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    Jose M Bruno-Barcena currently works at the Department of Plant and Microbial Biology, North Carolina State University. Jose does Process Development research in Biotechnology, Microbiology and Physiology. Current projects include Bioenergy and Beneficial modulators of the gut microbiome
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    Raleigh, North Carolina
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    Professor (Associate)
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    Aug 2014 - Sep 2016
    Associate Professor
    North Carolina State University · Plant and Microbial Biology
    Raleigh, United States
    Dec 2007 - Aug 2014
    Assistant Professor
    North Carolina State University · Department of Microbiology
    Raleigh, United States
    Mar 2004 - Aug 2007
    Research Assistant Professor
    North Carolina State University · Department of Microbiology
    Raleigh, United States
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    Modulators of the gut microbiome can influence the microbial composition and function, as well as host gene expression; however, understanding of the ecological principles applied to exogenously introduced elements in gut ecosystems is lacking. Specifically, dietary interventions can have profound effects on the intestinal microbiota, affecting transit time, luminal pH, and production of microbial metabolites (e.g., butyrate). In particular, diets containing bioactive compounds like prebiotics (functional foods that stimulate growth of intestinal native beneficial bacteria) have been identified as potential interventions for gut disorders due to their capacity to modulate the gut microbiota and act as soluble decoy receptors, preventing pathogen attachment to mucosal surfaces and lowering the risk for bacterial infections. Both, probiotic and prebiotic modulators of the gut microbiota have been shown to assist in host recovery from microbial imbalances (dysbiosis), and thus are extremely relevant options for treatment and prevention of disease.
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    Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States, and, even though 5-15% of the total CRC cases can be attributed to individual genetic predisposition, environmental factors could be considered major factors in susceptibility to CRC. Lifestyle factors increasing the risks of CRC include elevated body mass index, obesity, and reduced physical activity. Additionally, a number of dietary elements have been associated with higher or lower incidence of CRC. In this context, it has been suggested that diets high in fruit and low in meat might have a protective effect, reducing the incidence of colorectal adenomas by modulating the composition of the normal nonpathogenic commensal microbiota. In addition, it has been demonstrated that changes in abundance of taxonomic groups have a profound impact on the gastrointestinal physiology, and an increasing number of studies are proposing that the microbiota mediates the generation of dietary factors triggering colon cancer. High-throughput sequencing and molecular taxonomic technologies are rapidly filling the knowledge gaps left by conventional microbiology techniques to obtain a comprehensive catalog of the human intestinal microbiota and their associated metabolic repertoire. The information provided by these studies will be essential to identify agents capable of modulating the massive amount of gut bacteria in safe noninvasive manners to prevent CRC. Probiotics, defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (219), are capable of transient modulation of the microbiota, and their beneficial effects include reinforcement of the natural defense mechanisms and protection against gastrointestinal disorders. Probiotics have been successfully used to manage infant diarrhea, food allergies, and inflammatory bowel disease; hence, the purpose of this review was to examine probiotic metabolic activities that may have an effect on the prevention of CRC by scavenging toxic compounds or preventing their generation in situ. Additionally, a brief consideration is given to safety evaluation and production methods in the context of probiotics efficacy.
    Growth in aerobic environments has been shown to generate reactive oxygen species (ROS) and to cause oxidative stress in most organisms. Antioxidant enzymes (i.e., superoxide dismutases and hydroperoxidases) and DNA repair mechanisms provide protection against ROS. Acid stress has been shown to be associated with the induction of Mn superoxide dismutase (MnSOD) in Lactococcus lactis and Staphylococcus aureus. However, the relationship between acid stress and oxidative stress is not well understood. In the present study, we showed that mutations in the gene coding for MnSOD (sodA) increased the toxicity of lactic acid at pH 3.5 in Streptococcus thermophilus. The inclusion of the iron chelators 2,2′-dipyridyl (DIP), diethienetriamine-pentaacetic acid (DTPA), and O-phenanthroline (O-Phe) provided partial protection against 330 mM lactic acid at pH 3.5. The results suggested that acid stress triggers an iron-mediated oxidative stress that can be ameliorated by MnSOD and iron chelators. These findings were further validated in Escherichia coli strains lacking both MnSOD and iron SOD (FeSOD) but expressing a heterologous MnSOD from S. thermophilus. We also found that, in E. coli, FeSOD did not provide the same protection afforded by MnSOD and that hydroperoxidases are equally important in protecting the cells against acid stress. These findings may explain the ability of some microorganisms to survive better in acidified environments, as in acid foods, during fermentation and accumulation of lactic acid or during passage through the low pH of the stomach.
    In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O2·− and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.
