Production of propionate by fed-batch fermentation of Propionibacterium acidipropionici using mixed feed of lactate and glucose

Propionibacterium acidipropionici was grown in a fed-batch culture, fed with glucose or lactate, or mixtures of lactate and glucose. Lactate and glucose were always simultaneously consumed. As co-substrate, glucose modified the propionate:acetate molar ratio (P/A) and increased the fraction of carbon used for biomass production. A P/A of 7.63 was obtained with a lactate:glucose molar ratio of 4; a P/A value of 1.34 was obtained with lactate alone and 1.85 with glucose alone. The fraction of carbon recovered in biomass was 0.09 for glucose, 0.12 for lactate, and 0.21 for a lactate:glucose molar ratio of 4.     

Fermentation of triacetin and glycerol by Acetobacterium sp. No energy is conserved by acetate excretion

Two strains of homoacetogenic bacteria similar to Acetobacterium carbinolicum were enriched and isolated from freshwater and marine sediment samples with triacetin (glycerol triacetylester) as sole carbon and energy source. Also the type strains of A. carbinolicum and A. woodii were found to be able to grow with triacetin, and to convert it nearly exclusively to acetate. The triacetin-hydrolyzing enzyme was inducible, and was localized in the cytoplasmic fraction of both species at an activity of 0.21–0.26 U mg protein^-1. During fermentation of glycerol, varying amounts of 1,3-propranediol were produced which could be kept at a minimum in a glycerol-limited chemostat. Growth yields in batch and continuous culture experiments varied between 9.2 and 10.9 g mol glycerol^-1 and 6.5 and 7.6 g mol triacetin^-1 with five strains of homoacetogenic bacteria tested. These results indicate that excretion of acetate across the cytoplasmic membrane does not contribute to the energy conservation budget of these homoacetogenic bacteria.Dedicated to Prof. Dr. Holger W. Jannasch on occasion of his 60th birthday     

Green and economical production of propionic acid by Propionibacterium freudenreichii CCTCC M207015 in plant fibrous-bed bioreactor

Propionic acid production by Propionibacterium freudenreichii from molasses and waste propionibacterium cells was studied in plant fibrous-bed bioreactor (PFB). With non-treated molasses as carbon source, 12.69 ± 0.40 g l^-1 of propionic acid was attained at 120 h in free-cell fermentation, whereas the PFB fermentation yielded 41.22 ± 2.06 g l^-1 at 120 h and faster cells growth was observed. In order to optimize the fermentation outcomes, fed-batch fermentation was performed with hydrolyzed molasses in PFB, giving 91.89 ± 4.59 g l^-1 of propionic acid at 254 h. Further studies were carried out using hydrolyzed waste propionibacterium cells as substitute nitrogen source, resulting in a propionic acid concentration of 79.81 ± 3.99 g l^-1 at 302 h. The present study suggests that the low-cost molasses and waste propionibacterium cells can be utilized for the green and economical production of propionic acid by P. freudenreichii.     

Manganese and Arsenic Oxidation Performance of Bacterium-Yunotaki 86 (BY86) from Hokkaido,Japan, and the Bacterium's Phylogeny

We examined the Mn(II) oxidation performance of a bacterium, BY86, collected at Yunotaki Falls Hokkaido, Japan. The bacterium showed rapid oxidation of Mn(II), and brown precipitates containing Mn formed within a few days of incubation. The presence of higher oxidation states of Mn than Mn(II) was ascertained by the UV-vis and XANES sutdy. This bacterium did not oxidize As(III) to As(V) in the absence of Mn. In the presence of Mn, however, As(III) was rapidly oxidized to As(V) on the cell surfaces. These findings indicate that BY86 does not have the ability to directly oxidize As(III) to As(V) within a short period of contact, but indirectly oxidizes it by the Mn oxides generated on the cell surfaces. A phylogenetical study disclosed that BY86 was most closely related to Bacillus cereus with an identity of 99.90%. It is expected that our findings in this study will contribute to the study of Mn(II)-oxidizing bacteria, which play an important role in the biogeochemical cycling of Mn as well as other trace elements including As.     

