Predisposing factors for positive D-Xylose breath test for evaluation of small intestinal bacterial overgrowth:A retrospective study of 932 patients

AIM: To investigate, in the largest cohort to date, patient characteristics and associated risk factors for developing small intestinal bacterial overgrowth(SIBO) using the D-Xylose breath test(XBT).METHODS: We performed a retrospective crosssectional study to analyze patient characteristics who underwent the XBT for evaluation of SIBO. Diagnostic testing with the XBT was performed based on a clinical suspicion for SIBO in patients with symptoms of bloating, abdominal pain, abdominal distension, weight loss, diarrhea, and/or constipation. Consecutive electronicmedical records of 932 patients who completed the XBT at the University of Florida between 2005 and 2009 were reviewed. A two-way Analysis of Variance(ANOVA) was used to test for several associations including age, gender, and body mass index(BMI) with a +XBT. A two-way ANOVA was also performed to control for the differences and interaction with age and between genders. A similar analysis was repeated for BMI. Associations between medical conditions and prior surgical histories were conducted using the Mantel-Haenszel method for 2 by 2 contingency tables, stratified for gender. Reported odds ratio estimates reflect the odds of the prevalence of a condition within the +XBT group to that of the-XBT group. P values of less than 0.05(two-sided) were considered statistically significant.RESULTS: In the 932 consecutive eligible subjects studied, 513 had a positive XBT. A positive association was found between female gender and a positive XBT(P = 0.0025), and females with a positive test were, on average, greater than 5 years older than those with a negative test(P = 0.024). The mean BMI of positive XBT subjects was normal(24.5) and significantly lower than the subjects with a negative XBT(29.5)(P = 0.0050). A positive XBT was associated with gastroesophageal reflux disease(GERD)(OR = 1.35; 95%CI: 1.02-1.80, P = 0.04), peptic ulcer disease(PUD)(OR = 2.61; 95%CI: 1.48-4.59, P < 0.01), gastroparesis(GP)(OR = 2.04; 95%CI: 1.21-3.41, P < 0.01) and steroid use(OR = 1.35; 95%CI: 1.02-1.80, P = 0.01). Irritable bowel syndrome, independent protonpump inhibitor(PPI) usage, or previous abdominal surgery was not significantly associated with a positive XBT. No single subdivision by gender or PPI use was associated with a significant difference in the odds ratios between any of the subsets. CONCLUSION: Female gender, lower BMI, steroid use, PUD, GERD(independent of PPI use), and GP were more prevalent in patients with SIBO, determined by a positive XBT. Increasing age was associated with SIBO in females, but not in males.

Dehydration of xylose to furfural over niobium phosphate catalyst in biphasic solvent system

Phosphoric acid treated niobic acid(NbP)was used for the dehydration of xylose to furfural in biphasic solvent system,which was found to exhibit the best performance among the tested catalysts.The excellent performance of NbP could be explained by the better synergistic cooperation between Bro¨nsted and Lewis acid sites.Moreover,NbP showed good stability and no obvious deactivation or leaching of Nb could be observed after six continuous recycles.

Enhancement of Simultaneous Xylose and Glucose Utilization by Regulating ZWF1 and PGI1 in Saccharomyces Cerevisiae

Xylose utilization is one of the key issues in lignocellulose bioconversion.Because of glucose repression,in most engineered yeast with heterogeneous xylose metabolic pathway,xylose is not consumed until glucose is completely utilized.Although simultaneous glucose and xylose utilization have been achieved in yeast by RPE1 deletion,we regulated ZWF1 and PGI1 transcription to improve simultaneous xylose and glucose utilization by controlling the metabolic flux from glucose into the PP pathway.Xylose and glucose consumption increased by approximately 80 and 72%,respectively,whereas ZWF1 was overexpressed by multi-copy plasmids with a strong transcriptional promoter.PGI1 expression was knocked down by promoter replacement; the glucose and xylose metabolism increased when PGI1p was replaced by weak promoters,SSA1p and PDA1p.ZWF1 overexpression decreased while PGI1 down-regulation increased the ethanol yield to some extent in the recombinant strains.

