D-HT207, and several key downstream metabolites of L-phenylalanine were associated with inhibition of S. aureus including tyrosine, capsaicin, etc. Therefore, we speculated that the different antimicrobial activities between P. megaterium P-NA14 and P. megaterium D-HT207 were possibly related to the content of L-phenylalanine and its metabolites. This study preliminarily explored why the same strains isolated from different hosts exhibit different activities from the perspective of metabolomics.

Study on the mechanism of biochar affecting the effectiveness of phosphate solubilizing bacteria.

Low phosphorus utilization and phosphorus fertilizer pollution are serious issues primarily affecting soil health. To investigate the effects of biochar on the growth, phosphorus solubilization, and metabolites of phosphorus-solubilizing bacteria (PSB), rice husk biochar (RH) and rice straw biochar (RS) were incubated with Bacillus megatherium (BM1) and Bacillus mucilaginosus (BM2), respectively. The highest phosphorus solubilization was observed in BM2 following the addition of RS. The dissolved amount of phosphorus was 244.99 mg/L, which was 43.86% higher than that of the control group. Hence, biochar can improve the phosphorus solubilization capacity of PSB by affecting the organic acid and polysaccharide contents, and phosphatase activity secreted by the PSB, as the porous structure and surface characteristics of biochar ensured the adsorption of PSB. This study can help improve the functional activity of PSB and provide basis for improving the utilization of soil phosphorus, which in turn, aid in the development of biochar-based microbial fertilizers.

Degradation of alkane hydrocarbons by Priestia megaterium ZS16 and sediments consortia with special reference to toxicity and oxidative stress induced by the sediments in the vicinity of an oil refinery.

Petroleum hydrocarbon is a critical ecological issue with impact on ecosystems through bioaccumulation. It poses significant risks to human health. Due to the extent of alkane hydrocarbon pollution in some environments, biosurfactants are considered as a new multifunctional technology for the efficient removal of petroleum-based contaminants. To this end, Yamuna river sediments were collected at different sites in the vicinity of Mathura oil refinery, UP (India). They were analysed by atomic absorption spectrophotometry and gas chromatography-mass spectrometry (GC-MS) for heavy metals and organic pollutants. Heptadecane, nonadecane, oleic acid ester and phthalic acid were detected. In total 107 bacteria were isolated from the sediments and screened for biosurfactant production. The most efficient biosurfactant producing strain was tested for its capability to degrade hexadecane efficiently at different time intervals (0 h, 7 d, 14 d and 21 d). FT-IR analysis defined the biosurfactant as lipopeptide. 16S rRNA gene sequencing identified the bacterium as Priestia megaterium. The strain lacks resistance to common antibiotics thus making it an important candidate for remediation. The microbial consortia present in the sediments were also investigated for their capability to degrade C16, C17 and C18 alkane hydrocarbons. By using gas chromatography-mass spectrophotometry the metabolites were identified as 1-docosanol, dodecanoic acid, 7-hexadecenal, (Z)-, hexadecanoic acid, docosanoic acid, 1-hexacosanal, 9-octadecenoic acid, 3-octanone, Z,Z-6,28-heptatriactontadien-2-one, heptacosyl pentafluoropropionate, 1,30-triacontanediol and decyl octadecyl ester. Oxidative stress in Vigna radiata L. roots was observed by using Confocal Laser Scanning Microscopy. A strong reduction in seed germination and radicle and plumule length was observed when Vigna radiata L. was treated with different concentrations of sediment extracts, possibly due to the toxic effects of the pollutants in the river sediments. Thus, this study is significant since it considers the toxicological effects of hydrocarbons and to degrade them in an environmentally friendly manner.

Synbiotic Compositions of Bacillus megaterium and Polyunsaturated Fatty Acid Salt Enable Self-Sufficient Production of Specialized Pro-Resolving Mediators.