Development of mediated amperometric biosensors for hypoxanthine, glucose and lactate: a new format

Electrochemical growth was used to form the organic conducting salt of tetrathiafulvalene (TTF) and tetracyanoquinodimethane (TCNQ) on platinum wires inserted in a glass capillary. Glucose oxidase, lactate oxidase and xanthine oxidase were deposited and crosslinked on the salt structure to produce mediated biosensors responsive to the corresponding analytes. Reliability, stability, interference, and the effect of oxygen on the electrode's response were studied. Among three common electroactive interfering substances tested, ascorbic acid was very active at the TTF-TCNQ structure and the highest response was exhibited by the enzyme-free electrode. Acetaminophen and uric acid displayed similar behaviour at a lower magnitude. The presence of oxygen significantly decreased the current responses of all electrodes.

The xanthine oxidase-bearing mediated electrodes were able to assay the hypoxanthine content of either the fish extract, fish homogenate or slurry of manually ground tissue, yielding results in good agreement with conventional enzymatic assays. The electrodes were stable more than 120 days and could be reused more than 30 times without losing their original activities.     

Anaerovibrio glycerini sp. nov., an anaerobic bacterium fermenting glycerol to propionate,cell matter,and hydrogen

A strictly anaerobic, Gram-negative bacterium was isolated in continuous culture from black freshwater sediment with glycerol as sole source of carbon and energy. It was present in such sediments at 10⁸ cells per ml. The isolate was highly specialized and used only glycerol and the glycerol residue of diolein as substrate, and fermented it quantitatively to propionate. During growth in mineral medium, small amounts of hydrogen were produced which corresponded exactly to the calculated amount of electrons released in cell matter formation from glycerol. Yeast extract enhanced cell yields with glycerol, but did not support growth itself. In cell-free extracts, benzylviologen-dependent hydrogenase activity as well as a b-type cytochrome and some of the enzymes of the methylmalonylCoA pathway were found. The guanine-plus-cytosine content of the DNA was 34.3±1.0 mol% and corresponded well with that of Anaerovibrio lipolytica which was found to be 31.4 mol%. The consequences of the electron balance of this glycerol fermentation are discussed with respect to glycerol fermentation by other propionic acid-forming bacteria.     

不同条件下两株溶磷菌溶磷量及葡萄糖脱氢酶基因表达与酶活分析北大核心CSCD

【目的】获得大豆根际土壤中溶磷能力较强的菌株,明确在菌株溶磷过程中葡萄糖脱氢酶(GDH)的作用特点及其基因的表达水平。【方法】利用溶磷圈方法分离与纯化溶磷菌株,采用Vitek 2系统和16S r RNA序列分析菌株的分类地位;测定2菌株的溶磷量、GDH活性,并根据GDH基因的保守区序列设计引物,克隆GDH基因,利用实时荧光定量PCR测定不同条件下基因的相对表达量。【结果】筛选出2株具有较强溶磷能力的溶磷菌,分别鉴定为Pseudomonas sp.和Enterobacter sp.,2菌株最高溶磷量分别为558μg/m L和478μg/m L;成功地克隆了2株溶磷菌的GDH基因,片段大小分别为2007 bp和2066 bp;2菌株在不同磷源、不同p H值培养基中GDH活性及基因表达量不同,菌株wj1在高磷条件下基因表达量最高,磷胁迫条件下基因表达量较低,而wj3在不同磷源条件下GDH基因表达量都较低。且GDH基因表达量及酶活的变化与wj3菌株溶磷量没有直接的关系。【结论】从大豆根际土壤中分离获得溶磷能力较强的菌株Pseudomonas sp.wj1和Enterobacter sp.Wj3,GDH活性及基因表达在2株菌溶磷过程中具有不同的作用特点,2菌株溶磷机制不完全相同。     

Novel Leuconostoc citreum starter culture system for the fermentation of kimchi, a fermented cabbage product

