Rumen fermentation and epithelial gene expression responses to diet ingredients designed to differ in ruminally degradable protein and fiber supplies.

Although numerous studies exist relating ruminal volatile fatty acid (VFA) concentrations to diet composition and animal performance, minimal information is available describing how VFA dynamics respond to diets within the context of the whole rumen environment. The objective of this study was to characterize how protein and fiber sources affect dry matter intake, rumen pH, fluid dynamics, fermentation parameters, and epithelial gene expression. Four diet treatments (soybean meal or heat-treated soybean meal and beet pulp or timothy hay) were delivered to 10 wethers. The soybean meals served as crude protein (CP) sources while the beet pulp and timothy hay represented neutral detergent fiber (NDF) sources. Feed intake, rumen pH, fluid pool size, and fluid passage rate were unaffected by treatment. Butyrate synthesis and absorption were greater on the beet pulp treatment whereas synthesis and absorption of other VFA remained unchanged. Both CP and NDF treatment effects were associated with numerous VFA interconversions. Expression levels of rumen epithelial genes were not altered by diet treatment. These results indicate that rumen VFA dynamics are altered by changes in dietary sources of nutrients but that intake, rumen environmental parameters, and the rumen epithelium may be less responsive to such changes.

Thermal adaptation of acetic acid bacteria for practical high-temperature vinegar fermentation.

Thermotolerant microorganisms are useful for high-temperature fermentation. Several thermally adapted strains were previously obtained from Acetobacter pasteurianus in a nutrient-rich culture medium, while these adapted strains could not grow well at high temperature in the nutrient-poor practical culture medium, "rice moromi." In this study, A. pasteurianus K-1034 originally capable of performing acetic acid fermentation in rice moromi was thermally adapted by experimental evolution using a "pseudo" rice moromi culture. The adapted strains thus obtained were confirmed to grow well in such the nutrient-poor media in flask or jar-fermentor culture up to 40 or 39 °C; the mutation sites of the strains were also determined. The high-temperature fermentation ability was also shown to be comparable with a low-nutrient adapted strain previously obtained. Using the practical fermentation system, "Acetofermenter," acetic acid production was compared in the moromi culture; the results showed that the adapted strains efficiently perform practical vinegar production under high-temperature conditions.

Efficient expression of novel glutamate decarboxylases and high level production of γ-aminobutyric acid catalyzed by engineered Escherichia coli.

Glutamate decarboxylase (GAD) has the potential of converting L-glutamate to gamma-aminobutyric acid (GABA), which is an important non-proteinogenic amino acid that has a potential use as food additive or dietary supplement for its physiological functions. A novel pyridoxal 5'-phosphate (PLP)-dependent glutamate decarboxylase (LsGAD) was cloned from GRAS (generally recognized as safe) Lactobacillus senmaizukei by genome mining and efficiently expressed in Escherichia coli BL21. The LsGAD displayed excellent temperature property, pH property and kinetic parameters compared with the probe LbGAD and the other GADs. By increasing the copy number of the LsGAD encoding gene, the expression level of LsGAD and the biosynthesis yield of GABA were increased, which was near to 2 times of that was expressed in single copy. These results established a solid foundation for increasing the added value of L-glutamate and the biosynthesis of GABA.

Transcriptome analysis reveals the protection mechanism of proanthocyanidins for Saccharomyces cerevisiae during wine fermentation.

Grape-derived proanthocyanidins could act as a protector against various environmental stresses for Saccharomyces cerevisiae during wine fermentation, resulting in the increased physiological activity, fermentation efficiency and improved wine quality. In order to explore the possible protection mechanism of proanthocyanidins globally, RNA-seq analysis for wine yeast AWRI R2 cultivated with 0 g/L (group A), 0.1 g/L (group B), 1.0 g/L (group C) proanthocyanidins were applied in this study. Differentially expressed genes were enriched into six metabolic pathways including vitamin B6, thiamine, amino acids, aminoacyl-tRNA, carbohydrate and steroid based on KEGG enrichment analysis. Four key genes (SNZ2, THI6, THI21 and THI80), participated in the biosynthesis of vitamin B6 and thiamine, were up-regulated significantly in proanthocyanidins treated yeast cells and the gene expression levels were verified by RT-qPCR. Yeast cells supplemented with proanthocyanidins performed increased intracellular levels of vitamin B6 and thiamine and higher cell viability compared to the control group. In addition, the composition of intracellular fatty acids showed an obvious alternation in proanthocyanidins-treated yeast cells, in which the UFAs content increased whereas the SFA content decreased. In general, we provided an indirect protection effect of proanthocyanidins on the yeast cells to alleviate environmental stresses during wine fermentation.

