Strain G1 Research Articles

Fermentation performance optimization in an ectopic fermentation system

Ectopic fermentation systems (EFSs) were developed for wastewater treatment. Previous studies have investigated the ability of thermophilic bacteria to improve fermentation performance in EFS. Continuing this research, we evaluated EFS performance using principle component analysis and investigated the addition of different proportions of cow dung. Viable bacteria communities were clustered and identified using BOX-AIR-based repetitive extragenic palindromic-PCR and 16S rDNA analysis. The results revealed optimal conditions for the padding were maize straw inoculated with thermophilic bacteria. Adding 20% cow dung yielded the best pH values (6.94–8.56), higher temperatures, increased wastewater absorption, improved litter quality, and greater microbial quantities. The viable bacteria groups were enriched by the addition of thermophilic consortium, and exogenous strains G21, G14, G4-1, and CR-15 were detected in fermentation process. The proportion of Bacillus species in treatment groups reached 70.37% after fermentation, demonstrating that thermophilic bacteria, especially Bacillus, have an important role in EFS, supporting previous predictions.

ISOLATION, SCREENING AND THE INFLUENCE OF CULTIVATION FACTORS ON CELLULASE OF BACTERIA ISOLATED FROM TERMITES GUT

From five termites guts samples collected in different places in Ha Noi and Vinh, 11 isolates which formed halo on agar plates with CMC were isolated. Among them strain G4 possessed the highest CMCase activity. Based on morphology and 16S rRNA gene sequences analysis, G4 was identified as Bacillus subtilis G4. The results from cultivation study revealed that M3 medium containing rice bran 1 %, soy flour 1 %, casein 1 % and 1 % NaCl was the best medium for cellulase production of B. subtilis G4. At optimal conditions for G4 which were inoculums ratio 1 %, cultivation temperature 37ºC, cultivation time 72 hours, shaking speed 150 rpm and initial pH medium 7, CMCase and FPase activity reached 3.36 U/ml and 0.35 U/ml, respectively.

CHARACTERIZATION OF CELLULASE PREPARATION OF BACILLUS SP.G4 ISOLATED FROM THE TERMITES GUT

The Bacillus sp. strain G4 isolated from termites gut can produce cellulase with high activity in rice bran medium after 72 h of incubation at 37 °C. The crude enzymewas collected by centrifugation at 6,000 rpmfor 15 minutes. The precipitation of cellulasewith ammonium sulfate 60 – 90 % saturation and acetone at the concentration 60 % – 90 % was studied. Results showed that ammonium sulfate 90 % saturation gave cellulase with highest purification factor 8.87 but the yield was only 59.9 %, whereas acetone at 90 % concentration gave highest yield 83.57 % with purification factor 4.38. CMCase activity of cellulasepreparation obtained by acetone precipitation at 90 % was optimum at pH 7 and 60 °C. Furthermore, CMCase was stable at pH 6.0 - 7.0 and at temperature lower than 50 °C. The CMCase was activated by ion Ca2+ but inhibited by Co2+, Zn2+, Cu2+, Fe2+, and was not affected by Mg2+ and Ba2+. The CMCase was inhibited by high concentration of SDS while EDTA and Tween 80 played activated role.

Poorly neutralizing polyclonal antibody in vitro against coxsackievirus A16 circulating strains can prevent a lethal challenge in vivo

ABSTRACTNeutralizing antibodies (NTAbs) is a major criterion for evaluation the immunogenicity of many vaccines, for example, poliovirus and EV71 vaccine. Here, we firstly discovered that polyclonal antibodies induced by inactivated CVA16 vaccine and lived CVA16 virus have poor ability to neutralize circulating CVA16 strains in vitro. However, the passive transfer of poorly neutralizing polyclonal antibodies can protect suckling mice from lethally challenged with circulating strains in vivo. In addition, the obvious dose response was found between the titer of antibodies and the survival rate. Interestingly, poorly neutralizing polyclonal antibodies against circulating CVA16 strains, have good ability to neutralize prototype strain G10 in vitro. Between G10 and circulating CVA16 strains, there are total 47 variant sites in capsid, which are near the interface of VP1, VP2, and VP3, and close to 2-fold axis. Based on the structure of CVA16, the obvious structural changes were observed in residue 213 of VP1 GH loop, residue 139 of VP2 EF loop, and residues 59, 182 and 183 of VP3 GH loop. What we found may provide a new sight for the development of CVA16 vaccine.

