Microbiology

Effects of pH value on the expression of key iron/sulfur oxidation genes during bioleaching of chalcopyrite on thermophilic condition Abstract Physicochemical factors such pH value would affect the microbial metabolism during chalcopyrite bioleaching. To this end, the effects of pH on the expression of critical functional genes during bioleaching were evaluated. A mixed culture of moderate thermophiles was used for chalcopyrite bioleaching at initial pH values of 1.0, 2.0, and 3.0, and bioleaching processes were monitored via measuring the physicochemical parameters. Quantitative real-time PCR assay was used to monitor the dynamics of microbial community structures and the expression of critical iron/sulfur oxidation genes (4Fe-4S ferredoxin and sulfate adenylyltransferase genes, respectively). Redundancy analysis and calculation of correlation coefficients were used to reveal linkages between gene expression and various physicochemical factors. The leaching processes at initial pH of 1.0 and 3.0 were prolonged compared with that at initial pH of 2.0. It was shown that Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus were the dominant species during the early stage in free and attached cells, respectively, while Ferroplasma thermophilum became predominant in the later phase. The gene expression in Sulfobacillus thermosulfidooxidans and Ferroplasma thermophilum was greatly affected by pH values. On the other hand, the relationship between pH and gene expression in Acidithiobacillus caldus was not significant. The study unraveled the importance of pH value on chalcopyrite bioleaching, and pH selectively influenced the expression of key functional genes of some specific species.

Probiotic lactobacilli mediated changes in global epigenetic signatures of human intestinal epithelial cells during Escherichia coli challenge Abstract Host genome environment association is critical for proper development and functioning of an individual. Microbiota and probiotics within the cellular vicinity may serve such critical stimuli that can bring out different epigenetic mediated host responses. The aim of present study was to explore the changes in epigenetic signatures of Caco-2 cells by probiotic strains (Lactobacillus rhamnosus MTCC 5897: LR, Lactobacillus fermentumMTCC 5898: LF and their mixture: RF), respectively, or during challenge with Escherichia coli (ATCC 14948) using exclusion, competition and displacement assays. Adenocarcinoma intestinal epithelial Caco-2 cells were treated with LR, LF, RF and E. coli for 6 h, respectively. Caco-2 cells were also challenged with E. coli and probiotic lactobacilli during exclusion, competition and displacement assays. Finally, global epigenetic modifications by acetylation of H4 and H3 histone proteins and DNA methylation patterns were determined. Probiotic-treated Caco-2 cells displayed significant (p < 0.01) reduction in percent global H4 and H3 acetylation, respectively, in contrast to their elevated (p < 0.05) levels after E. coli infection. On the other hand, a remarkable (p < 0.01) decrease in percent H4 and H3 acetylation were observed when E. coli were excluded, competed or displaced by lactobacilli strains. No changes in the global DNA methylation patterns were observed in Caco-2 cells after exposure to probiotic strains or E. coli, respectively, but surprisingly, their levels increased significantly (p < 0.05) when lactobacilli-treated cells were challenged with E. coli during exclusion or competition than displacement assays. Probiotic L. rhamnosus and L. fermentum modulated the host epigenetic signatures via global histone acetylation individually or during E. coli challenge by exclusion, competition and displacement assays. Whilst on the other hand changes in global DNA methylation patterns were obtained significantly during probiotic treatment with E. coli in exclusion and competition protocols.

Antibacterial activity of endophytic fungi isolated from Sceletium tortuosum L. (Kougoed) Abstract Endophytic fungi have the ability to co-exist with their host plants without causing any harm and are beneficial to both the plant and the fungi. The current study determined the antimicrobial properties and identify the chemical compounds of secondary metabolites produced by endophytic fungi isolated from Sceletium tortuosum L. A total of 60 endophytic fungi produced secondary metabolites that were detected after fermentation and extraction. Antibacterial properties of the secondary metabolites were determined using the disc diffusion assay against pathogenic environmental Gram-positive and Gram-negative bacteria as well as control stains. The chemical compounds were characterized by GC-MS. Overall, 15% of fungal extracts displayed narrow spectrum of activity against the bacteria strains. Despite this, none of the fungal extracts inhibited growth of Enterococcus faecalis (ATCC S1299) and Enterococcus gallinarum (ATCC 700425) while Bacillus cereus (ATCC 10876) was the most susceptible against the fungal extracts. Fusarium oxysporum (GG 008) with accession no. KJ774041.1 displayed significant antibacterial activity that was linked to high levels of 5-hydroxymethylfurfural (HMF) and octadecanoic acid as revealed by GC-MS. This study revealed the presence of bioactive secondary metabolites with antibacterial activities from fungi isolated from Sceletium tortuosum L.

