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1.  Purification and characterization of a novel lipopeptide from Streptomyces amritsarensis sp. nov. active against methicillin-resistant Staphylococcus aureus 
AMB Express  2014;4:50.
Nowadays antimicrobial lipopeptides are being widely exploited for developing potential therapeutic agents for treating bacterial infections. In the present study, we have purified and characterized an antimicrobial lipopeptide produced by Streptomyces amritsarensis sp. nov. (= MTCC 11845T = JCM 19660T). The lipopeptide was purified using silica gel chromatography, size exclusion chromatography and reverse phase- HPLC. The MS/MS analysis of the lipopeptide revealed that it has amino acid sequence as Ala-Thr-Gly-Ser-His-Gln and a long chain fatty acid tail with six times repeated the molecular mass of 161 Da which is corresponding to -C12H19. Based on the molecular mass (878.5 Da) and amino acid composition, the lipopeptide was identified as a novel lipopeptide. The MIC values of purified lipopeptide against Bacillus subtilis (MTCC 619), Staphylococcus epidermidis (MTCC 435), Mycobacterium smegmatis (MTCC 6) and clinical strain, Methicillin Resistant Staphylococcus aureus (MRSA) were found to be 10, 15, 25 and 45 μg/ml, respectively. It was completely stable at 70°C for 1 h and retained 81.8% activity after autoclaving (121°C for 15 min). It did not show any change in its activity profile between pH 5.0 - 9.0 and is stable to trypsin, proteinase K and lipase enzymes. It was found to be non-mutagenic against Salmonella typhimurium (TA98) and did not show cytotoxicity when checked against Chinese hamster ovary (CHO) cell line. In addition to antibacterial activity it also exhibits biosurfactant activity.
PMCID: PMC4077001
Antibacterial; Novel; Lipopeptide; Thermostable; Non-toxic
2.  Contamination of water resources by pathogenic bacteria 
AMB Express  2014;4:51.
Water-borne pathogen contamination in water resources and related diseases are a major water quality concern throughout the world. Increasing interest in controlling water-borne pathogens in water resources evidenced by a large number of recent publications clearly attests to the need for studies that synthesize knowledge from multiple fields covering comparative aspects of pathogen contamination, and unify them in a single place in order to present and address the problem as a whole. Providing a broader perceptive of pathogen contamination in freshwater (rivers, lakes, reservoirs, groundwater) and saline water (estuaries and coastal waters) resources, this review paper attempts to develop the first comprehensive single source of existing information on pathogen contamination in multiple types of water resources. In addition, a comprehensive discussion describes the challenges associated with using indicator organisms. Potential impacts of water resources development on pathogen contamination as well as challenges that lie ahead for addressing pathogen contamination are also discussed.
PMCID: PMC4077002
Pathogens; Contamination; Water resources; Watershed; Pathogens transport
3.  Assessing medium constituents for optimal heterologous production of anhydromevalonolactone in recombinant Aspergillus oryzae 
AMB Express  2014;4:52.
Anhydromevalonolactone (AMVL) is a bioactive natural product that arises from a molecular biology technique using Aspergillus oryzae as a heterologous host. AMVL has been used as a precursor for the synthesis of insect pest control reagents and has numerous applications in the biotechnological and medical industries. In this study, the Plackett-Burman Design and the Central Composite Design, which offer efficient and feasible approaches, were complemented to screen significant parameters and identify the optimal values for maximum AMVL production. The results suggested that sucrose, NaNO3, yeast extract and K2HPO4 were the key factors affecting AMVL production in a complex medium, whereas the major components required for a defined medium were NaNO3, K2HPO4, KH2PO4 and trace elements. These factors were subsequently optimized using the response surface methodology. Under optimal conditions, a maximum AMVL production of 250 mg/L in the complex medium and 200 mg/L in the defined medium was achieved, which represents an increase of approximately 3-4-fold compared to the commonly used malt extract medium.
PMCID: PMC4077012
Anhydromevalonolactone; Aspergillus oryzae; Plackett-Burman design; Central composite design; Response surface methodology
4.  Printed paper-based arrays as substrates for biofilm formation 
AMB Express  2014;4:32.