Management of late leaf spot (Phaeoisariopsis personata) and root rot (Macrophomina phaseolina) diseases of groundnut (Arachis hypogaea L.) with plant growth-promoting rhizobacteria, systemic acquired resistance inducers and plant extracts

Greenhouse experiments were conducted to study the efficacy of plant growth-promoting rhizobacteria (PGPR) for control of root rot (RR) in groundnut (Arachis hypogaea L.) caused by Macrophomina phaseolina and to test the ability of plant systemic acquired resistance (SAR) inducers and plant extracts to protect groundnut plants from late leaf spot (LLS) caused by Phaeoisariopsis personata. Seed treatment and soil application of a talc-based formulation of B. subtilis strain G1 significantly reduced the incidence of root rot under greenhouse conditions. In experiments with SAR inducers, foliar application of salicylic acid (SA) (7 mM) on 45 days after sowing significantly reduced LLS incidence and increased the pod yield. Foliar application of aqueous extract (10%) from leaves of Adhatoda vasica and zimmu (Allium sativum x A. cepa) on 45 days after sowing significantly decreased the LLS incidence and increased the pod yield compared with the untreated control. Field experiments were conducted to develop an integrated method for the management of LLS and RR of groundnut using the best performing PGPR, SAR inducer and plant extract in combinations. Combined application of B. subtilis strain G1 through seed (10 g/kg) and soil (2.5 kg/ha) followed by foliar application of A. vasica extract (10%) on 30, 45 and 60 days after sowing significantly reduced LLS and RR diseases in groundnut and increased the pod yield under field conditions. The above treatment resulted in significant reductions in the area under the disease progress curve (AUDPC) for LLS compared with that of untreated control.

Species A rotaviruses isolated from hospitalized patients over 5years of age in Kolkata, India, in 2012/13.

In hospital-based diarrhoeal disease surveillance at Infectious Diseases & Beliaghata-General Hospital (May-2012 to April-2013), Kolkata, India, stool samples were collected from patients<5years (n=830) and>5years of age (n=728) hospitalized with diarrhea. Group-A rotavirus (GARV) was identified by ELISA followed by multiplex RT-PCR. In children<5years of age, 53.4% of the samples were positive for GARV. In patients>5years to 90years old, only 6.04% (n=44) tested positive for GARV. G2P[4] strains (n=16 [36.36%]) were the most prevalent, followed by G9P[4] strains (n=13 [29.54%]), while P[4]-(n=30 [68.18%]) was most prevalent among the P genotypes. The GARV strains G2, G9 and P[4] detected in adults clustered together in the phylogenetic tree with the GARV strains identified in children (<5years) during the same period. Rotavirus positivity was high among female patients (75%), suggesting that caregivers (mother/grandmother/older-siblings) may get infected through young children or may act as carriers for transmission.

Effect of Media on Acetone-Butanol-Ethanol Fermentation by Isolated Clostridium spp.

Butanol is one of the important industrial chemicals and currently recognized as an alternative and renewable biofuel. It has been receiving an increased attention due to the energy crisis and the decrease of petroleum. Butanol can be obtained from several anaerobic microorganisms, which belong to genus Clostridium. The problem is the ability of Clostridium sp. to produce low concentration of butanol. Consequently, the screening and isolating for new Clostridium strains which can produce high amount of biobutanol has been required. Clostridium sp. strain G10 was isolated from marsh sediment in King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand. Its capability of butanol production was 5.89 g/L with a total acetone-butanol-ethanol (ABE) concentration of 8.48 g/L under anaerobic conditions. The medium composition significantly affected the performance of (ABE) production by strain G10. To investigate the effect of media on solvent production by isolated strain, four media, including RCM, T6, GYCC, and P2 medium (30 g/L glucose in each medium) were utilized to cultivate Clostridium sp. strain G10. The results revealed that, T6 medium could produce the highest biobutanol concentration (13.49 g/L) and ABE was 16.22 g/L (acetone 2.63 g/L and ethanol 0.10 g/L).