Biochemical analysis of elephant foot yam ( Amorphophallus paeoniifolius ) lacto-pickle with probiotic Lactobacillus plantarum Abstract Lacto-pickles are tangy, crisp, and definitely not intimidating to eat. We previously standardized fermentations of elephant foot yam (EFY) into lacto-pickle using different fermentation specifications. The aims of this work were to perform the effect of starter culture strain Lactobacillus plantarum (MTCC-1325) on chemical and biochemical properties of EFY lacto-pickle. In this study, we conducted the preprocessing of blanched EFY blocks/cubes in brine (NaCl, 8%, w/v) and inoculated with the starter culture (L. plantarum MTCC-1325). The impact of L. plantarum (MTCC-1325) during lacto-pickle fermentation and its effect on the antioxidant property, structural characterization (scanning electron microscopy (SEM) and infrared spectroscopy (FTIR)), sensory analysis, and volatilome profile (gas chromatography and mass spectrometry (GC-MS)) of EFY lacto-pickle were determined. Inoculated samples showed significantly higher acidification of the brine, reaching a pH of 2.67 and titratable acidity (TA) of 2.8 g/L within 42 days of fermentation, suggesting potential antioxidant activity. The FTIR technique revealed the changes/deformation of functional groups and SEM study suggested the interaction/adhesion between starter culture L. plantarum (MTCC 1325) on EFY lacto-pickle. The starter culture used clearly influenced volatile organic compounds (VOCs) profiles of EFY lacto-pickle. Moreover, EFY lacto-pickle produced with the starter culture significantly perceived a positive response in the overall acceptance. Overall results indicated the successful and accelerated EFY lacto-pickle was achieved when using L. plantarum (MTCC-1325) as starter culture and thus the possible use of this fortified product by the consumers in their diet.

Dynamics of bacterial communities in a river water treatment wetland Abstract Bacteria play important roles in the removal of inorganic and organic pollutants in constructed wetlands (CWs) used for surface water treatment, however, the environmental variables driving the change of bacterial community remain poorly understood. The present work explored seasonal and spatial dynamics of bacterial communities in a large CW used for river water treatment and the associated environmental variables. Quantitative PCR assay and high-throughput sequencing analysis were used to determine the abundance, richness, diversity, and structure of wetland bacterial community. Bacterial abundance was correlated to nitrite and temperature, while bacterial diversity could be influenced by pH as well as carbon, nitrogen, and phosphorus. Bacterial community structure might be shaped by nitrogen and phosphorus. A considerable difference in bacterial community composition existed between wetland soils and sediments. Proteobacteria (accounting for 33–60%) was the largest bacterial phylum, and Chloroflexi (5–24%) was also abundant. The abundance, richness, diversity, and composition of the bacterial community were influenced by wetland plant type and wetland trophic status.