Specialized pro-resolving mediators (SPM) have emerged as crucial lipid mediators that confer the inflammation-resolving effects of omega-3 polyunsaturated fatty acids (n-3 PUFA). Importantly, SPM biosynthesis is dysfunctional in various conditions, which may explain the inconclusive efficacy data from n-3 PUFA interventions. To overcome the limitations of conventional n-3 PUFA supplementation strategies, we devised a composition enabling the self-sufficient production of SPM in vivo. Bacillus megaterium strains were fed highly bioavailable n-3 PUFA, followed by metabololipidomics analysis and bioinformatic assessment of the microbial genomes. All 48 tested Bacillus megaterium strains fed with the n-3 PUFA formulation produced a broad range of SPM and precursors thereof in a strain-specific manner, which may be explained by the CYP102A1 gene polymorphisms that we detected. A pilot study was performed to test if a synbiotic Bacillus megaterium/n-3 PUFA formulation increases SPM levels in vivo. Supplementation with a synbiotic capsule product led to significantly increased plasma levels of hydroxy-eicosapentaenoic acids (5-HEPE, 15-HEPE, 18-HEPE) and hydroxy-docosahexaenoic acids (4-HDHA, 7-HDHA) as well as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in healthy humans. To the best of our knowledge, we report here for the first time the development and in vivo application of a self-sufficient SPM-producing formulation. Further investigations are warranted to confirm and expand these findings, which may create a new class of n-3 PUFA interventions targeting inflammation resolution.

Engineering precursor and co-factor supply to enhance D-pantothenic acid production in Bacillus megaterium.

High-yielding chemical and chemo-enzymatic methods of D-pantothenic acid (DPA) synthesis are limited by using poisonous chemicals and DL-pantolactone racemic mixture formation. Alternatively, the safe microbial fermentative route of DPA production was found promising but suffered from low productivity and precursor supplementation. In this study, Bacillus megaterium was metabolically engineered to produce DPA without precursor supplementation. In order to provide a higher supply of precursor D-pantoic acid, key genes involved in its synthesis are overexpressed, resulting strain was produced 0.53 ± 0.08 g/L DPA was attained in shake flasks. Cofactor CH2-THF was found to be vital for DPA biosynthesis and was regenerated through the serine-glycine degradation pathway. Enhanced supply of another precursor, ß-alanine was achieved by codon optimization and dosing of the limiting L-asparate-1-decarboxylase (ADC). Co-expression of Pantoate-ß-alanine ligase, ADC, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate ammonia-lyase enhanced DPA concentration to 2.56 ± 0.05 g/L at shake flasks level. Fed-batch fermentation in a bioreactor with and without the supplementation of ß-alanine increased DPA concentration to 19.52 ± 0.26 and 4.78 ± 0.53 g/L, respectively. This present study successfully demonstrated a rational approach combining precursor supply engineering with cofactor regeneration for the enhancement of DPA titer in recombinant B. megaterium.

Improved yield of theaflavin-3,3'-digallate from Bacillus megaterium tyrosinase via directed evolution.

Theaflavin-3,3'-digallate (TFDG) in black tea possesses several health benefits. However, low TFDG yields limit its application. Herein, tyrosinases from Bacillus megaterium (Bmtyrc) were used to synthesize TFDG. To improve the catalytic efficiency of tyrosinase, a directed evolution strategy and a high-throughput screening method was employed. Compared with the wild type, mutant Bmtyrc-3 (N205D/D166E/D167G/F197W) showed 6.46 and 4.91-folds higher specific activity and 51.97- and 1.95-folds higher Vmax values towards epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), respectively. Moreover, Bmtyrc-3 displayed significantly enhanced catalytic efficiencies, and the space-time yield of TFDG was 35.35 g L-1d-1. Bmtyrc-3 presents a broader substrate binding area, caused by a mutation (N205D) encompassing the active site. Changes in the potential of the substrate binding site and hydrogen bonds, and the electrostatic effect on the protein surface resulted in an increased activity of the substrates EGCG and ECG.

The "beauty in the beast"-the multiple uses of Priestia megaterium in biotechnology.

Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. KEY POINTS: • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.