Structural and functional characterization of mercuric reductase from Lysinibacillus sphaericus strain G1.

In response to the widespread presence of inorganic Hg in the environment, bacteria have evolved resistance systems with mercuric reductase (MerA) as the key enzyme. MerA enzymes have still not been well characterized from gram positive bacteria. Current study reports physico-chemical, kinetic and structural characterization of MerA from a multiple heavy metal resistant strain of Lysinibacillus sphaericus, and discusses its implications in bioremediation application. The enzyme was homodimeric with subunit molecular weight of about 60kDa. The Km and Vmax were found to be 32µM of HgCl2 and 18 units/mg respectively. The enzyme activity was enhanced by β-mercaptoethanol and NaCl up to concentrations of 500µM and 100mM respectively, followed by inhibition at higher concentrations. The enzyme showed maximum activity in the pH range of 7-7.5 and temperature range of 25-50°C, with melting temperature of 67°C. Cu2+ exhibited pronounced inhibition of the enzyme with mixed inhibition pattern. The enzyme contained FAD as the prosthetic group and used NADPH as the preferred electron donor, but it showed slight activity with NADH as well. Structural characterization was carried out by circular dichroism spectrophotometry and X-ray crystallography. X-ray confirmed the homodimeric structure of enzyme and gave an insight on the residues involved in catalytic binding. In conclusion, the investigated enzyme showed higher catalytic efficiency, temperature stability and salt tolerance as compared to MerA enzymes from other mesophiles. Therefore, it is proposed to be a promising candidate for Hg2+ bioremediation.

Ability of Soil Isolated Actinobacterial Strains to Prevent, Bind and Biodegrade Ochratoxin A

Ochratoxin A (OTA) is one of the most important mycotoxins, and contaminates several agricultural products, particularly cereals, grapes, maize, barley, spices and coffee. The aim of this project was to reduce the levels of OTA by supplementing the artificially contaminated solutions with seven strains of actinobacteria (AT10, AT8, SN7, MS1, ML5, G10 and PT1) in order to evaluate their capacity for binding and metabolizing the OTA, as well as their ability to reduce the expression of the genes responsible for its production in A. carbonarius. In the first part of this study, we evaluated the capacity of Streptomyces strains for binding OTA on their surfaces after 0, 30 and 60 min of incubation with PBS solution supplemented with OTA. In the second part, we tested the ability of these strains, as well as their supernatants, to detoxify the ISP2 medium. Finally, we studied the effect of the Streptomyces cocultured with Aspergillus carbonarius on the expression of OTA biosynthesis genes. Results showed that, among the strains co-cultured with A. carbonarius, the strain G10 was able to reduce the expression of acpks, acOTApks, acOTAnrps and vea genes, thus reducing OTA from solid PDA medium to 13.50% of reduction. This strain was remarkably able to detoxify and bind OTA up to 47.07%. Strain AT8 was stronger in detoxifying OTA (52.61%), but had no significant effect on the studied gene expression.

STR3 and CYS3 Contribute to 2-Furfurylthiol Biosynthesis in Chinese Sesame-Flavored Baijiu Yeast.