The suitability of paper-based arrays for biofilm formation studies by Staphylococcus aureus is demonstrated. Laboratory-coated papers with different physicochemical properties were used as substrates. The array platform was fabricated by patterning the coated papers with vinyl-substituted polydimethylsiloxane (PDMS) -based ink. The affinity of bacteria onto the flexographically printed hydrophobic and smooth PDMS film was very low whereas bacterial adhesion and biofilm formation occurred preferentially on the unprinted areas, i.e. in the reaction arrays. The concentration of the attached bacteria was quantified by determining the viable colony forming unit (CFU/cm2) numbers. The distribution and the extent of surface coverage of the biofilms were determined by atomic force microscopy. In static conditions, the highest bacterial concentration and most highly organized biofilms were observed on substrates with high polarity. On a rough paper surface with low polarity, the biofilm formation was most hindered. Biofilms were effectively removed from a polar substrate upon exposure to (+)-dehydroabietic acid, an anti-biofilm compound.
PMCID: PMC4077624
Biofilm formation; Staphylococcus aureus; Polarity; Surface roughness; AFM; PDMS
5.  Systematic analysis of the ability of Nitric Oxide donors to dislodge biofilms formed by Salmonella enterica and Escherichia coli O157:H7 
AMB Express  2014;4:42.
Biofilms in the industrial environment could be problematic. Encased in extracellular polymeric substances, pathogens within biofilms are significantly more resistant to chlorine and other disinfectants. Recent studies suggest that compounds capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of sessile bacteria and provide a foundation for novel approaches to controlling biofilms formed by some microorganisms. In this work, we compared the ability of five nitric oxide donors (molsidomine, MAHMA NONOate, diethylamine NONOate, diethylamine NONOate diethylammonium salt, spermine NONOate) to dislodge biofilms formed by non-typhoidal Salmonella enterica and pathogenic E. coli on plastic and stainless steel surfaces at different temperatures. All five nitric oxide donors induced significant (35-80%) dispersal of biofilms, however, the degree of dispersal and the optimal dispersal conditions varied. MAHMA NONOate and molsidomine were strong dispersants of the Salmonella biofilms formed on polystyrene. Importantly, molsidomine induced dispersal of up to 50% of the pre-formed Salmonella biofilm at 4°C, suggesting that it could be effective even under refrigerated conditions. Biofilms formed by E. coli O157:H7 were also significantly dispersed. Nitric oxide donor molecules were highly active within 6 hours of application. To better understand mode of action of these compounds, we identified Salmonella genomic region recA-hydN, deletion of which led to an insensitivity to the nitric oxide donors.
PMCID: PMC4070026  PMID: 24995149
Biofilm control; Bacterial signaling; Food-borne pathogens; Nitric oxide
6.  Physiological and growth response of rice plants (Oryza sativa L.) to Trichoderma spp. inoculants 
AMB Express  2014;4:45.
Trichoderma spp., a known beneficial fungus is reported to have several mechanisms to enhance plant growth. In this study, the effectiveness of seven isolates of Trichoderma spp. to promote growth and increase physiological performance in rice was evaluated experimentally using completely randomized design under greenhouse condition. This study indicated that all the Trichoderma spp. isolates tested were able to increase several rice physiological processes which include net photosynthetic rate, stomatal conductance, transpiration, internal CO2 concentration and water use efficiency. These Trichoderma spp. isolates were also able to enhance rice growth components including plant height, leaf number, tiller number, root length and root fresh weight. Among the Trichoderma spp. isolates, Trichoderma sp. SL2 inoculated rice plants exhibited greater net photosynthetic rate (8.66 μmolCO2 m−2 s−1), internal CO2 concentration (336.97 ppm), water use efficiency (1.15 μmoCO2/mmoH2O), plant height (70.47 cm), tiller number (12), root length (22.5 cm) and root fresh weight (15.21 g) compared to the plants treated with other Trichoderma isolates tested. We conclude that beneficial fungi can be used as a potential growth promoting agent in rice cultivation.
PMCID: PMC4052627  PMID: 24949276
Trichoderma spp; Rice; Physiological response; Growth response
7.  Screening of liquid media and fermentation of an endophytic Beauveria bassiana strain in a bioreactor 
AMB Express  2014;4:47.