Development of a light activatable lignin nanosphere based spray coating for bioimaging and antimicrobial photodynamic therapy.

Many coating materials are commercially available to combat microbial infections. However, these coatings are difficult to synthesize, and are mostly composed of toxic chemicals. Lignin is an under-explored natural biopolymer with multifaceted potential. Lignin, with adhesive, UV resistant, and antimicrobial properties, is a suitable candidate to develop coating materials. Here we report a smart method to fabricate a sustainable nanospray coating from lignin which does not require any toxic chemicals or additives during synthesis. Initially, we have developed stable lignin nanospheres in a single step in aqueous medium, which were later utilized as a lignin nanospray (LNSR). The LNSR was characterized by dynamic light scattering, scanning electron microscopy, FTIR and other analytical techniques. This LNSR showed remarkable UV blocking, antioxidant and light-activated antimicrobial properties. Interestingly, for the first time, the LNSR demonstrated photoluminescence, making it useful for bioimaging. Moreover, singlet oxygen generation potential was observed in the LNSR, which could render it useful in phototheranostic applications (i.e. light assisted imaging and photodynamic therapy). Further, the LNSR was directly utilized to fabricate a sustainable coating. The nanospray coating exhibited maximum light-induced cell killing when applied to common microbes as detected by live-dead cell imaging. Taken together, the lignin nanospray coating developed via a direct pathway holds great promise to disinfect microbes in the presence of light.

Using a novel polysaccharide BM2 produced by Bacillus megaterium strain PL8 as an efficient bioflocculant for wastewater treatment.

In this study, the purification and characterization of a novel polysaccharide-based bioflocculant BM2 produced by a bacterium Bacillus megaterium strain PL8 with self-flocculating property were investigated. The results showed that BM2 was an acidic polysaccharide composed of Gal, GalUA, Glc, GlcUA and Man at a molar ratio of 45.1: 33.8:9.3:9.2:2.4, respectively. The molecular weight of BM2 was 4.55 × 106 Da. BM2 had high flocculation efficiencies across a wide pH ranged from 4 to 11 and a wide temperature ranged from 20 to 100 °C towards kaolin clay. BM2 was a cation-independent bioflocculant which could achieve high flocculation activity without the addition of other cations. Adsorption bridging was the main mechanism in the flocculation process of BM2 towards kaolin clay. The BM2 also displayed a high removal efficiency in terms of Congo red (88.14%) and Pb2+ ions (82.64%). These results suggested that BM2 had a great potential as an efficient bioflocculant candidate in wastewater treatment.

Bacillus megaterium-induced biocorrosion on mild steel and the effect of Artemisia pallens methanolic extract as a natural corrosion inhibitor.

Methanolic extract of Artemisia pallens (MEAP) (Asteraceae) was explored as greenbiocorrosion inhibitor for mild steel 1010 in 1.5% sodium chloride environment. Bacillus megaterium SKR7 induces the development of biofilm on the metal surface and forms the pitting corrosion. MEAP was showed (25 ppm) optimum inhibition effect of biocorrosion and further corrosion rate was highly reduced (0.3335 mm/year) than the control system (0.009 mm/year). The electrochemical study has supported the results with a higher value of total resistance (34 Ω cm2) when compared to control systems. It reveals the formation of a protective layer on the metal surface and reduces the adsorption of biofilm. This was due to the antimicrobial effect of MEAP. Overall, the results recognized that MEAP used as a green corrosion inhibitor for MS 1010 with 83% inhibition efficiency.

Engineered P450 BM3 and cpADH5 coupled cascade reaction for ß-oxo fatty acid methyl ester production in whole cells.

Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product.

Responses of soil enzyme activities and plant growth in a eucalyptus seedling plantation amended with bacterial fertilizers.

