Influence of Reduced Electron Shuttling Compounds on Biological H<Subscript>2</Subscript> Production in the Fermentative Pure Culture <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Several reports suggest that extracellular electron shuttles influence fermentative metabolism in a beneficial manner for bioremediation and biotechnology strategies. The focus of this research was to characterize the effects of reduced electron shuttling molecules on fermentative H₂ production. Reduced electron shuttles may provide reducing equivalents to generate H₂, which influences alternate cellular processes. Electron shuttling compounds cycle between reduced-oxidized states and influence fermentative physiology. Clostridium beijerinckii fermentation was altered using a physiological approach that resulted in H₂ production with the reduced extracellular electron shuttle anthrahydroquinone-2,6,-disulfonate (AH₂QDS) and biologically reduced humic substances as the primary electron donors. Cells were suspended in a buffer with an excess of the biological electron transfer molecule NAD⁺, with AH₂QDS (100–1000 μM) or biologically reduced humic substances (0.01–0.025 g/L) as the sole electron source. Increasing concentrations of AH₂QDS and reduced humics increased H₂ production, while H₂ production was suppressed by Fe(III) hydroxides, which outcompeted the cells for electrons from the reduced shuttles, suggesting that the shuttles are in fact electron donors for H₂ production. Oxidized AQDS/humics did not increase H₂ production. Organic acid production shifted toward butyric acid in the presence of reduced electron shuttles, particularly with growing cells. Growth and hydrogen production rates in growing cells were initially faster in the presence of the reduced electron shuttles; however, the final biomass yield was inversely proportional to the starting AH₂QDS concentration, which suggests that reduced shuttles may compete with anabolic cell processes for available energetic resources or that the shift to excess butyrate becomes toxic to the cells.     

Interactions between Clostridium beijerinckii and Geobacter metallireducens in co‐culture fermentation with anthrahydroquinone‐2, 6‐disulfonate (AH2QDS) for enhanced biohydrogen production from xylose

To enhance biohydrogen production, Clostridium beijerinckii was co‐cultured with Geobacter metallireducens in the presence of the reduced extracellular electron shuttle anthrahydroquinone‐2, 6‐disulfonate (AH₂QDS). In the co‐culture system, increases of up to 52.3% for maximum cumulative hydrogen production, 38.4% for specific hydrogen production rate, 15.4% for substrate utilization rate, 39.0% for substrate utilization extent, and 34.8% for hydrogen molar yield in co‐culture fermentation were observed compared to a pure culture of C. beijerinckii without AH₂QDS. G. metallireducens grew in the co‐culture system, resulting in a decrease in acetate concentration under co‐culture conditions and a presumed regeneration of AH₂QDS from AQDS. These co‐culture results demonstrate metabolic crosstalk between the fermentative bacterium C. beijerinckii and the respiratory bacterium G. metallireducens and suggest a strategy for industrial biohydrogen production. Biotechnol. Bioeng. 2013; 110: 164–172. © 2012 Wiley Periodicals, Inc.     

H<Subscript>2</Subscript> synthesis from pentoses and biomass in <Emphasis Type="Italic">Thermotoga</Emphasis> spp.

We have investigated H₂ production on glucose, xylose, arabinose, and glycerol in Thermotoga maritima and T. neapolitana. Both species metabolised all sugars with hydrogen yields of 2.7–3.8 mol mol⁻¹ sugar. Both pentoses were at least comparable to glucose with respect to their qualities as substrates for hydrogen production, while glycerol was not metabolised by either species. Glycerol was also not metabolised by T. elfii. We also demonstrated that T. neapolitana can use wet oxidised wheat straws, in which most sugars are stored in glycoside polymers, for growth and efficient hydrogen production, while glucose, xylose and arabinose are consumed in parallel.     

