Heavy metals interact with the microbial community and affect biogas production in anaerobic digestion: A review.

Heavy metals (HMs), which accumulate in digestion substrates, such as plant residues and livestock manure, can affect biogas yields during anaerobic digestion (AD). Low concentration of Cu2+ (0-100 mg/L), Fe2+ (50-4000 mg/L), Ni2+ (0.8-50 mg/L), Cd2+ (0.1-0.3 mg/L), and Zn2+ (0-5 mg/kg) promote biogas production, while high concentrations inhibit AD. Trace amounts of HMs are necessary for the activity of some enzymes. For example, Cu2+ and Cd2+ serve as cofactors in the catalytic center of cellulase and stimulate enzyme activity. High contents of Cd2+ and Cu2+ inhibit enzyme activity by disrupting protein structures. Trace amounts of HMs stimulate the growth and activity of methanogens, while high levels have toxic effects on methanogens. HMs affect the hydrolysis, acidification, and other biochemical reactions of organics in AD by changing the enzyme structure and they also impact methanogen growth. A better understanding of the impact of HMs on AD can provide valuable insights for improving the digestion of poultry manure and plant residues contaminated with HMs, as well as help mitigate HMs pollution. Although several studies have been conducted in this field, few comprehensive reviews have examined the effect of many common HMs on AD. This review summarizes the effects of HMs on the biogas production efficiency of AD and also discusses the effects of HMs on the activities of enzymes and microbial communities.

Electrospun Microbial-Encapsulated Composite-Based Plasticized Seed Coat for Rhizosphere Stabilization and Sustainable Production of Canola ( Brassica napus L.).

Plant-growth-promoting bacteria show promises in crop production; nevertheless, innovation in their stable delivery is required for practical use by farmers. Herein, the composite of poly(vinyl alcohol)/poly(vinylpyrrolidone) plasticized with glycerol and loaded with the microbial consortium ( Bacillus subtilis plus Seratia marcescens) was fabricated and engineered onto canola ( Brassica napus L.) seed via electrospinning. Scanning electron microscopy showed that the biocomposite is a one-dimensional membrane, which encapsulated microbes in a multilayered nanostructure, and their interfacial behavior between microorganism and seed is beneficial for safer farming. A universal testing machine and thermogravimetric analysis demonstrated that the biocomposite holds sufficient thermomechanical properties for stable handling and practical management. A spectroscopic study resolved the living hybrid-polymer structure of the biocomposite and proved the plasticizing role of glycerol. A swelling study supports the degradation of the biocomposite in the hydrophilic environment as a result of the leaching of the plasticizer, which is important for the sustained release of microbial cells. A shelf life study supported that the biocomposite seed coat placed a threshold level of microbes [5.675 ± 0.48 log10 colony forming units (CFU)/seed] and maintained their satisfactory viability for 15 days at room temperature. An antifungal and nutrient-solubilizing study supported that the biocomposite seed coat could provide opportunities to biocontrol diseases and improve nutrient acquisition by the plant. A pot study documents the better performance of the biocomposite seed coat on seed germination, seedling growth, leaf area, plant dry biomass, and root system. A chemical and microbial study demonstrated that the biocomposite seed coat improved the effectiveness of the bioinoculant in the root-soil interface, where they survive, flourish, and increase the nutrient pool status. In particular, this study presents advances in the fabrication of the biocomposite for encapsulation, preservation, sustained release, and efficacious use of microorganisms onto seeds for precision farming.

Fabrication of Electrospun Probiotic Functionalized Nanocomposite Scaffolds for Infection Control and Dermal Burn Healing in a Mice Model.