    A strategy for functional gene replacement in the chromosome of Lactobacillus gasseri is described. The phospho-beta-galactosidase II gene (lacII) was functionally replaced by the manganese superoxide dismutase (MnSOD) gene (sodA) from Streptococcus thermophilus, by adapting the insertional inactivation method described for lactobacilli [Russell, W.M. and Klaenhammer, T.R. 2001 Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 67, 4361-4364]. L. gasseri carrying the heterologous sodA gene grew on lactose as efficiently as the wild-type parent. An active MnSOD was expressed in the transgenic strain, and the enzyme migrated on PAGE-SOD activity gels to the same position as that of MnSOD from S. thermophilus. The expression of MnSOD from a single copy of sodA integrated in the chromosome of L. gasseri provided enhanced tolerance to hydrogen peroxide, and extended the viability of carbon/energy starved cultures stored at 25 degrees C. This is the first report showing the successful utilization of the pORI plasmids system to generate marker-free gene integration in L. gasseri strains.
    Emerging evidence has implicated reactive oxygen species (ROS) in the pathogenesis of inflammatory bowel disease (IBD). Although intestinal epithelial cells produce the ROS-neutralizing enzyme superoxide dismutase (SOD), the protein and activity levels of copper/zinc (Cu/Zn) and manganese (Mn) SOD are perturbed in inflamed tissues of IBD patients. Thus we investigated the ability of MnSOD from Streptococcus thermophilus to reduce colitis symptoms in interleukin (IL) 10-deficient mice using Lactobacillus gasseri as a delivery vehicle. Cohorts of 13-15 IL-10-deficient mice were left untreated or supplemented with native L. gasseri or L. gasseri expressing MnSOD for 4 wk. Colonic tissue was collected and inflammation was histologically scored. The presence of innate immune cells was investigated by immunohistochemistry and the host antioxidant response was determined by quantitative PCR. It was demonstrated that L. gasseri was stably maintained in mice for at least 3 days. L. gasseri producing MnSOD significantly reduced inflammation in IL-10-deficient mice compared with untreated controls (P < 0.05), whereas the anti-inflammatory effects of both native and MnSOD producing L. gasseri were more pronounced in males. The anti-inflammatory effects of L. gasseri were associated with a reduction in the infiltration of neutrophils and macrophages. Transcripts of antioxidant genes were equivalent in colonic tissues obtained from control and probiotic-treated IL-10-deficient mice. This study demonstrates that L. gasseri producing MnSOD has significant anti-inflammatory activity that reduces the severity of colitis in the IL-10-deficient mouse.
    Recent efforts to combat increasing greenhouse gas emissions include their capture into advanced biofuels, such as butanol. Traditionally, biobutanol research has been centered solely on its generation from sugars. Our results show partial re-assimilation of CO2 and H2 by n-butanol-producer C. beijerinckii. This was detected as synchronous CO2/H2 oscillations by direct (real-time) monitoring of their fermentation gasses. Additional functional analysis demonstrated increased total carbon recovery above heterotrophic values associated to mixotrophic assimilation of synthesis gas (H2, CO2 and CO). This was further confirmed using ¹³C-Tracer experiments feeding ¹³CO2 and measuring the resulting labeled products. Genome- and transcriptome-wide analysis revealed transcription of key C-1 capture and additional energy conservation genes, including partial Wood-Ljungdahl and complete reversed pyruvate ferredoxin oxidoreductase / pyruvate-formate-lyase-dependent (rPFOR/Pfl) pathways. Therefore, this report provides direct genetic and physiological evidences of mixotrophic inorganic carbon-capture by C. beijerinckii.
    A Clostridium ljungdahlii lab-isolated spontaneous-mutant strain, OTA1, has been shown to produce twice as much ethanol as the C. ljungdahlii ATCC 55383 strain when cultured in a mixotrophic medium containing fructose and syngas. Whole-genome sequencing identified four unique single nucleotide polymorphisms (SNPs) in the C. ljungdahlii OTA1 genome. Among these, two SNPs were found in the gene coding for AcsA and HemL, enzymes involved in acetyl-CoA formation from CO/CO2. Homology models of the respective mutated enzymes revealed alterations in the size and hydrogen bonding of the amino acids in their active sites. Failed attempts to grow OTA1 autotrophically suggested that one or both of these mutated genes prevented acetyl-CoA synthesis from CO/CO2, demonstrating that its activity was required for autotrophic growth by C. ljungdahlii. An inoperable Wood-Ljungdahl pathway resulted in higher CO2 and ethanol yields and lower biomass and acetate yields compared to WT for multiple growth conditions including heterotrophic and mixotrophic conditions. The two other SNPs identified in the C. ljungdahlii OTA1 genome were in genes coding for transcriptional regulators (CLJU_c09320 and CLJU_c18110) and were found to be responsible for deregulated expression of co-localized arginine catabolism and 2-deoxy-d-ribose catabolism genes. Growth medium supplementation experiments suggested that increased arginine metabolism and 2-deoxy-d-ribose were likely to have minor effects on biomass and fermentation product yields. In addition, in silico flux balance analysis simulating mixotrophic and heterotrophic conditions showed no change in flux to ethanol when flux through HemL was changed whereas limited flux through AcsA increased the ethanol flux for both simulations. In characterizing the effects of the SNPs identified in the C. ljungdahlii OTA1 genome, a non-autotrophic hyper ethanol-producing strain of C. ljungdahlii was identified that has utility for further physiology and strain performance studies and as a biocatalyst for industrial applications.