A novel approach for biological control of insect pests could be the use of the endophytic entomopathogenic Beauveria bassiana isolate ATP-02. For the utilization of the endophyte as a commercial biocontrol agent, the fungus has to be mass-produced. B. bassiana was raised in shake flask cultures to produce high concentrations of total spores (TS), which include blastospores (BS) and submerged conidiospores (SCS). The highest concentration of 1.33×109 TS/mL and the highest yield of 5.32×1010 TS/g sucrose was obtained in the TKI broth with 5% sugar beet molasses which consists of 50% sucrose as a carbon source. In spite of the lower sugar concentration (2.5%) the amount of TS could be increased up to 11-times in contrast to the cultivation with 5% sucrose. The scale-up to a 2 L stirred tank reactor was carried out at 25°C, 200–600 rpm and 1 vvm at pH 5.5. A TS yield of 5.2×1010 TS/g sucrose corresponding to a SCS yield of 0.2×1010 SCS/g sucrose was obtained after 216 h. With regards to the culture medium the cost of 1012 TS amounts to 0.24 €. Plutella xylostella larvae, which were fed with oilseed rape leaves treated with spores from fermentation resulted in 77 ± 5% mortality. Moreover, spores from submerged cultivation were able to colonize oilseed rape leaves via leaf application. This is the first report of fermentation of an endophytic B. bassiana strain in a low-cost culture medium to very high yields of TS.
PMCID: PMC4052666  PMID: 24949278
Submerged culture; Fermentation; Beauveria bassiana; Endophyte; Blastospores; Submerged conidiospores; Biological control
8.  The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulose hydrolysates 
AMB Express  2014;4:46.
We investigated the severity of the inhibitory effects of 13 phenolic compounds usually found in spruce hydrolysates (4-hydroxy-3-methoxycinnamaldehyde, homovanilyl alcohol, vanillin, syringic acid, vanillic acid, gallic acid, dihydroferulic acid, p-coumaric acid, hydroquinone, ferulic acid, homovanillic acid, 4-hydroxybenzoic acid and vanillylidenacetone). The effects of the selected compounds on cell growth, biomass yield and ethanol yield were studied and the toxic concentration threshold was defined for each compound. Using Ethanol Red, the popular industrial strain of Saccharomyces cerevisiae, we found the most toxic compound to be 4-hydroxy-3-methoxycinnamaldehyde which inhibited growth at a concentration of 1.8 mM. We also observed that toxicity did not generally follow a trend based on the aldehyde, acid, ketone or alcohol classification of phenolic compounds, but rather that other structural properties such as additional functional groups attached to the compound may determine its toxicity. Three distinctive growth patterns that effectively clustered all the compounds involved in the screening into three categories. We suggest that the compounds have different cellular targets, and that. We suggest that the compounds have different cellular targets and inhibitory mechanisms in the cells, also compounds who share similar pattern on cell growth may have similar inhibitory effect and mechanisms of inhibition.
PMCID: PMC4052683  PMID: 24949277
Phenolics; Toxicity; Inhibition; Tolerance; Conversion; Saccharomyces cerevisiae
9.  Erythritol production on wheat straw using Trichoderma reesei 
AMB Express  2014;4:34.
We overexpressed the err1 gene in the Trichoderma reesei wild-type and in the cellulase hyperproducing, carbon catabolite derepressed strain Rut-C30 in order to investigate the possibility of producing erythritol with T. reesei. Two different promoters were used for err1 overexpression in both strains, a constitutive (the native pyruvat kinase (pki) promoter) and an inducible one (the native β-xylosidase (bxl1) promoter). The derived recombinant strains were precharacterized by analysis of err1 transcript formation on D-xylose and xylan. Based on this, one strain of each type was chosen for further investigation for erythritol production in shake flasks and in bioreactor experiments. For the latter, we used wheat straw pretreated by an alkaline organosolve process as lignocellulosic substrate. Shake flask experiments on D-xylose showed increased erythritol formation for both, the wild-type and the Rut-C30 overexpression strain compared to their respective parental strain. Bioreactor cultivations on wheat straw did not increase erythritol formation in the wild-type overexpression strain. However, err1 overexpression in Rut-C30 led to a clearly higher erythritol formation on wheat straw.
PMCID: PMC4052684  PMID: 24949268
Erythritol; Erythrose reductase; Trichoderma reesei; Wheat straw; Lignocellulose
10.  Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation 
AMB Express  2014;4:43.
Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi (“truffles”) with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites.