To determine the dominant microorganisms involved in kimchi fermentation and to examine their effect on kimchi fermentation, we randomly isolated and characterized 120 lactic acid bacteria from kimchi during a 5-day fermentation at 15°C. Leuconostoc citreum was dominant during the early and mid-phases of kimchi fermentation whereas Lactobacillus sake/Lactobacillus curvatus or Lactobacillus brevis were found during later stages. Eighty-two out of 120 isolates (68%) were identified as Leuconostoc citreum by means of a polyphasic method, including 16S rDNA sequencing and DNA/DNA hybridization. A few Weissella confusa-like strains were also isolated during the mid-phase of the fermentation. Strain IH22, one of the Leuconostoc citreum isolates from kimchi, was used as an additive to evaluate growth and acid production in kimchi fermentation. This strain was consistently over 95% of the population in IH22-treated kimchi over a 5-day fermentation, while heterogeneous lactic acid bacteria were observed in the control kimchi. The pH in IH22-treated kimchi dropped rapidly but was stably maintained for 5 days, compared to its slow and prolonged decrease in the control kimchi. These results indicate that Leuconostoc citreum IH22 dominates over and retards the growth of other lactic acid bacteria in kimchi, suggesting it can be used as a bacterial starter culture to maintain the quality of kimchi for prolonged periods. This revised version was published online in June 2006 with corrections to the Cover Date.     

Potential for plant growth promotion in groundnut (Arachis hypogaea L.) cv. ALR-2 by co-inoculation of sulfur-oxidizing bacteria and Rhizobium

The use of Rhizobium inoculant for groundnut is a common practice in India. Also, co-inoculation of Rhizobium with other plant growth-promoting bacteria received considerable attention in legume growth promotion. Hence, in the present study we investigated effects of co-inoculating the sulfur (S)-oxidizing bacterial strains with Rhizobium, a strain that had no S-oxidizing potential in groundnut. Chemolithotrophic S-oxidizing bacterial isolates from different sources by enrichment isolation technique included three autotrophic (LCH, SWA5 and SWA4) and one heterotrophic (SGA6) strains. All the four isolates decreased the pH of the growth medium through oxidation of elemental S to sulfuric acid. Characterization revealed that these isolates tentatively placed into the genus Thiobacillus. Clay-based pellet formulation (2.5 x 10(7) cf ug(-1) pellet) of the Thiobacillus strains were developed and their efficiency to promote plant growth was tested in groundnut under pot culture and field conditions with S-deficit soil. Experiments in pot culture yielded promising results on groundnut increasing the plant biomass, nodule number and dry weight, and pod yield. Co-inoculation of Thiobacillus sp. strain LCH (applied at 60 kg ha(-1)) with Rhizobium under field condition recorded significantly higher nodule number, nodule dry weight and plant biomass 136.9 plant(-1), 740.0mg plant(-1) and 15.0 g plant(-1), respectively, on 80 days after sowing and enhanced the pod yield by 18%. Also inoculation of S-oxidizing bacteria increased the soil available S from 7.4 to 8.43 kg ha(-1). These results suggest that inoculation of S-oxidizing bacteria along with rhizobia results in synergistic interactions promoting the yield and oil content of groundnut, in S-deficit soils.     

Biotransformation of glycerol to <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid by <Emphasis Type="Italic">Acetobacter tropicalis</Emphasis>

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3 g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20 g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7 g/l d-GA from 200 g/l glycerol during 4 days of incubation in a jar fermentor.     

Transient Accumulation of Metabolic Intermediates of p-Cresol in the Culture Medium by a Pseudomonas sp. Strain A Isolated from a Sewage Treatment Plant

Summary Activated sludge from a sewage treatment plant in Kanpur, India, was screened for bacterial strains metabolizing p-cresol exclusively under aerobic conditions. One such isolate was identified to be belonging to the genus Pseudomonas based on morphological and physiological criteria as well as 16S ribosomal RNA gene sequence analysis. Two intermediates were identified from the culture medium during the growth phase of Pseudomonas sp. strain A that indicated that the strain degraded p-cresol via the protocatechuate (PCA) pathway. p-Cresol was rapidly converted into p-hydroxybenzaldehyde (PHB) during early growth phase, which was later utilized after p-cresol depletion. p-Hydroxybenzoate (PHBA) accumulation was observed during the later stages of exponential growth phase. Kinetic constants for the degradation of p-cresol were determined using Haldane’s model. High μ_max and inhibitory constant (K_I) values along with the observed accumulation of significant amounts of PHB in culture filtrates seem to indicate that the isolated Pseudomonas sp. strain A may be of potential use in biotransformations.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Bacterial removal of chromium (VI) and (III) in a continuous system