    Prebiotics are selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health. The aim of this study was to evaluate the influence of a β(1-4)galacto-oligosaccharides (GOS) formulation consisting of 90% pure GOS (GOS90), on the composition and activity of the mouse gut microbiota. Germ-free mice were colonised with microbiota from four pathogen-free wt 129 mice donors (SPF), and stools were collected during a feeding trial in which GOS90 was delivered orally for 14 days. Pyrosequencing of 16S rDNA amplicons showed that Bifidobacterium and specific Lactobacillus, Bacteroides and Clostridiales were more prevalent in GOS90-fed mice after 14 days, although the prebiotic impact on Bifidobacterium varied among individual mice. Prebiotic feeding also resulted in decreased abundance of Bacteroidales, Helicobacter and Clostridium. High-throughput quantitative PCR showed an increased abundance of Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis and Bifidobacterium gallicum in the prebiotic-fed mice. Control female mice showed a higher diversity (phylogenetic diversity (PD) = 15.1±3.4 in stools and PD = 13.0±0.6 in intestinal contents) than control males (PD = 7.8±1.6 in stool samples and PD = 9.5±1.0 in intestinal contents). GOS90 did not modify inflammatory biomarkers (interleukin (IL)-6, IL-12, IL-1β, interferon gamma and tumour necrosis factor alpha). Decreased butyrate, acetate and lactate concentrations in stools of prebiotic fed mice suggested an increase in colonic absorption and reduced excretion. Overall, our results demonstrate that GOS90 is capable of modulating the intestinal microbiome resulting in expansion of the probiome (autochtonous commensal intestinal bacteria considered to have a beneficial influence on health).
    As obligate aerobic soil organisms, the ability of Azotobacter species to fix nitrogen is unusual given that the nitrogenase complex requires a reduced cellular environment. Molecular hydrogen is an unavoidable byproduct of the reduction of dinitrogen; at least one molecule of H2 is produced for each molecule of N2 fixed. This could be considered a fault in nitrogenase efficiency, essentially a waste of energy and reducing equivalents. Wild-type Azotobacter captures this hydrogen and oxidizes it with its membrane-bound uptake hydrogenase complex. Strains lacking an active hydrogenase complex have been investigated for their hydrogen production capacities. What is the role of H2 in the energy metabolism of nitrogen-fixing Azotobacter? Is hydrogen production involved in Azotobacter species' protection from or tolerance to oxygen, or vice versa? What yields of hydrogen can be expected from hydrogen-evolving strains? Can the yield of hydrogen be controlled or increased by changing genetic, environmental, or physiological conditions? We will address these questions in the following mini-review.