Plant growth-promoting rhizobacteria (PGPR) are widely used to improve plant nutrient uptake and assimilation and soil physicochemical properties. We investigated the effects of bacterial (Bacillus megaterium strain DU07) fertilizer applications in a eucalyptus (clone DH32-29) plantation in Guangxi, China in February 2011. We used two types of organic matter, i.e., fermented tapioca residue ("FTR") and filtered sludge from a sugar factory ("FS"). The following treatments were evaluated: (1) no PGPR and no organic matter applied (control), (2) 3 × 109 CFU/g (colony forming unit per gram) PGPR plus FS (bacterial fertilizer 1, hereafter referred to as BF1), (3) 4 × 109 CFU/g plus FS (BF2), (4) 9 × 109 CFU/g plus FS (BF3), (5) 9 × 109 CFU/g broth plus FTR (BF4). Soil and plant samples were collected 3 months (M3) and 6 months (M6) after the seedlings were planted. In general, bacterial fertilizer amendments significantly increased plant foliar total nitrogen (TN) and soil catalase activity in the short term (month 3, M3); whereas, it significantly increased foliar TN, chlorophyll concentration (Chl-ab), proline; plant height, diameter, and volume of timber; and soil urease activity, STN, and available N (Avail N) concentrations in the long term (month 6, M6). Redundancy analysis showed that soil available phosphorus was significantly positively correlated with plant growth in M3, and soil Avail N was negatively correlated with plant growth in M6. In M3, soil catalase was more closely correlated with plant parameters than other enzyme activities and soil nutrients, and in M6, soil urease, polyphenol oxidase, and peroxidase were more closely correlated with plant parameters than other environmental factors and soil enzyme activities. PCA results showed that soil enzyme activities were significantly improved under all treatments relative to the control. Hence, photosynthesis, plant growth, and soil N retention were positively affected by bacterial fertilizer in M6, and bacterial fertilizer applications had positive and significant influence on soil enzyme activities during the trial period. Thus, bacterial fertilizer is attractive for use as an environmentally friendly fertilizer in Eucalyptus plantations following proper field evaluation.

Polyhydroxyalkanoate (PHA) biosynthesis from directly valorized ragi husk and sesame oil cake by Bacillus megaterium strain Ti3: Statistical optimization and characterization.

Polyhydroxyalkanoates (PHAs) signify the most promising biological substitute to petrochemical plastics. Renewable and inexpensive agro-industrial by-products can be used as potent fermentation feedstocks for sustainable PHA biosynthesis. This study aimed at using a wild type B. megaterium strain Ti3 innate hydrolytic enzyme/s for eco-friendly valorization of 16 lignocellulosic agrowastes to PHA without pretreatments. Initial hydrolytic screening PHA concentration of (0.04-0.17 g/L), highlighted the strain's metabolic versatility. Pareto ranking of Taguchi orthogonal array (TOA) established ragi husk (RH), sesame oil cake (SOC) and KH2PO4 as the most influential factors (p < 0.05). The optimized and validated Response surface methodology (RSM) model (R2, 0.979; desirability, 1) resulted in 3.8 and 3.6 fold increased PHA production, 4.3 and 3.25 fold increased PHA productivity. A positive correlation (r2, 0.5-0.97) was observed amid the producer innate hydrolytic enzymes (lipase, amylase and cellulase) and PHA production. The PHA was characterized by 1H and 13C NMR, GPC, TGA. The polymer was identified as a scl-mcl copolyester with 92% 3HB (3-hydroxybutyrate) and 8% 3HHp (3-hydroxyheptanoate) monomers by NMR. This the first report on B. megaterium self-enzyme reliant non-food agrowastes bioconversion to PHA with 3HHp (3-hydroxyheptanoate) monomers excluding precursor addition, commercial enzymes, pure carbon and nitrogen sources.

Maytenin Plays a Special Role in the Regulation of the Endophytic Bacillus megaterium in Peritassa campestris Adventitious Roots.