PMCID: PMC4052689  PMID: 24949275
Tuber spp; Truffles; Agrobacterium tumefaciens-mediated transformation; T-DNA; binary plasmid; Green fluorescent protein; Hygromycin phosphotransferase; TAIL-PCR
11.  Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts 
AMB Express  2014;4:39.
Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnologically relevant conditions was examined. Next, the fermentation fitness and metabolic traits of eight selected strains with a unique phenotypic profile were evaluated in a high-sugar synthetic medium under two nitrogen regimes. Although the strains exhibited significant differences in nitrogen requirements and utilization rates, a direct relationship between nitrogen consumption, specific growth rate, cell biomass, cell viability, acetic acid and glycerol formation was only observed under high-nitrogen conditions. In contrast, the strains produced more succinic acid under the low-nitrogen regime, and a direct relationship with the final cell biomass was established. Glucose and fructose utilization patterns depended on both yeast strain and nitrogen availability. For low-nitrogen fermentation, three strains did not fully degrade the fructose.
This study validates phenotypic and metabolic diversity among commercial wine yeasts and contributes new findings on the relationship between nitrogen availability, yeast cell growth and sugar utilization. We suggest that measuring nitrogen during the stationary growth phase is important because yeast cells fermentative activity is not exclusively related to population size, as previously assumed, but it is also related to the quantity of nitrogen consumed during this growth phase.
PMCID: PMC4052691  PMID: 24949272
Yeast; Nitrogen requirements; Stress resistance; Alcoholic fermentation
12.  Identification and characterization of endophytic bacteria from corn (Zea mays L.) roots with biotechnological potential in agriculture 
AMB Express  2014;4:26.
Six endophytic bacteria of corn roots were identified as Bacillus sp. and as Enterobacter sp, by sequencing of the 16S rRNA gene. Four of the strains, CNPSo 2476, CNPSo 2477, CNPSo 2478 and CNPSo 2480 were positive for the nitrogen fixation ability evaluated through the acetylene reduction assay and amplification of nifH gene. Two Bacillus strains (CNPSo 2477 and CNPSo 2478) showed outstanding skills for the production of IAA, siderophores and lytic enzymes, but were not good candidates as growth promoters, because they reduced seed germination. However, the same strains were antagonists against the pathogenic fungi Fusarium verticillioides, Colletotrichum graminicola, Bipolaris maydis and Cercospora zea-maydis. As an indication of favorable bacterial action, Enterobacter sp. CNPSo 2480 and Bacillus sp. CNPSo 2481 increased the root volume by 44% and 39%, respectively, and the seed germination by 47% and 56%, respectively. Therefore, these two strains are good candidates for future testing as biological inoculants for corn.
PMCID: PMC4052694  PMID: 24949261
Molecular phylogeny; 16S rRNA; nifH; Plant growth promotion; Antagonism
13.  Response surface modeling for hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores 
AMB Express  2014;4:21.
Response surface methodology using a face-centered cube design was used to describe and predict spore inactivation of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure of six spore-contaminated materials to hot, humid air. For each strain/material pair, an attempt was made to fit a first or second order model. All three independent predictor variables (temperature, relative humidity, and time) were significant in the models except that time was not significant for B. thuringiensis Al Hakam on nylon. Modeling was unsuccessful for wiring insulation and wet spores because there was complete spore inactivation in the majority of the experimental space. In cases where a predictive equation could be fit, response surface plots with time set to four days were generated. The survival of highly purified Bacillus spores can be predicted for most materials tested when given the settings for temperature, relative humidity, and time. These predictions were cross-checked with spore inactivation measurements.
PMCID: PMC4052701  PMID: 24949256
Bacillus; Spore; Decontamination; Hot humid air; RSM
14.  Combining the effects of process design and pH for improved xylose conversion in high solid ethanol production from Arundo donax 
AMB Express  2014;4:41.