    Lactic acid bacteria (LAB) are important in food fermentations and in human health. Due to their physiological and ecological heterogeneity, they encounter a variety of stressors including acid, osmolality, heat, bile-salt, and oxygen. LAB are exposed to oxidative stress caused by the partially reduced reactive oxygen species (ROS) generated from endogenous sources as well as from the environment. Findings over the past fifty years have demonstrated that this group of organisms comprise a heterogeneous mixture of different genera, where some members of the group have the capacity to synthesis antioxidant enzymes like Mn-containing superoxide dismutase (MnSOD), non-heme catalases (i.e., Mn-containing catalase –MnKat), and heme catalases (when provided with exogenous source of heme). Furthermore, some members of the group can accumulate large intracellular concentrations of manganese to use in the detoxification of ROS. In this chapter, we discuss the natural defenses against ROS in LAB as well as the technological practices used in the food and nutraceutical industries to protect LAB
    The β-hexosyltransferase (BHT) from Sporobolomyces singularis is a membrane bound enzyme that catalyzes transgalactosylation reactions to synthesize galacto-oligosaccharides (GOS). To increase the secretion of this active soluble protein version, we examined the uncharacterized novel N-terminus region (amino acids 1-110) which included two predicted endogenous structural domains. The first domain (amino acids 1-22) may act as a classical leader while a non-classical signal was located within the remaining region (amino acids 23-110). A functional analysis of these domains was performed by evaluating the amounts of the rBHT forms secreted by recombinant P. pastoris strains carrying combinations of the predicted structural domains and the alpha mating factor (MFα) from Saccharomyces cerevisiae as positive control. Upon replacement of the leader domain (amino acids 1-22) by MFα (MFα-rBht(23-594)), protein secretion increased and enzymatic activity of both soluble and membrane bound was improved by 53- and 14-fold, respectively. Leader interference was demonstrated when MFα preceded the putative classical rBHT(1-22) leader (amino acids 1-22) explaining the limited secretion of soluble protein by Pichia pastoris (GS115::MFα-rBht(1-594)). To validate the role of the N-terminus domains in promoting protein secretion, we tested the domains using a non-secreted protein, the anti-β-galactosidase single chain variable antibody fragment scFv13R4. The recombinants carrying chimeras of the N-terminal 1-110 regions of rBHT preceding scFv13R4 correlated with the secretion strength of soluble protein observed with the rBHT recombinants. Finally, soluble bioactive HIS tagged and non-tagged rBHT (purified to homogeneity) obtained from the most efficient recombinants (GS115::MFα-rBht(23-594)-HIS and GS115::MFα-rBht(23-594)) showed comparable activity rates of GOS generation.
    Spore-forming solventogenic Clostridium spp. are receiving renewed attention due to their butanol production abilities. However, there is an absence of literature describing the preparation of dense, vigorous and homogeneous seed cultures of Clostridium spp., guaranteeing reproducibility during fermentation. Therefore, we performed series of growth experiments of Clostridium beijerinckii NCIMB 8052 and its offspring SA-1 to evaluate the influence of inoculum age (harvest time) on the subsequent population's maximum specific growth rate, as a signal of population homogeneity. The organisms were cultivated in Reinforced Clostridial Medium (RCM) and supplemented sweet sorghum juice (SSSJ). The best inoculum ages coincided with the late-exponential growth phase: between 9 and 11 h in the conditions tested. Additionally, the harvest time was delayed up to 4 h by pre-adapting the seed culture with 0.75 g/L butyric acid. These findings were validated by performing a series of bench-top batch fermentations showcasing reproducibility in growth kinetics with 95% confidence limits. Overall, these experiments allowed us to understand the transient nature of seed cultures of C. beijerinckii NCIMB 8052 and SA-1, while enabling reproducibility and consistent culture performance.
    The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a byproduct. In batch cultures, A. vinelandii strain CA6, a mutant of CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen production. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6's genome, encompassing genes related to molybdate and iron transport and hydrogen re-oxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6's yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    During the past decade, DNA sequencing output has been mostly dominated by the second generation sequencing platforms which are characterized by low cost, high throughput and shorter read lengths for example, Illumina. The emergence and development of so called third generation sequencing platforms such as PacBio has permitted exceptionally long reads (over 20 kb) to be generated. Due to read length increases, algorithm improvements and hybrid assembly approaches, the concept of one chromosome, one contig and automated finishing of microbial genomes is now a realistic and achievable task for many microbial laboratories. In this paper, we describe high quality sequence datasets which span three generations of sequencing technologies, containing six types of data from four NGS platforms and originating from a single microorganism, Clostridium autoethanogenum. The dataset reported here will be useful for the scientific community to evaluate upcoming NGS platforms, enabling comparison of existing and novel bioinformatics approaches and will encourage interest in the development of innovative experimental and computational methods for NGS data.
    Referencia OEPM: P200101757.-- Fecha de solicitud: 17/07/2001.-- Titulares: Universidad Politécnica de Valencia (UPV), Consejo Superior de Investigaciones Científicas (CSIC). Consiste en hacer pasar un primer haz de la luz de intensidad variable a través de una primera probeta por la que circula la sustancia a controlar, y en hacer pasar un segundo haz de luz de intensidad fija a través de una segunda probeta con una muestra de referencia. Se comparan las intensidades de dichos haces tras atravesar las probetas y se varía la intensidad del primer haz de manera que dichas intensidades sean iguales. Mediante el procesamiento de la señal que hace variar al primer haz se calcula el parámetro de interés en la primera probeta. Peer reviewed
    We report the complete genome sequence of Clostridium beijerinckii SA-1, derived by directed evolution from C. beijerinckii NCIMB 8052, selecting for enhanced solvent tolerance. This sequence allows for accurate placement of SA-1 as C. beijerinckii, permits functional analyses of mutant phenotypes, and suggests methods for distinguishing SA-1 from its parent. FOOTNOTES Address correspondence to José M. Bruno-Bárcena, jbbarcen{at}ncsu.edu. Citation Noar J, Makwana ST, Bruno-Bárcena JM. 2014. Complete genome sequence of solvent-tolerant Clostridium beijerinckii strain SA-1. Genome Announc. 2(6):e01310-14. doi:10.1128/genomeA.01310-14. Received 7 November 2014. Accepted 10 November 2014. Published 18 December 2014. Copyright © 2014 Noar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
    Question - Is there a reliable molecular target to differentitate between C. ljungdahlii, C. autoethanogenum and C. ragsdalei?