Evidence for Escherichia coli DcuD carrier dependent FOF1-ATPase activity during fermentation of glycerol.

During fermentation Escherichia coli excrete succinate mainly via Dcu family carriers. Current work reveals the total and N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity at pH 7.5 and 5.5 in E. coli wild type and dcu mutants upon glycerol fermentation. The overall ATPase activity was highest at pH 7.5 in dcuABCD mutant. In wild type cells 50% of the activity came from the FOF1-ATPase but in dcuD mutant it reached ~80%. K+ (100 mM) stimulate total but not DCCD inhibited ATPase activity 40% and 20% in wild type and dcuD mutant, respectively. 90% of overall ATPase activity was inhibited by DCCD at pH 5.5 only in dcuABC mutant. At pH 7.5 the H+ fluxes in E. coli wild type, dcuD and dcuABCD mutants was similar but in dcuABC triple mutant the H+ flux decreased 1.4 fold reaching 1.15 mM/min when glycerol was supplemented. In succinate assays the H+ flux was higher in the strains where DcuD is absent. No significant differences were determined in wild type and mutants specific growth rate except dcuD strain. Taken together it is suggested that during glycerol fermentation DcuD has impact on H+ fluxes, FOF1-ATPase activity and depends on potassium ions.

Overexpression of PkINO1 improves ethanol resistance of Pichia kudriavzevii N77-4 isolated from the Korean traditional fermentation starter nuruk.

The yeast Pichia kudriavzevii N77-4 was isolated from the Korean traditional fermentation starter nuruk. In this study, fermentation performance and stress resistance ability of N77-4 was analyzed. N77-4 displayed superior thermotolerance (up to 44°C) in addition to enhanced acetic acid resistance compared to Saccharomyces cerevisiae. Moreover, N77-4 produced 7.4 g/L of ethanol with an overall production yield of 0.37 g/g glucose in 20 g/L glucose medium. However, in 250 g/L glucose medium the growth of N77-4 slowed down when the concentration of ethanol reached 14 g/L or more and ethanol production yield also decreased to 0.30 g/g glucose. An ethanol sensitivity test indicated that N77-4 was sensitive to the presence of 1% ethanol, which was not the case for S. cerevisiae. Furthermore, N77-4 displayed a severe growth defect in the presence of 6% ethanol. Because inositol biosynthesis is critical for ethanol resistance, expression levels of the PkINO1 encoding a key enzyme for inositol biosynthesis was analyzed under ethanol stress conditions. We found that ethanol stress clearly repressed PkINO1 expression in a dose-dependent manner and overexpression of PkINO1 improved the growth of N77-4 by 19% in the presence of 6% ethanol. Furthermore, inositol supplementation also enhanced the growth by 13% under 6% ethanol condition. These findings indicate that preventing downregulation in PkINO1 expression caused by ethanol stress improves ethanol resistance and enhances the utility of P. kudriavzevii N77-4 in brewing and fermentation biotechnology.

Secretory overproduction of a raw starch-degrading glucoamylase in Penicillium oxalicum using strong promoter and signal peptide.