The impact of pH coupled to process design for the conversion of the energy crop Arundo donax to ethanol was assessed in the present study under industrially relevant solids loadings. Two main process strategies were investigated, i.e. the traditional simultaneous saccharification and co-fermentation (SSCF) and a HYBRID design, where a long high temperature enzymatic hydrolysis step was carried out prior to continued low temperature SSCF, keeping the same total reaction time. Since acetic acid was identified as the major inhibitor in the slurry, the scenarios were investigated under different fermentation pH in order to alleviate the inhibitory effect on, in particular, xylose conversion. The results show that, regardless of fermentation pH, a higher glucan conversion could be achieved with the HYBRID approach compared to SSCF. Furthermore, it was found that increasing the pH from 5.0 to 5.5 for the fermentation phase had a large positive effect on xylose consumption for both process designs, although the SSCF design was more favored. With the high sugar concentrations available at the start of fermentation during the HYBRID design, the ethanol yield was reduced in favor of cell growth and glycerol production. This finding was confirmed in shake flask fermentations where an increase in pH enhanced both glucose and xylose consumption, but also cell growth and cell yield with the overall effect being a reduced ethanol yield. In conclusion this resulted in similar overall ethanol yields at the different pH values for the HYBRID design, despite the improved xylose uptake, whereas a significant increase in overall ethanol yield was found with the SSCF design.
PMCID: PMC4052779  PMID: 24949274
Bioethanol; High solids loading; Xylose fermentation; SSCF; Enzymatic hydrolysis
15.  Assessment of packed bed bioreactor systems in the production of viral vaccines 
AMB Express  2014;4:25.
Vaccination is believed to be the most effective method for the prevention of infectious diseases. Thus it is imperative to develop cost effective and scalable process for the production of vaccines so as to make them affordable for mass use. In this study, performance of a novel disposable iCELLis fixed bed bioreactor system was investigated for the production of some viral vaccines like Rabies, Hepatitis-A and Chikungunya vaccines in comparison to conventional systems like the commercially available packed bed system and roller bottle system. Vero and MRC-5 cell substrates were evaluated for growth parameters in all the three systems maintaining similar seeding density, multiplicity of infection (MOI) and media components. It was observed that Vero cells showed similar growth in all the three bioreactors whereas MRC-5 cells showed better growth in iCELLis Nano system and roller bottle system. Subsequently, the virus infection and antigen production studies also revealed that for Hepatitis-A and Chikungunya iCELLis Nano bioreactor system was better to the commercial packed bed bioreactor and roller bottle systems. Although for rabies antigen production commercially available packed bed bioreactor system was found to be better. This study shows that different bioreactor platforms may be employed for viral vaccine production and iCELLis Nano is one of such new convenient and a stable platform for production of human viral vaccines.
PMCID: PMC4052670  PMID: 24949260
Vaccine production; Rabies virus; Chikungunya virus; Hepatitis-A virus; Packed bed bioreactor; iCELLis Nano bioreactor
16.  Family 1 carbohydrate binding-modules enhance saccharification rates 
AMB Express  2014;4:36.
Cellulose degrading enzymes usually have a two-domain structure consisting of a catalytic domain and a non-catalytic carbohydrate-binding module. Although it is well known the importance of those modules in cell wall degrading process, their function is not yet fully understood. Here, we analyze the cellulose-hydrolysis activity enhancement promoted by the cellobiohydrolase I carbohydrate-binding module from Trichoderma harzianum. It was cloned, expressed, purified and used in combination with either a commercial cellulase preparation, T. reesei cellobiohydrolase I or its separate catalytic domain to hydrolyze filter paper. In all cases the amount of glucose released was increased, reaching up to 30% gain when the carbohydrate-binding module was added to the reaction. We also show that this effect seems to be mediated by a decrease in the recalcitrance of the cellulosic substrate. This effect was observed both for crystalline cellulose samples which underwent incubation with the CBM prior to application of cellulases and for the ones incubated simultaneously. Our studies demonstrate that family 1 carbohydrate-binding modules are able to potentiate the enzymatic degradation of the polysaccharides and their application might contribute to diminishing the currently prohibitive costs of the lignocellulose saccharification process.
PMCID: PMC4052752  PMID: 24949270
Carbohydrate binding-module; Cellulose binding-domain; Enzymatic hydrolysis; Cellulosic ethanol; Amorphogenesis
17.  Effects of biosurfactants on the viability and proliferation of human breast cancer cells 
AMB Express  2014;4:40.