Preliminary Characterization of Xylose Reductase Partially Purified by Reversed Micelles from <i>Candida tropicalis</i>IEC5-ITV, an Indigenous Xylitol-Producing Strain

Xylose reductase (EC 1.1.1.21) of Candida tropicalis IEC5-ITV, an indigenous xylitol-producing strain, was partially purified by reversed micelles and characterized, an 8.1 fold purification factor being obtained. The XR present in the crude extract exhibited its highest specific activity at pH 6.0 and 40℃, while in that obtained by reverse micelles, this occurs at pH 6.0 and 30℃. XR before and after extraction is stable within a range of 30 to 40℃, pH 7 after one hour of incubation under these conditions. After two months’storage at –18℃, the enzyme obtained by reverse micelles lost 76.60% specific activity. The estimated molecular weight by PAGE-SDS was 32.42 kD. KM for xylose was higher for the XR extracted by reverse micelles (0.026 M) than that obtained for the enzyme before extraction (0.0059 M), while KM for cofactor NADPH was lower after than before extraction (1.85 mM to 12.0 mM respectively). There was no activity with NADH as a cofactor. Variations in pH and temperature optima, as well as kinetic parameters before and after partial XR purification by reverse micelles are probably due to an alteration in enzyme molecule structure caused by the solvents used during extraction.

ETHANOL FERMENTATION OF MIXED GLUCOSE AND XYLOSE BY PICHIA STIPITIS

Pichia stipitis CBS 5773 yeast cells were used to ferment the mixed substrates consisted of glucose andxylose to produce ethanol.The effects of aeration rate,initial substrate concentration and pH on substrateutilization and ethanol yield were evaluated.During batch fermentation,the oscillation phenomena in cell growthwere observed at low aeration rate,whereas the diauxic growth at high aeration rate.The substrate utilizationratio and ethanol yield reached 95％ and 0.46g/g respectively under appropriate operation conditions.Amodified unstructural model was proposed to simulate the diauxic cell growth,substrate consumption andproduct formation.

Effect of Dissolved Oxygen and Inoculum Concentration on Xylose Reductase Production from <i>Candida guilliermondii</i>Using Sugarcane Bagasse Hemicellulosic Hydrolysate

This work evaluated the effect of dissolved oxygen and the initial inoculum concentration on xylose reductase (XR) production by Candida guilliermondii from sugarcane bagasse hemicellulosic hydrolysate. Both the parameters were studied under an experimental design 22 with triplicate at central point. The statistical analysis of the results indicated a significant negative effect on XR production from the variable inoculum. The variable dissolved oxygen also showed a negative effect on XR production. We found the maximum enzyme activity (2.5 U?mg?1) when both the factors were applied at their lowest levels. The yeast showed to be potentially capable for xylose reductase production when sugarcane bagasse hemicellulosic hydrolysate was used as carbon source. Also, the results presented important information for further optimization of xylose reductase attainment.

Development on ethanol production from xylose by recombinant Saccharomyces cerevisiae

Xylose is the second major fermentable sugar present in lignocellulosic hydrolysates, so its fermentation is essential for the economic con- version of lignocellulose to ethanol. However, the traditional ethanol production strain Sacch- aromyces cerevisiae does not naturally use xy-lose as a substrate. A number of different ap-proaches have been used to engineer yeasts to reconstruct the gene background of S. cerevi- siae in recent years. The recombinant strains showed better xylose fermentation quality by comparison with the natural strains. This review examines the research on S. cerevisiae strains that have been genetically modified or adapted to ferment xylose to ethanol from three aspects including construction of xylose transportation, xylose-metabolic pathway and inhibitor toler-ance improvement of S. cerevisiae.