Raw starch-degrading enzymes (RSDEs) are capable of directly degrading raw starch granules below the gelatinization temperature of starch, which may significantly reduce the cost of starch-based biorefining. However, low yields of natural RSDEs from filamentous fungi limit their industrial application. In this study, transcriptomic and secretomic profiling was employed to screen strongest promoters and signal peptides for use in overexpression of a RSDE gene in Penicillium oxalicum. Top five strong promoters and three signal peptides were detected. Using a green fluorescent protein (GFP) as the reporter, the inducible promoter pPoxEgCel5B of an endoglucanase gene PoxEgCel5B and the signal peptide spPoxGA15A of a raw starch-degrading glucoamylase PoxGA15A were respectively identified as driving the highest GFP production in P. oxalicum. PoxGA15A-overexpressed P. oxalicum strain OXPoxGA15A, which was constructed based on both pPoxEgCel5B and spPoxGA15A, produced significantly higher amounts of recombinant PoxGA15A than the parental strain ∆PoxKu70. Furthermore, crude enzyme from the OXPoxGA15A strain exhibited high activities towards raw starch from cassava, potato, and uncooked soluble starch. Specifically, raw cassava starch-degrading enzyme activity reached 241.6 U/mL in the OXPoxGA15A, which was 3.4-fold higher than that of the ∆PoxKu70. This work provides a feasible method for hyperproduction of RSDEs in P. oxalicum.

Characterization of microbial community structure and metabolic potential using Illumina MiSeq platform during the black garlic processing.

Black garlic is a distinctive garlic deep-processed product made from fresh garlic at high temperature and controlled humidity. To explore microbial community structure, diversity and metabolic potential during the 12days of the black garlic processing, Illumina MiSeq sequencing technology was performed to sequence the 16S rRNA V3-V4 hypervariable region of bacteria. A total of 677,917 high quality reads were yielded with an average read length of 416bp. Operational taxonomic units (OTU) clustering analysis showed that the number of species OTUs ranged from 148 to 1974, with alpha diversity increasing remarkably, indicating the high microbial community abundance and diversity. Taxonomic analysis indicated that bacterial community was classified into 45 phyla and 1125 distinct genera, and the microbiome of black garlic samples based on phylogenetic analysis was dominated by distinct populations of four genera: Thermus, Corynebacterium, Streptococcus and Brevundimonas. The metabolic pathways were predicted for 16S rRNA marker gene sequences based on Kyoto Encyclopedia of Genes and Genomes (KEGG), indicating that amino acid metabolism, carbohydrate metabolism and membrane transport were important for the black garlic fermentation process. Overall, the study was the first to reveal microbial community structure and speculate the composition of functional genes in black garlic samples. The results contributed to further analysis of the interaction between microbial community and black garlic components at different stages, which was of great significance to study the formation mechanism and quality improvement of black garlic in the future.

Use of a wine yeast deletion collection reveals genes that influence fermentation performance under low-nitrogen conditions.

A deficiency of nitrogenous nutrients in grape juice can cause stuck and sluggish alcoholic fermentation, which has long been a problem in winemaking. Nitrogen requirements vary between wine yeast strains, and the ability of yeast to assimilate nitrogen depends on the nature and concentration of nitrogen present in the medium. In this study, a wine yeast gene deletion collection (1844 deletants in the haploid AWRI1631 background) was screened to identify genes whose deletion resulted in a reduction in the time taken to utilise all sugars when grown in a chemically defined grape juice medium supplemented with limited nitrogen (75 mg L-1 as a free amino acid mixture). Through micro-scale and laboratory-scale fermentations, 15 deletants were identified that completed fermentation in a shorter time than the wildtype (c.a. 15%-59% time reduction). This group of genes was annotated to biological processes including protein modification, transport, metabolism and ubiquitination (UBC13, MMS2, UBP7, UBI4, BRO1, TPK2, EAR1, MRP17, MFA2 and MVB12), signalling (MFA2) and amino acid metabolism (AAT2). Deletion of MFA2, encoding mating factor-a, resulted in a 55% decrease in fermentation duration. Mfa2Δ was chosen for further investigation to understand how this gene deletion conferred fermentation efficiency in limited nitrogen conditions.

Fermented Papaya Preparation Restores Age-Related Reductions in Peripheral Blood Mononuclear Cell Cytolytic Activity in Tube-Fed Patients.