2-Furfurylthiol is an important aroma compound with characteristic sesame flavor. It was recently identified as the representative aroma compound of Chinese sesame-flavored Baijiu. But its formation mechanism is unclear. In this study, the dominant yeast species Saccharomyces cerevisiae, Pichia kudriavzevii, and Wickerhamomyces anomalus were isolated from samples of Chinese sesame-flavored fermentation and were screened for the capacity of 2-furfurylthiol biosynthesis. Some isolates were found to be capable of generating 2-furfurylthiol using furfural and l-cysteine as precursors, among which S. cerevisiae G20 exhibited the strongest capacity with a yield of 3.03 mg/L. Furthermore, the genes STR3 and CYS3 from S. cerevisiae were cloned and overexpressed in the reference strain S. cerevisiae S288c and the isolate strain G20, respectively, which resulted in a significant increase in 2-furfurylthiol release in the two strains. Moreover, inactivation of STR3 gene in S288c and G20 led to obvious reduction in 2-furfurylthiol production, demonstrating that STR3 got involved in 2-furfurylthiol biosynthesis in S. cerevisiae. It is the first time that the yeast isolates with high capacity of 2-furfurylthiol biosynthesis were found during Chinese sesame-flavored Baijiu fermentation and confirmed that the genes STR3 and CYS3 were closely relevant to 2-furfurylthiol biosynthesis.

Catalytic hydrolysis of starch for biohydrogen production by using a newly identified amylase from a marine bacterium Catenovulum sp. X3

An identified cold-adaptive, organic solvents-tolerant alkaline α-amylase (HP664) from Catenovulum sp. strain X3 was heterologously expressed and characterized in E. coli, and it was further applied to starch saccharification for biohydrogen production. The recombinant HP664 belongs to a member of glycoside hydrolase family 13 (GH13), with a molecular weight of 69.6kDa without signal peptides, and also shares a relatively low similarity (49%) to other reported amylases. Biochemical characterization demonstrated that the maximal enzymatic activity of HP664 was observed at 35°C and pH 9.0. Most metal ions inhibited its activity; however, low polar organic solvents (e.g., benzene and n-hexane) could enhance the activity by 35–50%. Additionally, HP664 also exhibited the catalytic capability on various polysaccharides, including potato starch, amylopectin, dextrin and agar. In order to increase the bioavailability of starch for H2 production, HP664 was utilized to elevate fermentable oligosaccharide level, and the results revealed that the maximal hydrolytic percentage of starch was up to 44% with 12h of hydrolysis using 5.63U of HP664. Biohydrogen fermentation of the starch hydrolysate by Clostridium sp. strain G1 yielded 297.7mL of H2 after 84h of fermentation, which is 3.73-fold higher than the control without enzymatic treatment of HP664.

Cloning of Partial β-Mannanase Gene from Indonesia Marine Bacteria Bacillus Subtilis LBF-005

The strain LBF-005 from marine bacteria have already isolated and screened for mannanase degrading enzyme in submerged fermentation process. This strain was further identified by using 16S rRNA showed that bacterium is belong to Bacillus subtilis that could produce mannanase with activity around 9.5 U/mL. The optimum pH and temperature for the activity of crude enzyme for mannanase was 6.0 and 50 oC. Cloning of mannanase gene from B. subtilis was conducted using six primers set designed based on the homology analysis conserve region several mannanase from bacteria (Bacillus sp.) glycosyl hydrolase (GH) family 26. Optimization of PCR conditions was performed by gradient PCR to obtained PCR product of b-mannanase gene. The PCR product was obtained by third primer combination and was estimated to be around 972-bp. Analysis of the nucleotide sequence showed that sequences has similarity with mananase gene from other Bacillus sp., such as the B. subtilis strain WLY-12, B. subtilis strain WL-8, B. subtilis strain CICC 10260, B.subtilis strain CD-25, B.subtilis strain G1, and Bacillus sp. SWU60 were about 98%, 98%, 98%, 98%, 97% and 95%, respectively. The next research will to obtain a whole gene encoding β-mannanase by using in vitro cloning method, characterization of recombinant mannanase and application this enzyme for mannooligosaccharides production.