Low-temperature caproate production,microbial diversity,and metabolic pathway in xylose anaerobic fermentation

Mixed culture fermentation(MCF)is challenged by the unqualified activity of enriched bacteria and unwanted methane dissolution under low temperatures.In this work,caproate production from xylose was investigated by MCF at a low temperature(20°C).The results showed that a 9 d long hydraulic retention time(HRT)in a continuously stirred tank reactor was necessary for caproate production(~0.3 g/L,equal to 0.6 g COD/L)from xylose(10 g/L).The caproate concentration in the batch mode was further increased to 1.6 g/L.However,changing the substrate to ethanol did not promote caproate production,resulting in~1.0 g/L after 45 d of operation.Four genera,Bifidobacterium,Caproiciproducens,Actinomyces,and Clostridium_sensu_stricto_12,were identified as the enriched caproate-producing bacteria.The enzymes in the fatty acid biosynthesis(FAB)pathway for caproate production were identified via metagenomic analysis.The enzymes for the conversion of(C_(n+2))-2,3-Dehydroxyacyl-CoA to(C_(n+2))-Acyl-CoA(i.e.,EC 1.3.1.8 and EC 1.3.1.38)in the reverseβ-oxidation(RBO)pathway were not identified.These results could extend the understanding of low-temperature caproate production.

Engineering of Saccharomyces cerevisiae for co-fermentation of glucose and xylose:Current state and perspectives

The use of non-food lignocellulosic biomass to produce ethanol fits into the strategy of a global circular economy with low dependence on fossil energy resources.Xylose is the second most abundant sugar in lignocellulosic hydrolysate,and its utilization in fermentation is a key issue in making the full use of raw plant materials for ethanol production and reduce production costs.Saccharomyces cerevisiae is the best ethanol producer but the organism is not a native xylose user.In recent years,great efforts have been made in the construction of xy-lose utilizing S.cerevisiae strains by metabolic and evolutionary engineering approaches.In addition,managing global transcriptional regulation works provides an effective means to increase the xylose utilization capacity of recombinant strains.Here we review the common strategies and research advances in the research field in order to facilitate the researches in xylose metabolism and xylose-based fermentation.

Development and characterization of Saccharomyces cerevisiae strains genetically modified to over-express the pentose phosphate pathway regulating transcription factor STB5 in the presence of xylose

Background Several enzymes in the pentose phosphate pathway of Saccharomyces cerevisiae have been identified as relating to the constraint of xylose consumption and conversion to ethanol.However,no strategy has been proposed for simultaneous regulation of all contributing enzymes.If multiple enzymes contribute to constraint,over expression of a native transcription factor controlling the entire constraining pathway may provide optimal pathway wide regulation.Further characterization of this strain on both pure sugars and lignocellulosic hydrolysates would provide an opportunity to identify additional bot-tlenecks not addressed by the modification of the pentose phosphate pathway expression pattern.Results A series of strains were developed expressing STB5 and PGI1 under the control of a novel xylose inducible promoter.Increased transcription of STB5 and its regulatory targets was verified via qRT-PCR.No statistically significant difference was found in terms of xylose consumption or ethanol yield in these strains versus control strains.Xylose consumption through both the fermentative and respiratory pathways appeared to be related to oxygen availability and culture density with high-density(low oxygen)cultures consuming xylose more slowly than low-density cultures.The maximum specific consumption rate for high-density cultures was 0.21 g xylose/gDCW/h versus 0.41 g xylose/gDCW/h in lower density cultures.Statistically similar ethanol yields at high and low density(approximately 0.25 g ethanol/g xylose)suggest that the maximum rate of fermentation is linked to the rate of respiration in a stoichiometric fashion.Conclusion This study did not find evidence supporting the pentose phosphate pathway constraint identified in other works.Instead,NAD+availability mediated by oxygen availability and citric acid cycle flux was suggested to limit fermentation.While increased aeration could provide increased conversion of NAD+to NADH(and a stoichiometric increase in fermen-tation flux),this increase would not be expected improve ethanol yield beyond 50%of the theoretical maximum.Based on these findings,future work in Saccharomyces cerevisiae development for fermentation of lignocellulosic hydrolysates should focus on balancing NAD+/NADH availability through non-respiratory pathways.