MALDI-TOF/TOF mass spectrometry for determination of yeast diversity in traditional cornelian cherry tarhana produced with different cereal/pseudocereal flours Abstract Purpose This study is to determine the dynamics in the microflora of cornelian cherry tarhana (CCT), a traditional food of Turkey, by MALDI-TOF/TOF MS. Methods Non-fermented and fermented CCT productions were performed by using flours of bread wheat, wholegrain hull-less barley, buckwheat, and clear flour. Identification of the isolates obtained from raw materials and various production steps of the CCT was performed by MALDI-TOF/TOF MS. Dendograms were prepared by cluster analysis and the distances between the strains isolated during production stages and from cornelian cherry puree were determined. Results Totally, 231 isolates were obtained and 45.5% of them were identified at species level, 30.3% at genus level while 24.2% of the isolates could not be identified. It was found that microflora of cornelian cherry tarhana is mainly composed of yeasts. Thirty-two percent of the identified yeast isolates were Hanseniaspora uvarum and the others were Saccharomycescerevisiae (19.6%), Torulaspora delbrueckii (18.6%), Candida krusei (11.3%), Candida lusitaniae (9.3%), Metschnikowia pulcherrima (3.1%), Wickerhamomyces anomalus (2.1%), Candida kefyr (2.1%), Cyberlindnera fabianii (1%), and Candida parapsilosis (1%). Only two lactic acid bacteria could be isolated, which were Lactobacillus reuteri and Enterococcus spp., originating from buckwheat flour and clear flour, respectively. Dendograms revealed that some of the yeast strains isolated during production were originating from cornelian cherry. Conclusions Microflora of CCT was investigated for the first time. This is one of the few studies using MALDI-TOF/TOF MS for identification of food originated yeasts. Novel species–identified, endogenic yeasts which could have potential technological characteristics were introduced.

Inulinase hyperproduction by Kluyveromyces marxianus through codon optimization, selection of the promoter, and high-cell-density fermentation for efficient inulin hydrolysis Abstract This study aimed to overexpress an inulinase gene in Kluyveromyces marxianus to achieve the inulinase overproduction and preparation of ultra-high-fructose syrup. First, the inulinase gene (INU1 gene) was overexpressed through codon optimization and selection of a suitable promoter. Then, the inulinase was overproduced by high-cell-density fermentation. Finally, ultra-high-fructose syrup was prepared. It was found that optimization of the codons of the native INU1 gene encoding inulinase from Kluyveromyces marxianus KM-0 made a recombinant strain KM-N70 carrying the optimized INU1N gene produce 251.4 U/mL of the inulinase activity. Furthermore, inulinase activity produced by a recombinant KM-KN16 strain carrying the optimized INU1N gene directed by the native TPS1 promoter from K. marxianus KM-0 reached 338.5 U/mL and expression level of the optimized INU1N gene in the recombinant KM-KN16 strain was also greatly enhanced. During a 10-L fermentation, the recombinant KM-KN16 strain could produce 374.3 U/mL of inulinase activity within 24 h, while during a high-cell-density fed-batch fermentation, the recombinant KM-KN16 strain could produce 896.1 U/mL of inulinase activity and OD600nm value of its culture reached 108. The crude inulinase preparation obtained in this study had an inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation. It was found that 90.3% of 332.4 g/L of inulin was hydrolyzed to produce 41.0 g/L of glucose and 256.0 g/L of fructose and 91.1% of 328.2 g/L of inulin in the extract of the tubers of Jerusalem artichoke was hydrolyzed to produce 48.3 g/L of glucose and 250.5 g/L of fructose by the crude inulinase preparation (75 U/g of the substrate) within 8 h. The hydrolysates contained major monosaccharides and a trace amount of trisaccharides and the monosaccharides were composed of around 85% fructose and 15% glucose. So far, any other yeasts available have produced only up to 120 U/mL of inulinase activity. Together, this made the recombinant KM-KN16 strain be the best inulinase producer at this moment. The inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation and the ultra-high-fructose syrup with 41.0 g/L of glucose and 256.0 g/L of fructose were obtained. The inulinase activity obtained in this study was the highest among all the inulinase activities produced by yeast, fungal, and bacterial strains obtained so far.