Biosurfactants are molecules with surface activity produced by microorganisms that can be used in many biomedical applications. The anti-tumour potential of these molecules is being studied, although results are still scarce and few data are available regarding the mechanisms underlying such activity. In this work, the anti-tumour activity of a surfactin produced by Bacillus subtilis 573 and a glycoprotein (BioEG) produced by Lactobacillus paracasei subsp. paracasei A20 was evaluated. Both biosurfactants were tested against two breast cancer cell lines, T47D and MDA-MB-231, and a non-tumour fibroblast cell line (MC-3 T3-E1), specifically regarding cell viability and proliferation. Surfactin was found to decrease viability of both breast cancer cell lines studied. A 24 h exposure to 0.05 g l-1 surfactin led to inhibition of cell proliferation as shown by cell cycle arrest at G1 phase. Similarly, exposure of cells to 0.15 g l-1 BioEG for 48 h decreased cancer cells’ viability, without affecting normal fibroblasts. Moreover, BioEG induced the cell cycle arrest at G1 for both breast cancer cell lines. The biosurfactant BioEG was shown to be more active than surfactin against the studied breast cancer cells. The results gathered in this work are very promising regarding the biosurfactants potential for breast cancer treatment and encourage further work with the BioEG glycoprotein.
PMCID: PMC4052778  PMID: 24949273
Surfactin lipopeptide; Lactobacillus paracasei glycoprotein; Cell viability; Cell cycle; Breast cancer
18.  Absolute Quantification of Individual Biomass Concentrations in a Methanogenic Coculture 
AMB Express  2014;4:35.
Identification of individual biomass concentrations is a crucial step towards an improved understanding of anaerobic digestion processes and mixed microbial conversions in general. The knowledge of individual biomass concentrations allows for the calculation of biomass specific conversion rates which form the basis of anaerobic digestion models. Only few attempts addressed the absolute quantification of individual biomass concentrations in methanogenic microbial ecosystems which has so far impaired the calculation of biomass specific conversion rates and thus model validation. This study proposes a quantitative PCR (qPCR) approach for the direct determination of individual biomass concentrations in methanogenic microbial associations by correlating the native qPCR signal (cycle threshold, Ct) to individual biomass concentrations (mg dry matter/L). Unlike existing methods, the proposed approach circumvents error-prone conversion factors that are typically used to convert gene copy numbers or cell concentrations into actual biomass concentrations. The newly developed method was assessed and deemed suitable for the determination of individual biomass concentrations in a defined coculture of Desulfovibrio sp. G11 and Methanospirillum hungatei JF1. The obtained calibration curves showed high accuracy, indicating that the new approach is well suited for any engineering applications where the knowledge of individual biomass concentrations is required.
PMCID: PMC4052637  PMID: 24949269
Anaerobic digestion; qPCR; Individual biomass concentration; Biomass specific conversion rates
19.  Study of CFU for individual microorganisms in mixed cultures with a known ratio using MBRT 
AMB Express  2014;4:38.
Determination of metabolically active cell count is an important step in designing, operating and controlling fermentation processes. It’s particularly relevant in processes involving mixed cultures, where multiple species contribute to the total growth. The motivation for the current study is to develop a methodology to estimate metabolically active cell counts for the individual species in a mixed culture with approximate equal numbers. Further, the methodology should indicate the presence of a contaminant in short time periods since in the agar plate methods used frequently it takes about 24 h. We present a methodology based on the rate of Methylene blue (MB) reduction to evaluate total count of metabolically active cells. The standard curve relating the slope of MB reduction and CFU of the individual species could be used to measure the metabolic activity of each species in the mixed culture. The slope of MB reduction could also be used to obtain the growth rate of individual species in a mixed culture and that of the total cell count. These measurements were achieved in less than 6 minutes during the growth of the cells. Evaluating the metabolic activity of individual species in a mixed culture is tedious, difficult and time consuming. The Methylene Blue dye Reduction Test (MBRT) presented here is capable of quickly estimating colony forming units (CFU) of individual species in a mixed culture if the ratio of the numbers of cells is known. The method was used to dynamically detect the occurrence of a contaminating microorganism during fermentation. The protocol developed here can be adapted to applications in processes involving mixed cultures.
PMCID: PMC4052774  PMID: 24949271
Colony forming units (CFU); metabolic active cell; Methylene blue dye; Escherichia coli; Bacillus subtilis
20.  Xylanase (GH11) from Acremonium cellulolyticus: homologous expression and characterization 
AMB Express  2014;4:27.