Tube-fed elderly patients are generally supplied with the same type of nutrition over long periods, resulting in an increased risk for micronutrient deficiencies. Dietary polyphenols promote immunity and have anti-inflammatory, anti-carcinogenic, and anti-oxidative properties. Carica papaya Linn. is rich in several polyphenols; however, these polyphenols are poorly absorbed from the digestive tract in their original polymerized form. Therefore, we determined the molecular components of a fermented Carica papaya Linn. preparation, as well as its effects on immunity and the composition of gut microbiota in tube-fed patients. Different doses of the fermented C. papaya L. preparation were administered to three groups of tube-fed patients for 30 days. Its effects on fecal microbiota composition and immunity were assessed by 16S rRNA gene sequencing and immune-marker analysis, respectively. The chemical composition of the fermented C. papaya L. preparation was analyzed by capillary electrophoresis- and liquid chromatography- time of flight mass spectrometry. The fermented C. papaya L. preparation restored peripheral blood mononuclear cell (PBMC) cytolytic activity; however, no other biomarkers of immunity were observed. Treatment with the preparation (9 g/day) significantly reduced the abundance of Firmicutes in the fecal microbiota. In particular, treatment reduced Clostridium scindens and Eggerthella lenta in most patients receiving 9 g/day. Chemical analysis identified low-molecular-weight phenolic acids as polyphenol metabolites; however, no polymerized, large-molecular-weight molecules were detected. Our study indicates that elderly patients who are tube-fed over the long-term have decreased PBMC cytolytic activity. In addition, low-molecular-weight polyphenol metabolites fermented from polymerized polyphenols restore PBMC cytolytic activity and modulate the composition of gut microbiota in tube-fed patients.

Transcriptional analysis of micronutrient zinc-associated response for enhanced carbohydrate utilization and earlier solventogenesis in Clostridium acetobutylicum.

The micronutrient zinc plays vital roles in ABE fermentation by Clostridium acetobutylicum. In order to elucidate the zinc-associated response for enhanced glucose utilization and earlier solventogenesis, transcriptional analysis was performed on cells grown in glucose medium at the exponential growth phase of 16 h without/with supplementary zinc. Correspondingly, the gene glcG (CAC0570) encoding a glucose-specific PTS was significantly upregulated accompanied with the other two genes CAC1353 and CAC1354 for glucose transport in the presence of zinc. Additionally, genes involved in the metabolisms of six other carbohydrates (maltose, cellobiose, fructose, mannose, xylose and arabinose) were differentially expressed, indicating that the regulatory effect of micronutrient zinc is carbohydrate-specific with respects to the improved/inhibited carbohydrate utilization. More importantly, multiple genes responsible for glycolysis (glcK and pykA), acidogenesis (thlA, crt, etfA, etfB and bcd) and solventogenesis (ctfB and bdhA) of C. acetobutylicum prominently responded to the supplementary zinc at differential expression levels. Comparative analysis of intracellular metabolites revealed that the branch node intermediates such as acetyl-CoA, acetoacetyl-CoA, butyl-CoA, and reducing power NADH remained relatively lower whereas more ATP was generated due to enhanced glycolysis pathway and earlier initiation of solventogenesis, suggesting that the micronutrient zinc-associated response for the selected intracellular metabolisms is significantly pleiotropic.

Fermentanomics: Relating quality attributes of a monoclonal antibody to cell culture process variables and raw materials using multivariate data analysis.

Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real-time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration-related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile.

Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis.

In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.

Effect of zinc supplementation on acetone-butanol-ethanol fermentation by Clostridium acetobutylicum.

In this article, effect of zinc supplementation on acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum was studied. It was found that when 0.001 g/L ZnSO4·7H2O was supplemented into the medium, solventogenesis was initiated earlier, with 21.0 g/L ABE (12.6 g/L butanol, 6.7 g/L acetone and 1.7 g/L ethanol) produced with a fermentation time of 40 h, compared to 19.4 g/L ABE (11.7 g/L butanol, 6.4 g/L acetone and 1.3g/L ethanol) produced with a fermentation time of 64 h in the control without zinc supplementation, and correspondingly ABE and butanol productivities were increased to 0.53 and 0.32 g/L/h from 0.30 and 0.18 g/L/h, increases of 76.7% and 77.8%, respectively, but their yields were not compromised. The reason for this phenomenon was attributed to rapid acids re-assimilation for more efficient ABE production, which was in accordance with relatively high pH and ORP levels maintained during the fermentation process. The maximum cell density increased by 23.8%, indicating that zinc supplementation stimulated cell growth, and consequently facilitated glucose utilization. However, more zinc supplementation exhibited an inhibitory effect, indicating that zinc supplementation at very low levels such as 0.001 g/L ZnSO4·7H2O will be an economically competitive strategy for improving butanol production.