The importance of microbiota paves the way to use microbial cells as medicines to treat pathobiomic diseases. This study reported the fabrication of probiotic (Enterococcus mundtii QAUEM2808)-functionalized nanocomposite scaffolds of poly(vinyl alcohol)/poly(vinylpyrrolidone)/glycerol via electrospinning. Scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis resolved the living composite structure and supported the encapsulation of E. mundtii throughout the nanostructured (318 ± 12 nm) fibers of bioscaffold membranes. The shelf life evaluation of 4-week-old samples supported that bioscaffolds showed an enhancement in probiotic survival count by 2.78 ± 0.10 log10 colony-forming units (cfu) versus counterpart biodispersion. The swelling and antagonistic evaluation showed that a bioscaffold is degradable in a simulated wound fluid which is essential for activation of probiotic strains to antagonize infection-causing Gram-positive and Gram-negative pathogens. A second-degree contact burn was made on the dorsum of male BALB/c mice (n = 30). The wounds were left open for 2 days to mimic burn contamination, and the mice were randomized into negative (untreated), positive (silver sulfadiazine cream), vehicle (biodispersion and nanoscaffold), and experimental bioscaffold groups (n = 6/group). These treatments were applied on 2, 6, 10, and 14 days postburn. A comparative wound closure, histopathology, and wound microbial evaluation demonstrated that the bioscaffolds accelerate epithelialization, collagen deposition, and hair follicle formation, inhibit harmful bacteria, and provide interference benefits. In particular, the probiotic active bioscaffold membrane could serve as a novel candidate to control infections and speed up the healing of burn wounds.

Correction: Qualitative analysis of biosurfactants from Bacillus species exhibiting antifungal activity.

[This corrects the article DOI: 10.1371/journal.pone.0198107.].

Qualitative analysis of biosurfactants from Bacillus species exhibiting antifungal activity.

Bacillus spp. produce a broad spectrum of lipopeptide biosurfactants, among which surfactin, iturin and fengycin are widely studied families. The goals of this study were to characterize the biosurfactant activity of Bacillus spp. and to investigate their motility and biofilm formation capabilities. In addition, we extracted lipopeptides from these bacteria to assess their antifungal activities and analyzed these products by mass spectrometry (MS). B. amyloliquefaciens FZB42, Bacillus sp. NH 217 and B. subtilis NH-100 exhibited excellent biosurfactant and surface spreading activities, whereas B. atrophaeus 176s and Paenibacillus polymyxa C1225 showed moderate activity, and B. subtilis 168 showed no activity. Strains FZB42, NH-100, NH-217, 176s and CC125 exhibited excellent biofilm formation capabilities. Lipopeptide extracts displayed good antifungal activity against various phytopathogens and their associated diseases, such as Fusarium moniliforme (rice bakanae disease), Fusarium oxysporum (root rot), Fusarium solani (root rot) and Trichoderma atroviride (ear rot and root rot). Lipopeptide extracts of these strains also showed hemolytic activity, demonstrating their strong potential to produce surfactants. LCMS-ESI analyses identified the presence of surfactin, iturin and fengycin in the extracts of Bacillus strains. Thus, the strains assayed in this study show potential as biocontrol agents against various Fusarium and Trichoderma species.

Biocontrol activity of surfactin A purified from Bacillus NH-100 and NH-217 against rice bakanae disease.

The potential of the Bacillus genus to antagonize phytopathogens is associated with the production of cyclic lipopeptides. Depending upon the type of lipopeptide, they may serve as biocontrol agents that are eco-friendly alternatives to chemical fertilizers. This study evaluates the biocontrol activity of surfactin-producing Bacillus (SPB) strains NH-100 and NH-217 and purified surfactin A from these strains against rice bakanae disease. Biologically active surfactin fractions were purified by HPLC, and surfactin A variants with chain lengths from C12 to C16 were confirmed by LCMS-ESI. In hemolytic assays, a positive correlation between surfactin A production and halo zone formation was observed. The purified surfactin A had strong antifungal activity against Fusarium oxysporum, F. moniliforme, F. solani, Trichoderma atroviride and T. reesei. Maximum fungal growth suppression (84%) was recorded at 2000 ppm against F. moniliforme. Surfactin A retained antifungal activity at different pH levels (5-9) and temperatures (20, 50 and 121 °C). Hydroponic and pot experiments were conducted to determine the biocontrol activity of SPB strains and the purified surfactin A from these strains on Super Basmati rice. Surfactin production in the rice rhizosphere was detected by LCMS-ESI at early growth stages in hydroponics experiments inoculated with SPB strains. However, the maximum yield was observed with a consortium of SPB strains (T4) and purified surfactin A (T5) treatments in the pot experiment. The outcomes of the present study revealed that surfactin A significantly reduced rice bakanae disease by up to 80%. These findings suggest that purified surfactin A could be an effective biocontrol agent against bakanae disease in rice and should be incorporated into strategies for disease management.