Involvement of NADPH oxidase-mediated H<Subscript>2</Subscript>O<Subscript>2</Subscript> signaling in PB90-induced hypericin accumulation in <Emphasis Type="Italic">Hypericum perforatum</Emphasis> cells

PB90 is a novel protein elicitor isolated from Phytophthora boehmeriae. Here, we report that treatment of PB90 stimulates hypericin production and hydrogen peroxide (H₂O₂) generation in Hypericum perforatum L. cells and demonstrate that H₂O₂ is essential for PB90-induced hypericin production. To further study the source of PB90-triggered H₂O₂, we have investigated activities of plasma membrane NADPH oxidase in Hypericum perforatum L. cells subjected to PB90 treatment. It is revealed that treatment of the cells with PB90 significantly increases NADPH oxidase activity. NADPH oxidase inhibitors suppress not only the PB90-stimulated NADPH oxidase activity but also the PB90-triggered H₂O₂ generation and PB90-induced hypericin production, showing that NADPH oxidase is involved in PB90-triggered H₂O₂ generation and hypericin production. Moreover, the suppression of NADPH oxidase inhibitors on PB90-induced hypericin production can be reversed by H₂O₂, although H₂O₂ per se has no effects on hypericin production of the cells. Together, the data demonstrate that PB90 may induce hypericin production of H. perforatum cells through the NADPH oxidase-mediated H₂O₂ signaling pathway.     

Improved <Emphasis Type="Italic">p</Emphasis>-hydroxybenzoate production by engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12 by using a mixed-substrate feeding strategy

The key precursors for p-hydroxybenzoate production by engineered Pseudomonas putida S12 are phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), for which the pentose phosphate (PP) pathway is an important source. Since PP pathway fluxes are typically low in pseudomonads, E4P and PEP availability is a likely bottleneck for aromatics production which may be alleviated by stimulating PP pathway fluxes via co-feeding of pentoses in addition to glucose or glycerol. As P. putida S12 lacks the natural ability to utilize xylose, the xylose isomerase pathway from E. coli was introduced into the p-hydroxybenzoate producing strain P. putida S12palB2. The initially inefficient xylose utilization was improved by evolutionary selection after which the p-hydroxybenzoate production was evaluated. Even without xylose-co-feeding, p-hydroxybenzoate production was improved in the evolved xylose-utilizing strain, which may indicate an intrinsically elevated PP pathway activity. Xylose co-feeding further improved the p-hydroxybenzoate yield when co-fed with either glucose or glycerol, up to 16.3 Cmol% (0.1 g p-hydroxybenzoate/g substrate). The yield improvements were most pronounced with glycerol, which probably related to the availability of the PEP precursor glyceraldehyde-3-phosphate (GAP). Thus, it was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways. Moreover, this approach opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks such as lignocellulose that typically has a high content of both glucose and xylose and (crude) glycerol.     

Expression of NAD<Superscript>+</Superscript>-dependent formate dehydrogenase in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> and its involvement in anaerobic metabolism and H<Subscript>2</Subscript> production

An expression system for NAD⁺-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H₂ yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate ratio in the fdhl-expressed strain showed that increased reducing power arose from the reconstruction of NADH regeneration pathway from formate thereby contributing to the improved H₂ production.     

Growth,net photosynthesis and leaf nutrient status of <Emphasis Type="Italic">Fagus crenata</Emphasis> seedlings grown in brown forest soil acidified with H<Subscript>2</Subscript>SO<Subscript>4</Subscript> or HNO<Subscript>3</Subscript> solution