New xylose transporters support the simultaneous consumption of glucose and xylose in Escherichia coli

Glucose and xylose are two major components of lignocellulose.Simultaneous consumption of glucose and xylose is critical for engineered microorganisms to produce fuels and chemicals from lignocellulosic biomass.Although many production limitations have been resolved,glucose‐induced inhibition of xylose transport remains a challenge.In this study,a cell growthbased screening strategy was designed to identify xylose transporters uninhibited by glucose.The glucose pathway was genetically blocked in Escherichia coli so that glucose functions only as an inhibitor and cells need xylose as the carbon source for survival.Through adaptive evolution,omics analysis and reverse metabolic engineering,a new phosphoenolpyruvate:carbohydrate phosphotransferase system(PTS)galactitol transporter(GalABC,encoded by EcolC_1640,EcolC_1641,and EcolC_1642 genes)that is not inhibited by glucose was identified.Inactivation of adenylate cyclase led to increased expression of the EcolC_1642 gene,and a point mutation in gene EcolC_1642(N13S)further enhanced xylose transport.During the second round of gene mining,AraE and a new ABC transporter(AraFGH)of xylose were identified.A point mutation in the transcription regulator araC(L156I)caused increased expression of araE and araFGH genes without arabinose induction,and a point mutation in araE(D223Y)further enhanced xylose transport.These newly identified xylose transporters can support the simultaneous consumption of glucose and xylose and have potential use in producing chemicals from lignocellulose.

High performance removal of sulfamethoxazole using large specific area of biochar derived from corncob xylose residue

To remove antibiotics from waste water,an alkali active porous biochar,850BC,was prepared from corncob xylose residue.In preparation,NaOH dipping was used for silicon removal and KOH activation was operated at 850℃.Further characterization containing BET,SEM,and FTIR were confirmed.850BC possessed a huge specific surface area of 3043 m^(2)·g^(−1),developed pore structure and abundant oxygen functional groups.The adsorption performance of sulfamethoxazole on 850BC was quick and efficient,and the adsorption capacity reached 1429 mg·g^(−1),which was significantly higher than other adsorbents reported previously.While pseudo-second-order kinetic model and Langmuir model could better describe the adsorption,chemisorp-tion dominated the SMX adsorption onto 850BC.In virtue of pore-filling andπ-πinteraction as major mechanism,a large surface area and rich oxygen-containing functional groups led to an excellent adsorption performance.Thus,this preparation method provided a biochar-based adsorbent with enhanced specific surface for efficient removal of antibiotic pollutants.

Effect of heat treatment on amino acids and volatile compounds of enzymatic pork trimmings hydrolysate supplemented with xylose and cysteine

In this research,the effects of heat treatment together with xylose and cysteine addition on the amino acid changes and volatile compound generation in non-enzymatic pork trimmings extract(NPE)or enzymatic pork trimmings hydrolysate(EPH)were studied.Enzymatic hydrolysis significantly increased the total amino acid content and provided necessary precursors for the thermal reactions.The addition of xylose assisted the browning process while the cysteine addition partially inhibited the formation of brown colour in both NPE and EPH.The addition of both cysteine and xylose significantly increased the formation of sulfur-containing volatile flavour compounds in EPH samples,but not in NPE samples.The characteristic“roasted meat”like aromatic volatile compounds,in particular,2-furfurylthiol,were only detected in EPH samples at 8.13 ng/mL,when both xylose(35 mg/mL)and cysteine(10 mg/mL)were added.

Crystal structure of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase

The crystal structures of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase (SDXyI) have been analysed and refined at 0.19nm. The crystal space group is I222, with unit cell dimensions of a=9.884 ran, b=9.393nm and c=8.798nm. Based on the coordinates of the Streptomyces rubiginosus xylose isomerase (SRXyI), the initial model of SDXyl was built up by the dose packing analysing and R-factor searching and refined by PROLSQ to a final R-factor of 0.177 with the rms deviations of bond lengths and bond angles of 0.001 9nm and 2.1°, respectively. No significant global conformation change existed between SRXyI and SDXyI except the local conformation in the active site.