    Answer
    C. ljungdahlii and C. autoethanogenum are basically the same organisms the second carries multiple deletions in the chromosome. I don't know about C. ragsdalei but you may want to check the chaperonin 60 (cpn60) sequences
    Hill et al 2004 cpnDB: a chaperonin sequence database. Genome Res 14: 1669–1675.
    Jian et al 2001. Int J Syst Evol Microbiol 51:1633–1638.
    Verbeke et al 2011 Syst Appl Microbiol 34: 171–179.
    Goh 1997 J Clin Microbiol 35: 3116–3121.
    Suerte
    Production of butanol by solventogenic Clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress directed evolution methodology was used two decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon and energy limiting substrates, sucrose and the products of its hydrolysis D-glucose and D-fructose and only D-fructose. Comparative small scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g/L of butanol and 26.3 g/L of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 g/g and 0.49 g/g, respectively (63 % and 65 % increase compared to NCIMB8052). SA-1 showed a deficiency in D-fructose transport as suggested by its 7 h generation time compared to 1 h for the NCIMB 8052. To potentially correlate physiological behavior with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyze the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants (SNV), one small deletion, and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769 may possibly explain some of the observed physiological behavior such as, rerouting of the metabolic carbon flow, deregulation of the D-fructose PTS transport system, and delayed sporulation.
    Clostridium autoethanogenum is an anaerobic, autotrophic acetogen that is capable of converting CO and CO2 into ethanol and acetate. Here we report the draft genome sequence of C. autoethanogenum JA1-1 strain DSM 10061 (4.5 Mbp; G+C content, 37.5%) and the findings obtained from annotation of the genome sequence.
    We report the complete genome sequences of Azotobacter vinelandii mutant strain CA6 and its parent wild-type strain, CA. When fixing nitrogen, strain CA6 displays slow growth and impaired molybdate uptake, tolerance to tungstates, and production of hydrogen gas, compared to results for strain CA. Comparing these genome sequences may provide a genetic basis for these mutant phenotypes.
    Galacto-oligosaccharides (GOS) are indigestible dietary fibers that are able to reach the lower gastrointestinal tract to be selectively fermented by health-promoting bacteria. In this report, we describe the heterologous expression of an optimized synthetically produced version of the β-hexosyltransferase gene (Bht) from Sporobolomyces singularis. The Bht gene encodes a glycosyl hydrolase (EC 3.2.1.21) that acts as galactosyltransferase, able to catalyze a one-step conversion of lactose to GOS. Expression of the enzyme in Escherichia coli yielded an inactive insoluble protein, while the methylotrophic yeast Pichia pastoris GS115 produced a bioactive β-hexosyltransferase (rBHT). The enzyme exhibited faster kinetics at pHs between 3.5 and 6 and at temperatures between 40 and 50°C. Enzyme stability improved at temperatures lower than 40°C, and glucose was found to be a competitive inhibitor of enzymatic activity. P. pastoris secreted a fraction of the bioactive rBHT into the fermentation broth, while the majority of the enzyme remained associated with the outer membrane. Both the secreted and the membrane-associated forms were able to efficiently convert lactose to GOS. Additionally, resting cells with membrane-bound enzyme converted 90% of the initial lactose into GOS at 68% yield (g/g) (the maximum theoretical is 75%) with no secondary residual (glucose or galactose) products. This is the first report of a bioactive BHT from S. singularis that has been heterologously expressed.