To obtain basic information for evaluating critical loads of acid deposition for protecting Japanese beech forests, growth, net photosynthesis and leaf nutrient status of Fagus crenata seedlings grown for two growing seasons in brown forest soil acidified with H₂SO₄ or HNO₃ solution were investigated. The whole-plant dry mass of the seedlings grown in the soil acidified by the addition of H₂SO₄ or HNO₃ solution was significantly less than that of the seedlings grown in the control soil not supplemented with H⁺ as H₂SO₄ or HNO₃ solution. However, the degrees of reduction in the whole-plant dry mass and net photosynthetic rate of the seedlings grown in the soil acidified by the addition of H⁺ as H₂SO₄ solution at 100 mg l^–1 on the basis of air-dried soil volume (S-100 treatment) were greater than those of the seedlings grown in the soil acidified by the addition of H⁺ as HNO₃ solution at 100 mg l^–1 (N-100 treatment). The concentrations of Al and Mn in the leaves of the seedlings grown in the S-100 treatment were significantly higher than those in the N-100 treatment. A positive correlation was obtained between the molar ratio of (Ca+Mg+K)/(Al+Mn) in the soil solution and the relative whole-plant dry mass of the seedlings grown in the acidified soils to that of the seedlings grown in the control soil. Based on the results, we concluded that the negative effects of soil acidification due to sulfate deposition are greater than those of soil acidification due to nitrate deposition on growth, net photosynthesis and leaf nutrient status of F. crenata, and that the molar ratio of (Ca+Mg+K)/(Al+Mn) in soil solution is a suitable soil parameter for evaluating critical loads of acid deposition in efforts to protect F. crenata forests in Japan.     

C<Subscript>1</Subscript> compounds as auxiliary substrate for engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C₁ compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.     

Improved homo <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid fermentation from xylose by abolishment of the phosphoketolase pathway and enhancement of the pentose phosphate pathway in genetically modified xylose-assimilating <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant strain that disrupted its phosphokeolase gene (ptk) was constructed. The Δptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1 g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose.     

Thermophilic,anaerobic co-digestion of microalgal biomass and cellulose for H<Subscript>2</Subscript> production

Microalgal biomass has been a focus in the sustainable energy field, especially biodiesel production. The purpose of this study was to assess the feasibility of treating microalgal biomass and cellulose by anaerobic digestion for H₂ production. A microbial consortium, TC60, known to degrade cellulose and other plant polymers, was enriched on a mixture of cellulose and green microalgal biomass of Dunaliella tertiolecta, a marine species, or Chlorella vulgaris, a freshwater species. After five enrichment steps at 60°C, hydrogen yields increased at least 10% under all conditions. Anaerobic digestion of D. tertiolecta and cellulose by TC60 produced 7.7 mmol H₂/g volatile solids (VS) which were higher than the levels (2.9–4.2 mmol/g VS) obtained with cellulose and C. vulgaris biomass. Both microalgal slurries contained satellite prokaryotes. The C. vulgaris slurry, without TC60 inoculation, generated H₂ levels on par with that of TC60 on cellulose alone. The biomass-fed anaerobic digestion resulted in large shifts in short chain fatty acid concentrations and increased ammonium levels. Growth and H₂ production increased when TC60 was grown on a combination of D. tertiolecta and cellulose due to nutrients released from algal cells via lysis. The results indicated that satellite heterotrophs from C. vulgaris produced H₂ but the Chlorella biomass was not substantially degraded by TC60. To date, this is the first study to examine H₂ production by anaerobic digestion of microalgal biomass. The results indicate that H₂ production is feasible but higher yields could be achieved by optimization of the bioprocess conditions including biomass pretreatment.     

A novel isolate of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for efficient butyric acid production by xylose fermentation

Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell growth rate (μ _m ) and the specific butyric acid yield were 0.1466 h^?1 and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose.     

Enhancement of artemisinin content and biomass in <Emphasis Type="Italic">Artemisia annua</Emphasis> by exogenous GA<Subscript>3</Subscript> treatment