Peritassa campestris (Celastraceae) root bark accumulates potent antitumor quinonemethide triterpenes (QMTs). When grown in their natural habitat, plants of the family Celastraceae produce different QMTs such as celastrol (3) and pristimerin (4). However, when they are inserted in in vitro culture systems, they accumulate maytenin (1) as the main compound. Recently, Bacillus megaterium was detected as an endophytic microorganism (EM) living inside P. campestris roots cultured in vitro. We hypothesized that compound (1) controls EM growth more efficiently, and that the presence of EMs in the root culture causes compound (1) to accumulate. For the first time, this work has explored plant-microorganism interaction in a species of the family Celastraceae by co-culture with an EM. Live endophytic bacteria were used, and QMT accumulation in P. campestris adventitious roots was our main focus. The antimicrobial activity of the main QMTs against endophytic B. megaterium was also evaluated. Our results showed that compound (1) and maytenol (5) were more effective than their precursors QMTs (3) and (4) in controlling the EM. Co-culture of B. megaterium with roots significantly reduced bacterial growth whereas root development remained unaffected. Compound (1) production was 24 times higher after 48 hr in the presence of the highest B. megaterium concentration as compared to the control. Therefore, P. campestris adventitious roots affect the development of the endophyte B. megaterium through production of QMTs, which in turn can modulate production of compound (1).

Antibacterial and Antispore Activities of Isolated Compounds from Piper cubeba L.

Piper cubeba L. is the berry of a shrub that is indigenous to Java, Southern Borneo, Sumatra, and other islands in the Indian Ocean. The plant is usually used in folk traditional medicine and is an important ingredient in cooking. The purpose of this study was to isolate and purify the bioactive compounds from P. cubeba L. fractions. In addition, the isolated compounds were tested for their antibacterial and antispore activities against vegetative cells and spores of Bacillus cereus ATCC33019, B. subtilis ATCC6633, B. pumilus ATCC14884, and B. megaterium ATCC14581. The phytochemical investigation of the DCM fraction yielded two known compounds: ß-asarone (1), and asaronaldehyde (2) were successfully isolated and identified from the methanol extract and its fractions of P. cubeba L. Results showed that exposing the vegetative cells of Bacillus sp. to isolated compounds resulted in an inhibition zone with a large diameter ranging between 7.21 to 9.61 mm. The range of the minimum inhibitory concentration (MIC) was between 63.0 to 125.0 µg/mL and had minimum bactericidal concentration (MBC) at 250.0 to 500.0 µg/mL against Bacillus sp. Isolated compounds at a concentration of 0.05% inactivated more than 3-Log10 (90.99%) of the spores of Bacillus sp. after an incubation period of four hours, and all the spores were killed at a concentration of 0.1%. The structures were recognizably elucidated based on 1D and 2D-NMR analyses (1H, 13C, COSY, HSQC, and HMBC) and mass spectrometry data. Compounds 1, and 2 were isolated for the first time from this plant. In conclusion, the two compounds show a promising potential of antibacterial and sporicidal activities against Bacillus sp. and thus can be developed as an anti-Bacillus agent.

Integrated Effects of Co-Inoculation with Phosphate-Solubilizing Bacteria and N2-Fixing Bacteria on Microbial Population and Soil Amendment Under C Deficiency.

The inoculation of beneficial microorganisms to improve plant growth and soil properties is a promising strategy in the soil amendment. However, the effects of co-inoculation with phosphate-solubilizing bacteria (PSB) and N2-fixing bacteria (NFB) on the soil properties of typical C-deficient soil remain unclear. Based on a controlled experiment and a pot experiment, we examined the effects of PSB (M: Bacillus megaterium and F: Pseudomonas fluorescens), NFB (C: Azotobacter chroococcum and B: Azospirillum brasilence), and combined PSB and NFB treatments on C, N, P availability, and enzyme activities in sterilized soil, as well as the growth of Cyclocarya Paliurus seedlings grow in unsterilized soil. During a 60-day culture, prominent increases in soil inorganic N and available P contents were detected after bacteria additions. Three patterns were observed for different additions according to the dynamic bacterial growth. Synergistic effects between NFB and PSB were obvious, co-inoculations with NFB enhanced the accumulation of available P. However, decreases in soil available P and N were observed on the 60th day, which was induced by the decreases in bacterial quantities under C deficiency. Besides, co-inoculations with PSB and NFB resulted in greater performance in plant growth promotion. Aimed at amending soil with a C supply shortage, combined PSB and NFB treatments are more appropriate for practical fertilization at intervals of 30-45 days. The results demonstrate that co-inoculations could have synergistic interactions during culture and application, which may help with understanding the possible mechanism of soil amendment driven by microorganisms under C deficiency, thereby providing an alternative option for amending such soil.