Performance of a newly isolated salt-tolerant yeast strain Pichia occidentalis G1 for degrading and detoxifying azo dyes

A salt-tolerant yeast named G1 which could decolorize various azo dyes was recently isolated and identified as Pichia occidentalis. Systematic researches on characterization, degradation pathway, detoxification effects and enzymes analysis of this yeast were done. The results showed that the optimal metabolism and growth parameters for strain G1 were: 2.0gL−1 glucose, 0.6gL−1 ammonium sulfate, 0.08gL−1 yeast extract, 30gL−1 NaCl, 160rmin−1, 30°C and pH 5.0. More than 98% of 50mgL−1 Acid Red B (ARB) could be decolorized within 16h under the optimal conditions. Additionally, strain G1 degraded and obviously detoxified ARB through a possible pathway successively consisting of decolorization, deamination/desulfonation and TCA cycle processes. Moreover, NADH-DCIP reductase was estimated as the key reductase for decolorization and ligninases including lignin peroxidase, manganese peroxidase and laccase were important oxidoreductases for further degradation of decolorization intermediates.

Total phenolic contents and antioxidant properties of buckwheat fermented by three strains of Agaricus

The aim of this work was to evaluate the effects of solid-state fermentation (SSF) with three strains of Agaricus (Agaricus blazei Murrill SH26, Agaricus bisporus AS2796 and Agaricus bisporus G1) on the total phenolic contents and antioxidant properties of buckwheat (Fagopyrum esculentum Möench). The results showed that the total phenolic contents of the fermented buckwheat varied with fermentation time and the starter organisms. The total phenolic content of buckwheat fermented with strain SH26 reached its maximum value of 18.07 mg/g after the fermentation of 21 days, which was 3.96 times higher than that of control. The ethanolic extracts of buckwheat fermented by strains SH26 and AS2796 exhibited enhanced antioxidant properties in comparison with the control. However, the ethanolic extracts of buckwheat fermented by strain G1 exhibited less antioxidant properties in comparison with the control. The ethanolic extracts of buckwheat fermented with strain SH26 exhibited the highest reducing power, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity and ferrous ion chelating ability, respectively. The ethanolic extracts of buckwheat fermented with strain AS2796 exhibited the highest superoxide anion radical scavenging ability.

Молекулярная классификация штаммов Echinococcus granulosus от крупного рогатого скота в Ливии

This study demonstrates the distribution of various E. granulosus strains in sheep, cattle and camel. The common sheep strain G1 is mainly found in sheep and cattle, but also parasitized camels. In contrast, the camel strain G6 is found mainly in camels and rarely in sheep and cattle. However, the study also revealed the possible presence of cryptic species that are closely related to both genotypes in livestock of Libya as evident by high mutations in several specimens. Based on the occurrence of overlapping hosts of E. granulosus in Libya, more research on the transmission cycles and genotypes of E. granulosus in Libya is required. In addition, it is suggested that surveys on potential intermediate hosts, including in humans with dogs as the major final host in Libya using higher resolution molecular tools such as microsatellite markers is recommended.

In vitro screening and identification of plant growth-promoting rhizobacteria against pathogen causing Astragalus root rot

The antagonistic effect of Bacillus spp. against Fusarium solani was evaluated by living body dual culture and Oxford cup method. The plant growth promoting properties of those strains that had obvious and stable antifungal activity were then tested. The results showed that the living body and bacteria-free fermentation filtrate of strain G10 both had obvious and stable antifungal effect to F. solani. Besides, the strain possessed such growth promoting properties as phosphate solubilization, nitrogen fixation, and production of IAA, amylase and HCN. Strain G10 was classified and identified as B. subtilis by a combination of morphological, physiological and biochemical tests, 16 SrDNA gene sequence analysis and the BBL CrystalTM bacteria identification. In conclusion, B. subtilis G10 has the basic characteristics of multifunctional strains and could be one of the microbiological resources for developing special bio-control agent against Astragalus root rot.