Diversity of β-lactam resistance genes in gram-negative rods isolated from a municipal wastewater treatment plant Abstract Urban wastewater treatment plants (UWTPs) are considered hot spots for the accumulation and transfer of antibiotic resistant bacteria and their genes. We investigated the prevalence, diversity, and genomic localization of β-lactamase resistance genes in wastewater from different parts of an UWTP, such as raw wastewater, wastewater from biological reactor, and treated wastewater, collected from the UWTP of Warsaw, Poland. In this study, we focused on gram-negative rods, mainly Enterobacteriaceae, and used Multiplex PCR to identify various β-lactamase resistance genes (bla). Susceptibility to a number of antibiotics and genomic localization of β-lactamase resistance genes were then determined using disc-diffusion and Southern hybridization methods, respectively. No differences in the frequency of different types of bla genes between the sampling points were discerned. Three new variants of β-lactamase genes (blaCMY-157, blaMOX-13, and blaFOX-15) were identified. Four bla genes (blaTEM-12, blaTEM-30, blaTEM-47/68, blaACT) that had never been found in UWTPs were identified in this study. Nine of the identified bla genes had been found in the same environment previously. The blaFOX-15 variant on a Kluyvera sp. plasmid and blaGES type on Raoultella spp. plasmids were observed for the first time in these genera. There was a decrease in the number of multidrug resistant strains in the effluent compared to the influent. Finally, a significantly higher number of cefotaxime and carbapenem unsusceptible strains were detected in the influent than in the effluent. The results strongly support the hypothesis of UWTPs as a hot spot for antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) accumulation and indicate that β-lactamase genes are widely disseminated among gram-negative rods isolated from this environment.

Capability of plant growth-promoting bacteria in chromium-contaminated soil after application of composted tannery sludge Abstract The aim of this study was to select bacterial strains with biochemical capability to tolerate high concentration of chromium (Cr) in soils. Plant growth-promoting bacteria (PGPB) were isolated from the root nodules of Phaseolus lunatus and grown in Cr-contaminated soil with the application of composted tannery sludge. Soils were collected from the experimental field with the application of composted tannery sludge (CTS) in five rates: 0, 2.5, 5, 10, and 20 t ha−1 CTS. Bacterial strains were isolated and evaluated for their biochemical capabilities for production of urease, protease, amylase, lipase, catalase, gelatinase, and indole-3-acetic acid, P solubilization, and Cr tolerance. A total of 54 PGPB were isolated from the nodules, being 40%, 37%, 13%, and 10% found in the treatments with 2.5, 5, 10, and 20 t ha−1, respectively. The majority of these isolates presented positive responses for the tests of urease, catalase, and phosphate solubilization, while some isolates were positive for the test of protease, lipase, carboxymethyl cellulose, gelatinase, and amylase. We also observed a decrease in the number of isolates able to tolerate high concentration of Cr. Three strains (UFPI-LCC61, UFPI-LCC64, and UFPI-LCC87) presented high biochemical capability and tolerance to Cr. However, the isolate UFPI-LCC87 showed high biochemical capability and tolerance to the highest concentration of Cr. Our results indicated bacterial strains that present potential to be used in soils contaminated with Cr and also for promoting plant growth.

Lichens and other lithobionts on the carbonate rock surfaces of the heritage site of the tomb of Lazarus (Palestinian territories): diversity, biodeterioration, and control issues in a semi-arid environment Abstract Purpose Investigations on the lithobiontic colonization of the stone cultural heritage in (semi-)arid regions are needed to address conservation strategies. In this work, lithobiontic communities were examined on the carbonate rock surfaces of the heritage site of the Tomb of Lazarus. We aimed to evaluate their distribution and interaction with the lithic substrate, together with the efficacy of biocidal treatments for their control. Methods Diversity and abundance of lithobionts were surveyed on the Jerusalem stone blocks of three architectural elements. Observations at the lichen-rock interface were carried out by reflected light and scanning electron microscopy. The efficacy against lichens of the widely used biocide benzalkonium chloride (BZC) was compared for different concentrations and application methods, and evaluated by epifluorescence microscopy. Results Chlorolichens were the dominant component of lithobiontic communities, more thoroughly adapted to the semi-arid conditions of the site than mosses and black biofilms of cyanobacteria and dematiaceous fungi. A different structural organization, in terms of thallus thickness and depth of the hyphal penetration component, characterized epilithic and endolithic lichen species, responsible for different deteriogenic activities. Biocidal assays showed that even the methodologies that are usually effective in temperate conditions (as the application of BZC 1.5% by poultice) may not completely devitalize lichens adapted to the stress conditions of semi-arid climates, unless a pervasive biocide diffusion through metabolically active thalli is carefully guaranteed. Conclusion Lithobionts act as biodeteriogens on the semi-arid surfaces of the investigated heritage site. Their removal is thus recommendable, but it needs to be adequately supported with a careful calibration of devitalization strategies.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com