    Recent advances in systems biology, omics, and computational studies allow us to carry out data mining for improving biofuel production bioprocesses. Of particular interest are bioprocesses that center on microbial capabilities to biotransform both the hexose and pentose fractions present in crop residues. This called for a systematic exploration of the components of the media to obtain higher-density cultures and more-productive fermentation operations than are currently found. By using a meta-analysis approach of the transcriptional responses to butanol stress, we identified the nutritional requirements of solvent-tolerant strain Clostridium beijerinckii SA-1 (ATCC 35702). The nutritional requirements identified were later validated using the chemostat pulse-and-shift technique. C. beijerinckii SA-1 was cultivated in a two-stage single-feed-stream continuous production system to test the proposed validated medium formulation, and the coutilization of D-glucose and D-xylose was evaluated by taking advantage of the well-known ability of solventogenic clostridia to utilize a large variety of carbon sources such as mono-, oligo-, and polysaccharides containing pentose and hexose sugars. Our results indicated that C. beijerinckii SA-1 was able to coferment hexose/pentose sugar mixtures in the absence of a glucose repression effect. In addition, our analysis suggests that the solvent and acid resistance mechanisms found in this strain are differentially regulated compared to strain NRRL B-527 and are outlined as the basis of the analysis toward optimizing butanol production.
    Interest in natural cell immobilization or biofilms for lactic acid fermentation has developed considerably over the last few decades. Many studies report the benefits associated with biofilms as industrial methods for food production and for wastewater treatment, since the formation represents a protective means of microbial growth offering survival advantages to cells in toxic environments. The formation of biofilms is a natural process in which microbial cells adsorb to a support without chemicals or polymers that entrap the cells and is dependent on the reactor environment, microorganism, and characteristics of the support. These unique characteristics enable biofilms to cause chronic infections, disease, food spoilage, and devastating effects as in microbial corrosion. Their distinct resistance to toxicity, high biomass potential, and improved stability over cells in suspension make biofilms a good tool for improving the industrial economics of biological lactic acid production. Lactic acid bacteria and specific filamentous fungi are the main sources of biological lactic acid. Over the past two decades, studies have focused on improving the lactic acid volumetric productivity through reactor design development, new support materials, and improvements in microbial production strains. To illustrate the operational designs applied to the natural immobilization of lactic acid producing microorganisms, this chapter presents the results of a search for optimum parameters and how they are affected by the physical, chemical, and biological variables of the process. We will place particular emphasis upon the relationship between lactic acid productivity attained by various types of reactors, supports, media formulations, and lactic acid producing microorganisms.
    Interest in natural cell immobilization or biofilms for lactic acid fermentation has developed considerably over the last few decades. Many studies report the benefits associated with biofilms as industrial methods for food production and for wastewater treatment, since the formation represents a protective means of microbial growth offering survival advantages to cells in toxic environments. The formation of biofilms is a natural process in which microbial cells adsorb to a support without chemicals or polymers that entrap the cells and is dependent on the reactor environment, microorganism, and characteristics of the support. These unique characteristics enable biofilms to cause chronic infections, disease, food spoilage, and devastating effects as in microbial corrosion. Their distinct resistance to toxicity, high biomass potential, and improved stability over cells in suspension make biofilms a good tool for improving the industrial economics of biological lactic acid production. Lactic acid bacteria and specific filamentous fungi are the main sources of biological lactic acid. Over the past two decades, studies have focused on improving the lactic acid volumetric productivity through reactor design development, new support materials, and improvements in microbial production strains. To illustrate the operational designs applied to the natural immobilization of lactic acid producing microorganisms, this chapter presents the results of a search for optimum parameters and how they are affected by the physical, chemical, and biological variables of the process. We will place particular emphasis upon the relationship between lactic acid productivity attained by various types of reactors, supports, media formulations, and lactic acid producing microorganisms.
    This article addresses the computation of invariant control laws [A. Fradkov, I. Miroshnik, V. Nikiforov, Nonlinear and Adaptive Control of Complex Systems, Kluwer, 1999] for fed-batch fermenters represented by two standard models. It will be shown how to derive partial state feedbacks that, assuming ideal conditions and perfect model, keep the specific growth rate μ constant provided the initial conditions are adequate. The invariant control law is the closed loop version of the exponential feeding already suggested in several references as shown later. The paper presents an analysis of invariance and a study of global stability within the framework of partial stability. That is, stability with respect to some of the state variables. This enables us to treat the case with Haldane-like or non-monotonous kinetics.
    Oxalic acid is found in dietary sources (such as coffee, tea, and chocolate) or is produced by the intestinal microflora from metabolic precursors, like ascorbic acid. In the human intestine, oxalate may combine with calcium, sodium, magnesium, or potassium to form less soluble salts, which can cause pathological disorders such as hyperoxaluria, urolithiasis, and renal failure in humans. In this study, an operon containing genes homologous to a formyl coenzyme A transferase gene (frc) and an oxalyl coenzyme A decarboxylase gene (oxc) was identified in the genome of the probiotic bacterium Lactobacillus acidophilus. Physiological analysis of a mutant harboring a deleted version of the frc gene confirmed that frc expression specifically improves survival in the presence of oxalic acid at pH 3.5 compared with the survival of the wild-type strain. Moreover, the frc mutant was unable to degrade oxalate. These genes, which have not previously been described in lactobacilli, appear to be responsible for oxalate degradation in this organism. Transcriptional analysis using cDNA microarrays and reverse transcription-quantitative PCR revealed that mildly acidic conditions were a prerequisite for frc and oxc transcription. As a consequence, oxalate-dependent induction of these genes occurred only in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 5.5. Where genome information was available, other lactic acid bacteria were screened for frc and oxc genes. With the exception of Lactobacillus gasseri and Bifidobacterium lactis, none of the other strains harbored genes for oxalate utilization.