Construction of a recombinant yeast strain converting xylose and glucose to ethanol

Candida shehatae gene xyl1 and Pichia stipitis gene xyl2,encoding xylose reductase(XR)and xylitol dehydrogenase(XD)respectively,were amplified by PCR.The genes xyl1 and xyl2 were placed under the control of promoter GAL in vector pYES2 to construct the recombinant expression vector pYES2-P12.Subsequently the vector pYES2-P12 was transformed into S.cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-12.The alcoholic ferment indicated that the recombinant yeast YS58-12 could convert xylose to ethanol with the xylose consumption rate of 81.3%.

Using high-throughput data and dynamic flux balance modeling techniques to identify points of constraint in xylose utilization in Saccharomyces cerevisiae

Background Several enzymes and cofactors have been identified as contributing to the slow utilization of xylose by xylose-fermenting strains of Saccharomyces cerevisiae.However,there has been no consensus on which of these possible bottle-necks are the most important to address.A previous strain characterization study from our lab suggested that insufficient NAD+limits fermentation and may be the most important bottleneck affecting utilization of xylose for the production of ethanol.The development and validation of a genome scale dynamic flux balance model would help to verify the existence and extent of this and other metabolic bottlenecks and suggest solutions to guide future strain development thereby minimiz-ing bottleneck impact on process economics.Results A dynamic flux balance model was developed to identify bottlenecks in several strains of S.cerevisiae,both with wild-type pentose phosphate pathway expression and with the pathway over expressed.ZWF1 was found to be limiting in the oxidative portion of the pentose phosphate pathway under oxygen replete conditions.This pathway is used to regenerate NADPH.Under oxygen limiting conditions,respiration of xylose was limited by the lack of oxygen as a terminal electron acceptor.Ethanol production was also limited under these conditions due to the inability to balance NAD+/NADH.The model suggests the use of the anaplerotic glyoxylate pathway to improve NAD+/NADH balance,increasing ethanol produc-tion by 50%while producing succinate as a coproduct at upwards of 20 g/l.Conclusion In the production of high value chemicals from biomass,the use of the respiratory metabolism is a waste of feedstock carbon.Bottlenecks previously identified in the oxidative pentose phosphate pathway are currently only relevant under oxygen-replete conditions and cannot impact the partitioning of carbon between the respiratory and fermentative pathways.Focusing future efforts on the non-respiratory balancing of NAD+/NADH,perhaps through the glyoxylate pathway,would improve the economics of ethanol production both directly and through coproduct formation.

Coproduction of xylose and biobutanol from corn stover via recycling of sulfuric acid pretreatment solution

Sulfuric acid was used in the pretreatment of corn stover to obtain xylose as a value-added by-product,and the pretreated corn stover(Pre-CS)was hydrolyzed to produce glucose for butanol fermentation.The aim of this work is to achieve high xylose accumulation and reduced wastewater by recycling the pretreatment solution.The pretreatment conditions were optimized as follows:180°C,15 min,1:7 solid-liquid ratio(w/w),0.6%H_(2)SO_(4)(w/w,first batch)/0.9%H_(2)SO_(4)(w/w,second and third batches),in which pretreatment solution was recycled for three times.Under above conditions,pretreatment solution containing 56.3 g/L xylose and 4.5 g/L glucose was obtained.Pre-CS residue was further hydrolyzed by cellulase to achieve 35.7-39.9 g/L glucose.The condensed corn stover hydrolysate was subjected to simultaneous detoxification and sterilization using 1%(w/w)activated carbon and then applied in butanol fermentation.The highest butanol titer of 9.5 g/L was obtained in 72 h.The results provide a practical approach for coproducing xylose and biobutanol from corn stover.