Amino acid fermentation at the origin of the genetic code.

There is evidence that the genetic code was established prior to the existence of proteins, when metabolism was powered by ribozymes. Also, early proto-organisms had to rely on simple anaerobic bioenergetic processes. In this work I propose that amino acid fermentation powered metabolism in the RNA world, and that this was facilitated by proto-adapters, the precursors of the tRNAs. Amino acids were used as carbon sources rather than as catalytic or structural elements. In modern bacteria, amino acid fermentation is known as the Stickland reaction. This pathway involves two amino acids: the first undergoes oxidative deamination, and the second acts as an electron acceptor through reductive deamination. This redox reaction results in two keto acids that are employed to synthesise ATP via substrate-level phosphorylation. The Stickland reaction is the basic bioenergetic pathway of some bacteria of the genus Clostridium. Two other facts support Stickland fermentation in the RNA world. First, several Stickland amino acid pairs are synthesised in abiotic amino acid synthesis. This suggests that amino acids that could be used as an energy substrate were freely available. Second, anticodons that have complementary sequences often correspond to amino acids that form Stickland pairs. The main hypothesis of this paper is that pairs of complementary proto-adapters were assigned to Stickland amino acids pairs. There are signatures of this hypothesis in the genetic code. Furthermore, it is argued that the proto-adapters formed double strands that brought amino acid pairs into proximity to facilitate their mutual redox reaction, structurally constraining the anticodon pairs that are assigned to these amino acid pairs. Significance tests which randomise the code are performed to study the extent of the variability of the energetic (ATP) yield. Random assignments can lead to a substantial yield of ATP and maintain enough variability, thus selection can act and refine the assignments into a proto-code that optimises the energetic yield. Monte Carlo simulations are performed to evaluate the establishment of these simple proto-codes, based on amino acid substitutions and codon swapping. In all cases, donor amino acids are assigned to anticodons composed of U+G, and have low redundancy (1-2 codons), whereas acceptor amino acids are assigned to the the remaining codons. These bioenergetic and structural constraints allow for a metabolic role for amino acids before their co-option as catalyst cofactors.

Release of Pleurotus ostreatus versatile-peroxidase from Mn2+ repression enhances anthropogenic and natural substrate degradation.

The versatile-peroxidase (VP) encoded by mnp4 is one of the nine members of the manganese-peroxidase (MnP) gene family that constitutes part of the ligninolytic system of the white-rot basidiomycete Pleurotus ostreatus (oyster mushroom). VP enzymes exhibit dual activity on a wide range of substrates. As Mn(2+) supplement to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds and lignin, we examined the effect of Mn(2+) on the expression profile of the MnP gene family. In P. ostreatus (monokaryon PC9), mnp4 was found to be the predominantly expressed mnp in Mn(2+)-deficient media, whereas strongly repressed (to approximately 1%) in Mn(2+)-supplemented media. Accordingly, in-vitro Mn(2+)-independent activity was found to be negligible. We tested whether release of mnp4 from Mn(2+) repression alters the activity of the ligninolytic system. A transformant over-expressing mnp4 (designated OEmnp4) under the control of the ß-tubulin promoter was produced. Now, despite the presence of Mn(2+) in the medium, OEmnp4 produced mnp4 transcript as well as VP activity as early as 4 days after inoculation. The level of expression was constant throughout 10 days of incubation (about 0.4-fold relative to ß-tubulin) and the activity was comparable to the typical activity of PC9 in Mn(2+)-deficient media. In-vivo decolorization of the azo dyes Orange II, Reactive Black 5, and Amaranth by OEmnp4 preceded that of PC9. OEmnp4 and PC9 were grown for 2 weeks under solid-state fermentation conditions on cotton stalks as a lignocellulosic substrate. [(14)C]-lignin mineralization, in-vitro dry matter digestibility, and neutral detergent fiber digestibility were found to be significantly higher (about 25%) in OEmnp4-fermented substrate, relative to PC9. We conclude that releasing Mn(2+) suppression of VP4 by over-expression of the mnp4 gene in P. ostreatus improved its ligninolytic functionality.