    Physiological capabilities and fermentation performance of Saccharomyces cerevisiae strains to be employed during industrial wine fermentations are critical for the quality of the final product. During the process of biomass propagation, yeast cells are dynamically exposed to a mixed and interrelated group of known stresses such as osmotic, oxidative, thermic, and/or starvation. These stressing conditions can dramatically affect the parameters of the fermentation process and the technological abilities of the yeast, e.g., the biomass yield and its fermentative capacity. Although a good knowledge exists of the behavior of S. cerevisiae under laboratory conditions, insufficient knowledge is available about yeast stress responses under the specific media and growth conditions during industrial processes. We performed growth experiments using bench-top fermentors and employed a molecular marker approach (changes in expression levels of five stress-related genes) to investigate how the cells respond to environmental changes during the process of yeast biomass production. The data show that in addition to the general stress response pathway, using the HSP12 gene as a marker, other specific stress response pathways were induced, as indicated by the changes detected in the mRNA levels of two stress-related genes, GPD1 and TRX2. These results suggest that the cells were affected by osmotic and oxidative stresses, demonstrating that these are the major causes of the stress response throughout the process of wine yeast biomass production.
    The physiological response to ambient pH on the regulation of the production of the xylanolytic enzyme complex was investigated using two modified strains of Aspergillus nidulans. Transcriptional gene fusions were constructed between the promoters xlnA and xlnB (alkaline and acid expressed, respectively) and the Aspergillus niger goxC (encoding glucose oxidase), and A.nidulans transformants possessing single integrations at the argB locus were selected. Changing the pH from 5.5 to 4.5 or 7 after induction resulted in differential expression of the homologous and heterologous proteins. Thus, versatile industrial strains capable of differentially producing mixtures of extracellular enzymes in response to ambient pH can be produced.
    Abstract Prebiotics are ingredients selectively fermented by the intestinal microbiota that promote changes in its structure and/or metabolism, conferring health benefits to the host. Studies show that +¦ (1-4) galacto-oligosaccharides [+¦ (1-4) GOS], lactulose and fructo-oligosaccharides increase intestinal concentration of lactate and short chain fatty acids, and stool frequency and weight, and they decrease fecal concentration of secondary bile acids, fecal pH, and nitroreductase and +¦-glucuronidase activities suggesting a clear role in colorectal cancer (CRC) prevention. This review summarizes research on prebiotics bioassimilation, specifically +¦ (1-4) GOS, and their potential role in CRC. We also evaluate research that shows that the impact of prebiotics on host physiology can be direct or through modulation of the gut intestinal microbiome, specifically the probiome (autochtonous beneficial bacteria). We present studies on a potential role in CRC progression to finally describe the current state of +¦ (1-4) GOS generation for industrial production
    During the last few years we have seen a public debate, involving every social statement, concerning food products and ingredients (1). The appearance in the market of the first food products, or organisms, which have been improved by recombinant DNA technologies (rDNA), has been received by society in a negative manner. We are referring specifically to genetically modified (GM) food products. Several definitions can explain the concept of GM products or, in a general way, the imprecise word transgenic, a concept that comes from the seventies, even when the terms were not the same. Since then, scientists are capable of constructing recombinant DNA molecules and precisely moving them to another organism or to the original one, by direct genetic manipulation. People understand “transgenic” as food products or their ingredients, resulting from this kind of modification. In other words, “either add a gene (or a set of them) from a donor genome to a recipient one-” and you will obtain it (2).
    There is great commercial interest in using immobilization technology for fermentation processes. Microbial immobilization is one of the novel methods in fermentation technology, especially important in the food and beverage industry, which allows the use of increased cell concentrations in the bioreactor, reducing process cycle times and increasing volumetric bioreactor productivity as compared with traditional batch and chemostat methods of fermentation. Results from bioreactor studies have demonstrated that immobilized cells have advantages over free cells, such as protection from toxic substances, increased plasmid stability and increased catalytic activity (1). Because of these advantages, methods employing immobilized microbial cells are used extensively in many industrial applications.