The capacity of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans to reduce different concentrations of hexavalent chromium in shake flask cultures has been investigated. A. ferrooxidans reduces 100% of chromium (VI) at concentrations of 1, 2.5 and 5 ppm, but in the presence of 10 ppm only 42.9% of chromium (VI) was reduced after 11 days of incubation. A. thiooxidans showed a lower capacity to reduce this ion and total reduction of chromium (VI) was only obtained for concentrations of 1 and 2.5 ppm, whereas 64.7% and 30.5% was reached for 5 and 10 ppm, respectively, after 11 days. A continuous flow mode system was subsequently investigated, in which A. thiooxidans was immobilized on elemental sulphur and the acidic medium obtained was employed to solubilize chromium (III) and to reduce chromium (VI) present in a real electroplating waste [30% of chromium (III) and 0.1% of chromium (VI)]. The system enabled the reduction of 92.7% of hexavalent chromium and represents a promising way to treat this type of waste in the industry.     

Reduction of Cr(VI) by Desulfovibrio vulgaris and Microbacterium sp.

Many industrial wastes contain Cr(VI), a carcinogen and mutagen, the toxicity of which can be ameliorated by reduction to Cr(III). Microbacterium sp. NCIMB 13776 andDesulfovibrio vulgaris NCIMB 8303 reduced Cr(VI) to Cr(III) anoxically using 25 mM sodium citrate buffer (pH 7), with 25 mM sodium acetate and 25 mM sodium formate as electron donors at 30 °C, under which conditions the rates of reduction of 500 M sodium chromate were 77 and 6 nmol h^–1 mg dry cell wt for D. vulgaris and Microbacterium sp., respectively, these being increased to 127 and 17 nmol h^–1 mg dry cell wt in the presence of 20 mM MOPS/NaOH buffer.     

Sucrose-phosphate synthase is regulated via metabolites and protein phosphorylation in potato tubers,in a manner analogous to the enzyme in leaves

(i) Sucrose-phosphate synthase (SPS) was purified 40-fold from stored potato (Solanum tuberosum L.) tubers to a final specific activity of 33–70 nkat·(mg protein)^–1 via batch elution from diethylaminoethyl (DEAE)-sephacel, polyethylene glycol (PEG) precipitation and Mono Q anion-exchange chromatography. (ii) Immunoblotting revealed a major and a minor band with molecular weights of 124.8 kDa and 133.5 kDa, respectively. Both bands were also present in extracts prepared in boiling SDS to exclude proteolysis. No smaller polypeptides were seen, except when the preparations were incubated before application on a polyacrylamide gel. (iii) The enzyme preparation was activated by glucose-6-phosphate and inhibited by inorganic phosphate. Both effectors had a large effect on the K _m (fructose-6-phosphate) and the K _m (uridine-5-diphosphoglucose) with phosphate acting antagonistically to glucose-6-phosphate. (iv) Preincubation of potato slices with low concentrations of okadaic acid or microcystin resulted in a three- to fourfold decrease in the activity of SPS when the tissue was subsequently extracted and assayed. The decrease was especially marked when the assay contained low concentrations of substrates and glucose-6-phosphate, and inorganic phosphate was included. Preincubation with mannose or in high osmoticum resulted in an increase of SPS activity. (v) Analogous changes were observed in germinating Ricinus communis L. seedlings. After preincubation of the cotyledons in glucose, high SPS activity could be measured, whereas okadaic acid, omission of glucose, or addition of phosphate or sucrose led to a large decrease of SPS activity in the selective assay. (vi) It is argued that SPS from non-photosynthetic tissues is regulated by metabolites and by protein phosphorylation in an analogous manner to the leaf enzyme.Abbreviations Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - Pi inorganic phosphate - PGI phosphoglucose isomerase - PP2A phosphoprotein phosphatase 2A - PEG polyethyleneglycol - SPS sucrose-phosphate synthase - UDPGlc uridine-5-diphosphoglucose This work was supported by the Deutsche Forschungsgemeinschaft, the BMFT and Sandoz AG, Basel, Switzerland. We are grateful to Prof. E. Beck (Pflanzenphysiologie, Bayreuth, Germany) for providing us with laboratory facilities, and to Dr. U. Sonnewald (Institut für Genbiologische Forschung, Berlin, Germany) for many discussions and providing us with unpublished data.     