Mesophilic fermentation of renewable biomass: does hydraulic retention time regulate methanogen diversity?

The present long-term study (about 1,100 days) monitored the diversity of methanogens during the mesophilic, anaerobic digestion of beet silage. Six fermentor samples were analyzed by ribosomal RNA gene restriction analysis, fluorescence in situ hybridization, and fluorescence microscopy. Hydrogenotrophic methanogens dominated within the population in all samples analyzed. Multidimensional scaling revealed that a rapid decrease in hydraulic retention time resulted in increased species richness, which in turn led to slightly higher CH(4) yields.

Purification and characterization of an exo-polygalacturonase from Pycnoporus sanguineus.

The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50-60 degrees C, with some enzyme activity retained up to 80 degrees C. Its activation energy was 5.352calmol(-1). PGase I showed a higher affinity towards PGA than citric pectin (Km=0.55+/-0.02 and 0.72+/-0.02mgml(-1), respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.

Tetracycline resistance genes and tetracycline resistant lactose-fermenting Enterobacteriaceae in activated sludge of sewage treatment plants.

Activated sludges were sampled from five sewage treatment plants (STPs) distributed in three geographically isolated areas, i.e., Hong Kong (Shatin, Stanley), Shanghai (Minhang) in China, and the bay area in California (Palo Alto and San Jose) of the United States. Among the tested 14 tetracycline resistance (tet) genes, nine genes encompassing efflux pumps (tetA, tetC, tetE, and tetG), ribosomal protection proteins (tetM, tetO, tetQ, and tetS), and enzymatic modification (tetX) were commonly detected in the STP sludge samples, whereas five genes encompassing efflux pumps [tetB, tetD, tetL, tetK, and tetA(P)] were not detected in any sludge sample. Additionally, 109 lactose-fermenting Enterobacteriaceae (LFE) strains were isolated from the activated sludge of the Shatin STP. Tetracycline-resistant (TR) LFE accounted for 32% of the total 109 LFE strains. The occurrence frequencies of tet genes among all TR-LEF strains varied from 0 to 91%, i.e., tetC (91%), tetA (46%), tetE (9%), tetG (6%), and tetD (6%). Finally, quantitative real-time polymerase chain reaction was used to quantify the change of tetC and tetA genes as the indicator of TR-LEF in the Shatin and Stanley STPs. The results showed that the concentrations of tetC and tetA genes in STP effluent ranged from 10(4) to 10(5) copies/mL, significantly lower than those in the influent by 3 orders of magnitude.

Analysis of growth of Lactobacillus plantarum WCFS1 on a complex medium using a genome-scale metabolic model.

A genome-scale metabolic model of the lactic acid bacterium Lactobacillus plantarum WCFS1 was constructed based on genomic content and experimental data. The complete model includes 721 genes, 643 reactions, and 531 metabolites. Different stoichiometric modeling techniques were used for interpretation of complex fermentation data, as L. plantarum is adapted to nutrient-rich environments and only grows in media supplemented with vitamins and amino acids. (i) Based on experimental input and output fluxes, maximal ATP production was estimated and related to growth rate. (ii) Optimization of ATP production further identified amino acid catabolic pathways that were not previously associated with free-energy metabolism. (iii) Genome-scale elementary flux mode analysis identified 28 potential futile cycles. (iv) Flux variability analysis supplemented the elementary mode analysis in identifying parallel pathways, e.g. pathways with identical end products but different co-factor usage. Strongly increased flexibility in the metabolic network was observed when strict coupling between catabolic ATP production and anabolic consumption was relaxed. These results illustrate how a genome-scale metabolic model and associated constraint-based modeling techniques can be used to analyze the physiology of growth on a complex medium rather than a minimal salts medium. However, optimization of biomass formation using the Flux Balance Analysis approach, reported to successfully predict growth rate and by product formation in Escherichia coli and Saccharomyces cerevisiae, predicted too high biomass yields that were incompatible with the observed lactate production. The reason is that this approach assumes optimal efficiency of substrate to biomass conversion, and can therefore not predict the metabolically inefficient lactate formation.