    A two-stage two-stream chemostat system and a two-stage two-stream immobilized upflow packed-bed reactor system were used for the study of lactic acid production by Lactobacillus casei subsp casei. A mixing ratio of D 12/D 2 = 0.5 (D = dilution rate) resulted in optimum production, making it possible to generate continuously a broth with high lactic acid concentration (48 g l−1) and with a lowered overall content of initial yeast extract (5 g l−1), half the concentration supplied in the one-step process. In the two-stage chemostat system, with the first stage at pH 5.5 and 37 °C and a second stage at pH 6.0, a temperature change from 40 °C to 45 °C in the second stage resulted in a 100% substrate consumption at an overall dilution rate of 0.05 h−1. To increase the cell mass in the system, an adhesive strain of L. casei was used to inoculate two packed-bed reactors, which operated with two mixed feedstock streams at the optimal conditions found above. Lactic acid fermentation started after a lag period of cell growth over foam glass particles. No significant amount of free cells, compared with those adhering to the glass foam, was observed during continuous lactic acid production. The extreme values, 57.5 g l−1 for lactic acid concentration and 9.72 g l−1 h−1 for the volumetric productivity, in upflow packed-bed reactors were higher than those obtained for free cells (48 g l−1 and 2.42 g l−1 h−1) respectively and the highest overall l(+)-lactic acid purity (96.8%) was obtained in the two-chemostat system as compared with the immobilized-cell reactors (93%).
    A strong anionic exchange resin was used to recover lactic acid directly from fermentation in an upflow fluidized bed column, resulting in 0.18g lactic acid/g resin bound with a subsequent elution of 94%. When the culture broth was heated and adjusted pH to 8.0, 0.4g lactic acid was bound perg resin, with a subsequent elution of 90%. L(+) and D(–) lactic acid isomers distribution was analyzed in the elution product resulting in an increase of L(+) isomer concentration. The resin did not alter its binding capacity after 23 cycles.
    Plantaricin C, a bacteriocin synthesized by Lactobacillus plantarum LL441, was optimally produced in chemostats kept at pH 5.0, 30°C, 150 rpm, and a dilution rate of 0.05 h1 when glucose was used as carbon source and a dilution rate of 0.10 to 0.12 h1 when sucrose or fructose was used instead. Production was abolished at high dilution rates, i.e., when the cells grew rapidly in all carbon sources This work was supported by grants ALI93-0873-CO2 and BIOT-CT96-0402 from the Comisión Interministerial de Ciencia y Tecnología of Spain and the BIOTECH Program of the European Union, respectively. F.S. was the recipient of a visitor's grant from the University of Oviedo. We thank the Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT) and UNESCO on its 50th anniversary Peer reviewed
    Lactobacillus casei had a typical morphology (short rods) in complex medium but lost its rod form when grown in a minimal basal medium. Atypical morphologies were very fragile since they were lysed by treatments with 1% SDS and 1mg/ml proteinase K but they were efficiently used for plasmids isolation without treatment with phenol.
    The study of the interactions between the microorganism and its environment is what is usually recognised as microbial physiology. The environment of a microorganism can be the ecological niche where it is present in nature or the conditions under which it is cultivated in the laboratory or in an industrial installation. As the occurrence of a microbiological product is also the resultant of the interaction between the microorganism and its environment, we can see that production and microbial physiology are closely related or are the same thing. This fact is, in many occasions, overlooked by some microbiologists, who had been carried away by the advances in microbial genetics and molecular biology and by engineers who lack training in Biology, using microbiological systems for process development and applications. For example, waste water treatment, which for the engineers is a process of water purification involving a series of unitary operations, from the standpoint of microbial physiology, the process is one of biomass and product formation using the contents of the wastewater as substrate (Speece, 1994). The result of properly taking into account the needs of the microbiota involved in the process, is an efficient system of waste water treatment. The same principle can be applied to any biotechnological process involving microorganims. Lactic Acid Bacteria (LAB) have an increased interest, not only for traditional uses in food preservation (Stiles, 1996), but as probiotics (Salminen et al.,1996) and for the production of biodegradable plastics (Lipinski, E.S., 1981) of medical interest; for this last purpose, pure isomers are required.
    In aerated cultures of Lactobacillus reuteri using maltose/glycerol, lactate was the main product followed by acetate at all pH (4.7, 5.5 and 6.5) tested while anaerobic cultures produced 1,3-propanediol besides lactate, acetate and ethanol. 1,3-Propanediol was the main product at pH 5.5 and 6.5. The high amount of acetate and the low concentration of ethanol found in anaerobic cultures was closely related to the synthesis of 1,3-propanediol.
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