Antagonistic Bacillus spp. reduce blast incidence on rice and increase grain yield under field conditions.

Rice blast is a severe threat for agricultural production. Plant growth promoting rhizobacteria could be suitable biocontrol agents to reduce the disease incidence. In this study, Bacillus spp. KFP-5, KFP-7, KFP-17 significantly reduced disease severity by 40-52% with grain yield of 3.2-3.9 t ha-1 in two rice varieties i.e., basmati super and basmati 385. Bacillus spp. significantly colonized the rice rhizosphere with a cell population of 2.40E+06-5.6E+07CFU. Rice plants treated with antagonistic bacterial suspension followed by challenge inoculation with P. oryzae were found to have higher activities of antioxidant enzymes such as superoxide dismutase (308-266 Ug-1 FW), peroxidase (change in absorbance (ΔA) = 0.20-0.71 min-1 g-1 FW), polyphenol oxidase (ΔA = 0.29-0.58 min-1 g-1 FW) and phenylalanine ammonia lyase (ΔA = 0.32-0.59 min-1 g-1 FW). A consistency in the performance of strains was observed in the consecutive years 2013-2014. These findings suggest that indigenous Bacillus spp. could be a potential bio-inoculum for rice to control blast diseases and enhance yield.

Bacillus spp., a bio-control agent enhances the activity of antioxidant defense enzymes in rice against Pyricularia oryzae.

Plant growth promoting rhizobacteria (PGPR) are found to control the plant diseases by adopting various mechanisms. Induced systemic resistance (ISR) is an important defensive strategy manifested by plants against numerous pathogens especially infecting at aerial parts. Rhizobacteria elicit ISR by inducing different pathways in plants through production of various metabolites. In the present study, potential of Bacillus spp. KFP-5, KFP-7, KFP-17 was assessed to induce antioxidant enzymes against Pyricularia oryzae infection in rice. The antagonistic Bacillus spp. significantly induced antioxidant defense enzymes i-e superoxide dismutase (1.7-1.9-fold), peroxidase (3.5-4.1-fold), polyphenol oxidase (3.0-3.8-fold), phenylalanine ammonia-lyase (3.9-4.4-fold), in rice leaves and roots under hydroponic and soil conditions respectively. Furthermore, the antagonistic Bacillus spp significantly colonized the rice plants (2.0E+00-9.1E+08) and secreted multiple biocontrol determinants like protease (1.1-5.5 U/mg of soil or U/mL of hydroponic solution), glucanase, (1.0-1.3 U/mg of soil or U/mL of hydroponic solution), siderophores (6.5-42.8 µg/mL or mg) in the rhizosphere of different rice varieties. The results showed that treatment with Bacillus spp. enhanced the antioxidant defense activities in infected rice, thus alleviating P. oryzae induced oxidative damage and suppressing blast disease incidence.

Molecular Identification and Genetic Characterization of Macrophomina phaseolina Strains Causing Pathogenicity on Sunflower and Chickpea.

Macrophomina phaseolina is the most devastating pathogen which causes charcoal rot and root rot diseases in various economically important crops. Three strains M. phaseolina 1156, M. phaseolina 1160, and M. phaseolina PCMC/F1 were tested for their virulence on sunflower (Helianthus annuus L.) and chickpea (Cicer arietinum L.). The strains showed high virulence on both hosts with a disease score of 2 on chickpea and sunflower. The strains also increased the hydrogen per oxide (H2O2) content by 1.4- to 1.6-fold in root as well as shoot of chickpea and sunflower. A significant increase in antioxidant enzymes was observed in fungal infected plants which indicated prevalence of oxidative stress during pathogen propagation. The M. phaseolina strains also produced hydrolytic enzymes such as lipase, amylase, and protease with solubilization zone of 5-43 mm, 5-45 mm, and 12-35 mm, respectively. The M. phaseolina strains were identified by 18S rRNA and analyzed for genetic diversity by using random amplified polymorphic DNA (RAPD) markers. The findings based on RAPD markers and 18S rRNA sequence analysis clearly indicate genetic variation among the strains collected from different hosts. The genetically diverse strains were found to be pathogenic to sunflower and chickpea.