Artemisinin is a promising and potent antimalarial drug naturally produced by the plant Artemisia annua L. but in very low yield. Its artemisinin content is known to be greatly affected by both genotype and environmental factors. In this study, the production of artemisinin and leaf biomass in Artemisia annua L. was significantly increased by exogenous GA₃ treatment. The effect of GA₃ application on expression of proposed key enzymes involved in artemisinin yield was examined in both wild type (007) and FPS-overexpression (253-2) lines of A. annua. In the wild type (007) at 6 h post GA₃ application there was an abrupt rise in FPS, ADS and CYP71AV1 expression and at 24 h a temporary and significant peak in artemisinin (1.45-fold higher than the control). After GA₃ application in line 253-2, there was a dramatic rise in expression of FPS at 3 h, CYP71AV1 at 9 h and ADS at 72 h and accumulation of artemisinin after 7 days, which was a delay when compared with the wild type plant. Thus, increased artemisinin content from exogenous GA₃ treatment was associated with increased expression of key enzymes in the artemisinin biosynthesis pathway. Interestingly, exogenous GA₃ continuously enhanced artemisinin content from the vegetative stage to flower initiation in both plant lines and gave significantly higher leaf biomass than in control plants. Consequently, the artemisinin yield in GA₃-treated plants was much higher than in control plants. Although the maximum artemisinin content was found at the full blooming stage [2.1% dry weight (DW) in 007 and 2.4% DW in 253-2], the highest artemisinin yield in GA₃-treated plants was obtained during the flower initiation stage (2.4 mg/plant in 007 and 2.3 mg/plant in 235-2). This was 26.3 and 27.8% higher, respectively, than in non-treated plants 007 and 253-2. This study showed that exogenous GA₃ treatment enhanced artemisinin production in pot experiments and should be suitable for field application.     

Putrescine protects hulless barley from damage due to UV-B stress via H<Subscript>2</Subscript>S- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-mediated signaling pathways

Key message

In hulless barley, H ₂ S mediated increases in H ₂ O ₂ induced by putrescine, and their interaction enhanced tolerance to UV-B by maintaining redox homeostasis and promoting the accumulation of UV-absorbing compounds.

Abstract

This study investigated the possible relationship between putrescence (Put), hydrogen sulfide (H₂S) and hydrogen peroxide (H₂O₂) as well as the underlying mechanism of their interaction in reducing UV-B induced damage. UV-B radiation increased electrolyte leakage (EL) and the levels of malondialdehyde (MDA) and UV-absorbing compounds but reduced antioxidant enzyme activities and glutathione (GSH) and ascorbic acid (AsA) contents. Exogenous application of Put, H₂S or H₂O₂ reduced some of the above-mentioned negative effects, but were enhanced by the addition of Put, H₂S and H₂O₂ inhibitors. Moreover, the protective effect of Put against UV-B radiation-induced damage to hulless barley was diminished by dl-propargylglycine (PAG, a H₂S biosynthesis inhibitor), hydroxylamine (HT, a H₂S scavenger), diphenylene iodonium (DPI, a PM-NADPH oxidase inhibitor) and dimethylthiourea (DMTU, a ROS scavenger), and the effect of Put on H₂O₂ accumulation was abolished by HT. Taken together, as the downstream component of the Put signaling pathway, H₂S mediated H₂O₂ accumulation, and H₂O₂ induced the accumulation of UV-absorbing compounds and maintained redox homeostasis under UV-B stress, thereby increasing the tolerance of hulless barley seedlings to UV-B stress.

     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Partitioning of photosynthetic electron flow between CO<Subscript>2</Subscript> assimilation and O<Subscript>2</Subscript> reduction in sunflower plants under water deficit

In sunflower (Helianthus annuus L.) grown under controlled conditions and subjected to drought by withholding watering, net photosynthetic rate (P _N) and stomatal conductance (g _s) of attached leaves decreased as leaf water potential (Ψ_w) declined from −0.3 to −2.9 MPa. Although g _s decreased over the whole range of Ψ_w, nearly constant values in the intercellular CO₂ concentrations (C _i) were observed as Ψ_w decreased to −1.8 MPa, but C _i increased as Ψ_w decreased further. Relative quantum yield, photochemical quenching, and the apparent quantum yield of photosynthesis decreased with water deficit, whereas non-photochemical quenching (q_NP) increased progressively. A highly significant negative relationship between q_NP and ATP content was observed. Water deficit did not alter the pyridine nucleotide concentration but decreased ATP content suggesting metabolic impairment. At a photon flux density of 550 μmol m⁻² s⁻¹, the allocation of electrons from photosystem (PS) 2 to O₂ reduction was increased by 51 %, while the allocation to CO₂ assimilation was diminished by 32 %, as Ψ_w declined from −0.3 to −2.9 MPa. A significant linear relationship between mean P _N and the rate of total linear electron transport was observed in well watered plants, the correlation becoming curvilinear when water deficit increased. The maximum quantum yield of PS2 was not affected by water deficit, whereas q_P declined only at very severe stress and the excess photon energy was dissipated by increasing q_NP indicating that a greater proportion of the energy was thermally dissipated. This accounted for the apparent down-regulation of PS2 and supported the protective role of q_NP against photoinhibition in sunflower.     