A Statistical Approach to Optimize Xylitol Production by <i>Debaryomyces nepalensis</i>NCYC 3413 <i>in Vitro</i>

Debaryomyces nepalensis NCYC 3413, halotolerant yeast isolated from rotten apple, was capable of utilizing components of hemicellulose hydrolysate such as glucose, galactose, mannose, xylose and arabinose. The organism utilizes xylose as a sole carbon source and produces xylitol. The Plackett-Burman design was applied to determine the specific medium components affecting xylitol production and found that xylose, K2HPO4, and ZnSO4 were critical in augmenting xylitol production. These significant parameters were further optimized using response surface methodology. The optimum concentrations of xylose, K2HPO4, and ZnSO4 were found to be 100 g/l, 10.6 g/l and 8.9 mg/l respectively. Under these optimal conditions the xylitol production increased from 27 g/l to 36 g/l with a yield of 0.44 g/g (57% increase in total yield). In addition, formation of the by product (glycerol) was decreased under optimal conditions.

Detection of Pathogenic Bacteria <i>Staphylococcus aureus</i>and <i>Salmonella</i>sp. from Raw Milk Samples of Different Cities of Pakistan

Food-borne diseases are the main public health problem throughout the world. Milk is important component of human diet including fats, proteins, vitamins and minerals. It is a best source of calcium and phosphorus. Different types of pathogenic bacteria like S. aureus and Salmonella enter in milk and then multiply, after multiplication they become active in causing diseases. These bacteria create serious problems for human health. This study aimed to isolate and identify pathogenic bacteria Staphylococcus aureus and Salmonella from raw milk samples of different cities of Pakistan. Primary screening of raw milk samples was done on the basis of morphological, cultural and biochemical techniques. The final identification was made using 16SrRNA sequence analysis. A total of 200 raw milk samples were collected from different cities of Pakistan. Selective medium xylose lysine deoxycholate agar (XLD) and Mannitol salt agar were used for the identification of Salmonella sp. and S. aureus. Staphylococcus aureus produced yellow colonies with yellow zones on Mannitol salt agar. Staphylococcus aureus exhibited gram-positive character with purple coloration and it was detected as cocci-shaped. Biochemically 91 (45%) samples enhibited Catalase, Coagulase, DNase, Urease, Citrate, fermentation tests positive and indole, oxidase and H2S tests negative with nonmotile character, indicating the presence of Staphylococcus aureus. Salmonella sp. was detected as gram negative rods with pink coloration on gram staining. Biochemically 87 (43%) samples revealed catalase, citrate, H2S and fermentation tests positive while oxidase, DNase, Indole and urease tests negative, indicating the presence of Salmonella sp. in these samples. Of the 200 samples tested, 43% were positive for Salmonella, while 45% samples were contaminated with S. aureus. The 16SrRNA sequence analysis confirmed the results of biochemical and cultural characterization by depicting 99% identity of samples with S. aureus and 98% identity with Salmonella spp. The occurrence of high percentage of these pathogenic bacteria in raw milk may be linked to its contamination at the time of collection, processing, strorage and distribution. This quantitative data could be utilized to better establish the appropriate levels of protection for raw milk, dairy products and processing technologies.

Literature Review on Furfural Production from Lignocellulosic Biomass

The use of renewable sources for obtainment of chemicals, biofuels, materials and energy has become each time larger due to environmental, political and economical problems of non-renewable energies utilization. Among several products that can be obtained from lignocellulosic biomass, which is a renewable source, there is furfural, a chemical from which many other value added chemical products can be obtained. The main route for furfural production consists of an acid hydrolysis of hemicelluloses present in lignocellulosic biomass to obtain xylose, which goes through a dehydration reaction to produce furfural. Due to the presence of an aldehyde group and a conjugated system of double bounds, furfural can go through several reactions, allowing the production of a range of value added products. In this sense, this article performs a review about mechanisms of furfural production from lignocellulosic biomass, highlighting its chemical properties which enable its utilization in different industrial applications of economic interest.