Root Associated Bacillus sp. Improves Growth, Yield and Zinc Translocation for Basmati Rice (Oryza sativa) Varieties.

Plant associated rhizobacteria prevailing in different agro-ecosystems exhibit multiple traits which could be utilized in various aspect of sustainable agriculture. Two hundred thirty four isolates were obtained from the roots of basmati-385 and basmati super rice varieties growing in clay loam and saline soil at different locations of Punjab (Pakistan). Out of 234 isolates, 27 were able to solubilize zinc (Zn) from different Zn ores like zinc phosphate [Zn3 (PO4)2], zinc carbonate (ZnCO3) and zinc oxide (ZnO). The strain SH-10 with maximum Zn solubilization zone of 24 mm on Zn3 (PO4)2ore and strain SH-17 with maximum Zn solubilization zone of 14-15 mm on ZnO and ZnCO3ores were selected for further studies. These two strains solubilized phosphorous (P) and potassium (K) in vitro with a solubilization zone of 38-46 mm and 47-55 mm respectively. The strains also suppressed economically important rice pathogens Pyricularia oryzae and Fusarium moniliforme by 22-29% and produced various biocontrol determinants in vitro. The strains enhanced Zn translocation toward grains and increased yield of basmati-385 and super basmati rice varieties by 22-49% and 18-47% respectively. The Zn solubilizing strains were identified as Bacillus sp. and Bacillus cereus by 16S rRNA gene analysis.

Molecular characterization of the modular chitin binding protein Cbp50 from Bacillus thuringiensis serovar konkukian.

Bacillus thuringiensis is an insecticidal bacterium whose chitinolytic system may be exploited to improve the insecticidal system of Bt-crops. A nucleotide fragment of 1368 bp from B. thuringiensis serovar konkukian S4, containing the complete coding sequence of the chitin binding protein Cbp50, was cloned and sequenced. Analyses have shown the protein to contain a modular structure consisting of an N-terminal CBM33 domain, two copies of a fibronectin-like domain and a C-terminal chitin binding domain classified as CBM5. The Cbp50 protein was heterologously expressed in Escherichia coli, purified and assessed for chitin binding activity. A deletion mutant (CBD-N; containing only the N-terminal CBM33 domain) of Cbp50 was produced to determine the role of C-terminal domains in the binding activity of the protein. The full-length Cbp50 was shown to bind ß-chitin most efficiently followed by α-chitin, colloidal chitin and cellulose. The polysaccharide binding activity of CBD-N was drastically decreased. The data demonstrate that both the N-terminal and C-terminal domains of Cbp50 are essential for the efficient binding of chitin. The purified Cbp50 showed antifungal activity against the phytopathogenic fungus Fusarium oxysporum and the opportunistic human pathogen Aspergillus niger. This is the first report of a modular chitin binding protein in bacteria.

Dicistronic binary vector system-A versatile tool for gene expression studies in cell cultures and plants.

Dicistronic binary vector constructs based on pGreenII vectors for Agrobacterium mediated gene transfer alleviate the translational expression monitoring of a target gene in plants. The functionality of the transformation vectors was proven by marker gene constructs containing a mannopine synthase promoter (p-MAS) fused to a beta-glucuronidase (gus) gene followed by an internal ribosome entry site and a firefly luciferase (luc) gene. The cap-dependent translation of a physically independent target protein can be monitored by the cap-independently co-translated luciferase, because both mRNAs are located on the same strand. Among three different IRES elements, the tobamo IRES element showed highest activity in transient expression. As a proof of principle for physiological studies the gus gene was replaced by a sodium antiporter gene (Atnhx1). Comparative studies with Atnhx1 transgenic luc expressing tobacco cell cultures and pea plants (Pisum sativum L.) showed improved salt tolerance in relation to their wild type counterparts grown under corresponding conditions. A coincidence of the luc gene expression and increased sodium chloride tolerance is demonstrated by measurement of luminescence and cell growth.