Cellulosic materials constitute most of the biomass on earth, and can be converted into biofuel or bio-based materials if fermentable sugars can be released using cellulose-related enzymes. Acremonium cellulolyticus is a mesophilic fungus which produces a high amount of cellulose-related enzymes. In the genome sequence data of A. cellulolyticus, ORFs showing homology to GH10 and GH11 xylanases were found. The xylanases of A. cellulolyticus play an important role in cellulolytic biomass degradation. Search of a draft genome sequence of A. cellulolyticus for xylanase coding regions identified seven ORFs showing homology to GH 11 xylanase genes (xylA, xylB, xylC, xylD, xylE, xylF and xylG). These genes were cloned and their enzymes were prepared with a homologous expression system under the control of a glucoamylase promoter. Six of the seven recombinant enzymes were successfully expressed, prepared, and characterized. These enzymes exhibited optimal xylanase activity at pH 4.0 – 4.5. But this time, we found that only XylC had enormously higher relative activity (2947 U•mg −1) than the other xylanases at optimum pH. This result is surprising because XylC does not retain a carbohydrate-binding module 1 (CBM-1) that is necessary to bind tightly own substrate such as xylan. In this study, we discuss the relationship between activity, pH and sequence of seven xylanases in A. cellulolyticus.
PMCID: PMC4052667  PMID: 24949262
Acremonium cellulolyticus; Xylanase; Hemicellulose; Homologous expression; Biomass; Xylan
21.  Disruption of pknG enhances production of gamma-aminobutyric acid by Corynebacterium glutamicum expressing glutamate decarboxylase 
AMB Express  2014;4:20.
Gamma-aminobutyric acid (GABA), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by Corynebacterium glutamicum that expresses Escherichia coli glutamate decarboxylase (GAD) B encoded by gadB. This strain was engineered to produce GABA more efficiently from biomass-derived sugars. To enhance GABA production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pknG (encoding serine/threonine protein kinase G), which controls the activity of 2-oxoglutarate dehydrogenase (Odh) in the tricarboxylic acid cycle branch point leading to glutamate synthesis. We succeeded in expressing GadB in a C. glutamicum strain harboring a deletion of pknG. C. glutamicum strains GAD and GAD ∆pknG were cultured in GP2 medium containing 100 g L−1 glucose and 0.1 mM pyridoxal 5′-phosphate. Strain GAD∆pknG produced 31.1 ± 0.41 g L−1 (0.259 g L−1 h−1) of GABA in 120 hours, representing a 2.29-fold higher level compared with GAD. The production yield of GABA from glucose by GAD∆pknG reached 0.893 mol mol−1.
PMCID: PMC4052669  PMID: 24949255
Corynebacterium glutamicum; Gamma-aminobutyric acid; Glutamate decarboxylase; 2-oxoglutarate dehydrogenase; Protein kinase G
22.  Microbial community in microbial fuel cell (MFC) medium and effluent enriched with purple photosynthetic bacterium (Rhodopseudomonas sp.) 
AMB Express  2014;4:22.
High power densities have been obtained from MFC reactors having a purple color characteristic of Rhodopseudomonas. We investigated the microbial community structure and population in developed purple MFC medium (DPMM) and MFC effluent (DPME) using 16S rRNA pyrosequencing. In DPMM, dominant bacteria were Comamonas (44.6%), Rhodopseudomonas (19.5%) and Pseudomonas (17.2%). The bacterial community of DPME mainly consisted of bacteria related to Rhodopseudomonas (72.2%). Hydrogen oxidizing bacteria were identified in both purple-colored samples: Hydrogenophaga and Sphaerochaeta in the DPMM, and Arcobacter, unclassified Ignavibacteriaceae, Acinetobacter, Desulfovibrio and Wolinella in the DPME. The methanogenic community of both purple-colored samples was dominated by hydrogenotrophic methanogens including Methanobacterium, Methanobrevibacter and Methanocorpusculum with significantly lower numbers of Methanosarcina. These results suggeste that hydrogen is actively produced by Rhodopseudomonas that leads to the dominance of hydrogen consuming microorganisms in both purple-colored samples. The syntrophic relationship between Rhodopseudomonas and hydrogenotrophic microbes might be important for producing high power density in the acetate-fed MFC under light conditions.