Production of 2-(2-phenylethyl)chromones in <Emphasis Type="Italic">Aquilaria sinensis</Emphasis> calli under different treatments

2-(2-Phenylethyl)chromones are main classes of aromatic compounds isolated from agarwood, the resinous portion derived from Aquilaria sinensis used as traditional medicine, incense and perfume. However, the mechanisms of biosynthesis of 2-(2-phenylethyl)chromones and agarwood formation are still unknown. Previous studies indicated that 6,7-dimethoxy-2-[2-(4′-methoxyphenyl)ethyl]chromone (AH₈) and 6,7-dimethoxy-2-(2-phenylethyl)chromone (AH₆) were more sensitive to salt stress and can be used as markers to analyze the production of 2-(2-phenylethyl)chromones. In this study, the content of AH₆ and AH₈ was analyzed by an AB Sciex 5500 Qtrap mass spectrometer and the congeners of 2-(2-phenylethyl)chromones were detected by LC-DAD-IT-TOF-MS system in A. sinensis calli under various abiotic stresses and hormone treatments. Among the abiotic stresses, salt and drought stress remarkably increased the production of AH₆ and AH₈, and could induce 41 and 23 species of 2-(2-phenylethyl)chromones, respectively. However, cold treatment has little influence on the production of 2-(2-phenylethyl)chromones. Ion stresses induced by KNO₃, CaCl₂, and (NH₄)₂SO₄ also significantly increased the content of AH₆ and AH₈, and could induced 14, 18, and 14 congeners of 2-(2-phenylethyl)chromones, respectively. Exogenous hormones including MeJA, SA and ABA induced a small amount of 2-(2-phenylethyl)chromones in calli within 20 days, while 6, 5, and 7 species of 2-(2-phenylethyl)chromones were detected after MeJA, SA and ABA treatment, respectively. Our study would provide solid basis for further research on biosynthesis of 2-(2-phenylethyl)chromones and agarwood formation.     

H<Subscript>2</Subscript>-Producing bacterial communities from a heat-treated soil inoculum

Hydrogen gas (60% H₂) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l^–1). The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H₂ by methanogens. The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30°C and 37°C). At 30-h HRT, the H₂ production rate was 80 ml h^–1 and yield was 0.91 mol H₂/mol glucose. At 10-h HRT, the H₂ production rate was more than 5 times higher at 436 ml h^–1, and yield was 1.61 mol H₂/mol glucose. Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA). Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae. At 10-h HRT, only Clostridiaceae were detected. When the temperature of the 10-h HRT reactor was increased from 30°C to 37°C, the steady-state H₂ production rate increased slightly to 463 ml h^–1 and yield was 1.8 mol H₂/mol glucose. Compared to 30°C, RISA fingerprints at 37°C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib).     

Hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) supply significantly improves xanthan gum production mediated by <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> in vitro

To improve xanthan gum productivity, a strategy of adding hydrogen peroxide (H₂O₂) was studied. The method could intensify oxygen supply through degradation of H₂O₂ to oxygen (O₂). In shake flask testing, the xanthan gum yield reached 2.8% (improved by 39.4%) when adding 12.5 mM H₂O₂ after 24 h of fermentation. In fermentor testing, it was obvious that the oxygen conditions varied with the H₂O₂ addition time. Eventually, gum yield of 4.2% (w/w) was achieved (increased by 27.3%). Compared with the method of intense mixing and increasing the air flow rate, adding H₂O₂ to improve the dissolved oxygen concentration was more effective and much better. Moreover, addition of H₂O₂ improved the quality of xanthan gum; the pyruvate content of xanthan was 4.4% (w/w), higher than that of the control (3.2%).