Identification of sulfonylurea biodegradation pathways enabled by a novel nicosulfuron-transforming strain Pseudomonas fluorescens SG-1: Toxicity assessment and effect of formulation

Nicosulfuron is a selective herbicide belonging to the sulfonylurea family, commonly used on maize culture. A bacterial strain SG-1 was isolated from an agricultural soil previously treated with nicosulfuron. This strain was identified as Pseudomonas fluorescens and is able to quantitatively dissipate 77.5% of nicosulfuron (1mM) at 28°C in the presence of glucose within the first day of incubation. Four metabolites were identified among which ASDM (2-(aminosulfonyl)-N,N-dimethyl-3-pyridinecarboxamide) and ADMP (2-amino-4,6-dimethoxypyrimidine) in substantial proportions, corresponding to the hydrolytic sulfonylurea cleavage. Two-phase dissipation kinetics of nicosulfuron by SG-1 were observed at the highest concentrations tested (0.5 and 1mM) due to biosorption. The extend and rate of formulated nicosulfuron transformation were considerably reduced compared to those with the pure active ingredient (appearance of a lag phase, 30% dissipation after 10days of incubation instead of 100% with the pure herbicide) but the same metabolites were observed. The toxicity of metabolites (standardized Microtox® test) showed a 20-fold higher toxicity of ADMP than nicosulfuron. P. fluorescens strain SG-1 was also able to biotransform two other sulfonylureas (metsulfuron-methyl and tribenuron-methyl) with various novel pathways. These results provide new tools for a comprehensive picture of the sulfonylurea environmental fate and toxicity of nicosulfuron in the environment.

Differential expression and characterization of cypermethrin-degrading potential proteins in Bacillus thuringiensis strain, SG4.

A cypermethrin-degrading bacterium (SG4) was isolated from the pesticide-contaminated soil in the agricultural field of the crop research centre of the University, and characterized as Bacillus thuringiensis strain, SG4. The bacterium degraded 78.9 % of cypermethrin (50 ppm) in 15 days when grown in a minimal medium. To understand the functional proteins of cypermethrin degradation in Bacillus thuringiensis strain SG4, a comparative proteomic analysis was performed in the presence/absence of cypermethrin after 5 days of incubation in minimal medium. More than 450 spots corresponding to different proteins were recorded by 2D electrophoresis. We report expression of 223 and 250 unique proteins under normal and induced conditions (cypermethrin stress), respectively. Identified proteins were categorized into different functional groups on the basis of their biological functions, viz., catabolic enzymes, translational and stress proteins, etc. Characterization of cypermethrin-specific proteins in a bacterial strain will help in biodegradation practices in situ.

Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function.

Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.

Bioremediation of hexavalent chromium contaminated soil by a bioleaching system with weak magnetic fields

Strains G1 (Geotrichum sp.) and B2 (Bacillus sp.) were isolated from a hexavalent chromium (Cr(VI)) contaminated site and they could remove Cr(VI) efficiently. The mixed culture of G1 and B2 (1:1, v/v) was prepared to test remediation of Cr(VI) contaminated soil. The influences of pH, magnetic field, dissolved oxygen, carbon sources and solid/liquid ratio on Cr(VI) removal were investigated, as well as the effects of pH and magnetic field on Cr(VI) desorption. The results indicated that the desorption of Cr(VI) from the contaminated soil was strongly affected by pH of the leaching solution with optimal value of 7.0 based on the growth of both microorganisms and desorption efficiency. Weak magnetic field of 7.0 mT promoted both the Cr(VI) desorption and the growth of G1. Compared with anaerobic conditions, bioreduction rate of Cr(VI) under aerobic conditions was higher. The optimal carbon source and solid/liquid ratio were glucose (5 g l−1) and 1/15 (w/v), respectively. The associated Cr(VI) reduction efficiency was 94.8%, which was 68.8% higher than control. The high bioremediation efficiency of the mixed cultures of G1 and B2 could be attributed to their alternative growth patterns to enhance the performance of bioremediation of the heavy metals contaminated sites.