PMCID: PMC4052673  PMID: 24949257
Purple color characteristic of Rhodopseudomonas; Microbial community; Hydrogen oxidizing bacteria; Hydrogenotrophic methanogens; Syntrophic relationship
23.  Efficient fermentation of an improved synthetic grape must by enological and laboratory strains of Saccharomyces cerevisiae 
AMB Express  2014;4:16.
Grape must or freshly pressed grape juice is a complex chemical matrix that impacts the efficiency of yeast fermentation. The composition of natural grape must (NGM) can be variable; thus, to ensure reproducibility, a synthetic grape must (SGM) with defined composition is commonly used. The aim of this work was to create conditions to advance the use of Saccharomyces cerevisiae laboratory strains for wine fermentation studies, considering previous results obtained for enological strains fermenting NGM under simulated winery conditions. We designed a new SGM formulation, ISA-SGM, by introducing specific modifications to a commonly used formulation, putting together previous reports. We added glucose and fructose in equal amounts (125 g/l) and 50 parts per million (ppm) sulfur dioxide (SO2, corresponding to standard enological treatment), and we optimized the concentrations of malic acid (3 g/l), citric acid (0.3 g/l), and tartaric acid (3 g/l). Using ISA-SGM, we obtained similar fermentative profiles for the wine strain ISA1000, the prototrophic strain S288C, and its auxotrophic derivative BY4741. In this case, the concentrations of supplements were optimized to 120 mg/l L-uracil, 80 mg/l L-methionine, 400 mg/l L-leucine, and 100 mg/l L-histidine. All these strains tested in ISA-SGM presented a similar fermentative performance as ISA1000 in NGM. ISA-SGM formulation is a promising new tool to allow the use of the auxotrophic BY strains in the detailed assessment of the alcoholic fermentation process under simulated winery conditions, and it provides a foundation to extract relevant physiological conclusions in future research on enological yeast traits.
PMCID: PMC4052690  PMID: 24949253
Synthetic grape must; Natural grape must; Wine fermentation; BY auxotrophic mutant serie
24.  Mushroom as a product and their role in mycoremediation 
AMB Express  2014;4:29.
Mushroom has been used for consumption as product for a long time due to their flavor and richness in protein. Mushrooms are also known as mycoremediation tool because of their use in remediation of different types of pollutants. Mycoremediation relies on the efficient enzymes, produced by mushroom, for the degradation of various types of substrate and pollutants. Besides waste degradation, mushroom produced a vendible product for consumption. However, sometimes they absorb the pollutant in their mycelium (biosorption process) and cannot be consumed due to absorbed toxicants. This article reviews the achievement and current status of mycoremediation technology based on mushroom cultivation for the remediation of waste and also emphasizes on the importance of mushroom as product. This critical review is also focused on the safety aspects of mushroom cultivation on waste.
PMCID: PMC4052754  PMID: 24949264
Biodegradation; Bioremediation; Genotoxicity; Biosorption; Mushroom; Ames test; Product; Industrial waste; Agroindustrial waste; Bioconversion
25.  Cloning, expression and characterization of a versatile Baeyer-Villiger monooxygenase from Dietzia sp. D5 
AMB Express  2014;4:23.
A novel BVMO encoding gene was identified from a draft genome sequence of a newly isolated strain of Dietzia. Analysis of the protein sequence revealed that it belongs to a group of BVMOs whose most characterized member is cyclopentadecanone monooxygenase (CPDMO). The gene was PCR amplified, cloned and successfully expressed in E. coli. The expressed recombinant enzyme was purified using metal affinity chromatography. Characterization of the purified enzyme revealed that it has a broad substrate scope and oxidized different compounds including substituted and unsubstituted alicyclic, bicyclic-, aliphatic-ketones, ketones with an aromatic moiety, and sulfides. The highest activities were measured for 2- and 3-methylcyclohexanone, phenylacetone, bicyclo-[3.2.0]-hept-2-en-6-one and menthone. The enzyme was optimally active at pH 7.5 and 35°C, a temperature at which its half-life was about 20 hours. The stability studies have shown that this enzyme is more stable than all other reported BVMOs except the phenylacetone monooxygenase from the thermophilic organism Thermobifida fusca.
PMCID: PMC4052671  PMID: 24949258
Baeyer-Villiger monooxygenase; Biocatalysis; Enzyme stability; Protein expression

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