Biodegradation of hydrolyzed polyacrylamide by a Bacillus megaterium strain SZK-5: Functional enzymes and antioxidant defense mechanism.

Hydrolyzed polyacrylamide (HPAM) is the most widely used water-soluble linear polymer with high molecular weight in polymer flooding. Microbiological degradation is an environment-friendly and effective method of treating HPAM-containing oilfield produced water. In this study, a strain SZK-5 that could degrade HPAM was isolated from soil contaminated by oilfield produced water. Based on morphological, biochemical characteristics and 16S rDNA sequence homology analysis, the strain was identified as Bacillus megaterium. The biodegradation capability of strain SZK-5 was determined by incubation in a mineral salt medium (MSM) containing HPAM under different environmental conditions, showing 55.93% of the HPAM removed after 7 d of incubation under the optimum conditions ((NH4)2SO4 = 1667.9 mg L-1, temperature = 24.05 °C and pH = 8.19). Cytochrome P450 (CYP) and urease (URE) played significant roles in biological carbon and nitrogen removal, respectively. The strain SZK-5 could resist the damages caused by oxidative stress given by crude oil and HPAM. To our knowledge, this is the first report about the biodegradation of HPAM by B. megaterium. These results suggest that strain SZK-5 might be a new auxiliary microbiological resource for the biodegradation of HPAM residue in wastewater and soil.

In Vitro Antibacterial Evaluation of Terminalia chebula as an Alternative of Antibiotics against Bovine Subclinical Mastitis.

The extent of subclinical mastitis in three breeds of cattle, Kankrej, Gir, and Crossbred, was performed at cattle farms in Anand town of Gujarat State, India. The prevalence of subclinical mastitis in crossbred cattle was higher compared to local breed of cattle. Causative agents identified using 16S rDNA polymerase chain reaction (PCR)-based molecular method were Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus megaterium. In vitro antibacterial activity of ethyl acetate extract of plant Terminalia chebula (Combretaceae) was checked by agar well diffusion method against four isolated and molecularly identified microorganisms. Ethyl acetate extract shows antimicrobial activity with varying magnitudes against all identified isolates. Among the three different concentrations, 500 µg/mL conc. of extract is as effective as that of standard amoxicillin. In vitro results support the use of plant extract from T. chebula as an alternative to antibiotics therapy against bovine subclinical mastitis.

Phosphorus Solubilization by Bacillus Species.

Microbial solubilization applies the natural ability of a microorganism to liberate phosphorus from unavailable structures. The main mechanism recognized to be responsible for the solubilization of phosphorus is the production of different types of organic acids. Three kinds of Bacillus species and three types of raw materials (poultry bones, fish bones, and ash) were tested for solubilization. The following parameters were compared for all discussed cases: pH, specific growth rate, solubilization factor, released phosphorus concentration, and total and individual concentration of organic acids. Utilization of ash brought about the highest specific and maximum specific growth rates. A decrease in pH was observed in most of the discussed cases with the exception of fish bones. At the same time, fish bones had the highest concentration of released P2O5 and the highest total concentration of produced organic acids (gluconic, lactic, acetic, succinic, and propionic) in all discussed cases. The tested Bacillus species produced the mentioned acids with the exception of B. megaterium, where propionic acid was not present. The lactic and acetic acids were those produced in the highest amount. The kind of raw materials and type of Bacillus species used in solubilization had a strong influence on the kind of organic acids that were detected in the broth culture and its total concentration, which had a direct influence on the amount of released phosphorus. The combination of Bacillus megaterium with the fish bones at 5 g/L is proposed as the pair that gives the highest concentration of released phosphorus (483 ± 5 mg/L).