Plant and bacterial mucilages of the maize rhizosphere: Comparison of their soil binding properties and histochemistry in a model system

Mucilages from the root tips of axenically-grown maize and from a bacterium (Cytophaga sp.) isolated from the rhizosheaths of field-grown roots, were immobilized by drying onto nylon blotting membrane. The mucilage plaques remained in place through repeated rewettings and histochemical treatments. Staining of the plaques showed that both mucilages included acidic groups, and 1,2 diols (the latter notably fewer in bacterial mucilage). Bacterial mucilage plaques stained strongly for protein, plant mucilage was unstained. Plaques of both mucilages bound soil particles strongly if soil was applied to wet mucilage and then dried. Bound soil was not lost with rewetting. Dry weight and densitometer measurements showed that bacterial mucilage bound about 10% more soil than the same surface area of root-cap mucilage. Pretreatment of plaques with periodate oxidation eliminated most soil binding by root-cap mucilage but this was completely reversible by reduction with borohydride. Soil binding to bacterial mucilage was unaffected by periodate but much diminished by borohydride pretreatment (partially restored by subsequent oxidation). Neither pretreatment with cationic dyes nor preincubation in pectinase, pectin methylesterase or protease affected subsequent soil binding by the mucilage plaques. Pretreatment of root-cap mucilage plaques with lectins specific for component sugars also did not alter soil binding. It is concluded that mucilages of both plant and bacterial origin can contribute to the adhesion and cohesion of maize rhizosheaths, but each by a different mechanism. Binding by root-cap mucilage depends on 1,2 diol groups of component sugars, that of bacterial mucilage does not, and is likely to be protein mediated. ei]Section editor: R O D Dixon     

Enhanced catalase synthesis by a novel combined system of photocatalytic reactor and fermentor

A novel combined system of a photocatalytic reactor, with UV and titanium dioxide as photocatalyst, and a fermentor with Bacillus sp. F26 as catalase producer was developed. Production of catalase was enhanced by 14% to 18.5 U ml⁻¹ without affecting cell growth.Nomenclature q_s: specific glucose consumption rate; μ: specific growth rate; q_p: specific CAT production rate     

Enhancement of bioremediation by Ralstonia sp. HM-1 in sediment polluted by Cd and Zn

In this study, the potential for the application of the bioaugmentation to Cd and Zn contaminated sediment was investigated. A batch experiment was performed in the lake sediments augmented with Ralstonia sp. HM-1. The degradation capacity of 18.7 mg-DOC/l/day in the treatment group was bigger than that of the blank group (4.4 mg-DOC/l/day). It can be regarded as the result of the reduction of the metal concentration in the liquid phase due to adsorption into the sediments, with the increased alkalinity resulting from the reduction of sulfate by sulfate reducing bacteria (SRB). The removal efficiency of cadmium and zinc in the treatment group was both 99.7% after 35 days. Restrain of elution to water phase from sediment in the Ralstonia sp. HM-1 added treatment group was also shown. In particular, the observed reduction of the exchangeable fraction and an increase in the bound to organics or sulfide fraction in the treatment group indicate its role in the prevention of metal elution from the sediment. Therefore, for bioremediation and restrain of elution from the sediment polluted by metal, Ralstonia sp. augmentation with indigenous microorganism including SRB, sediment stabilization and restrain of elution to surface water is recommended.     

Selenomonas acidaminovorans sp. nov., a versatile thermophilic proton-reducing anaerobe able to grow by decarboxylation of succinate to propionate

A moderately thermophilic anaerobic bacterium (strain Su883), which decarboxylated succinate to propionate, was isolated from granular methanogenic sludge. The bacterium appeared to ferment a number of amino acids including glutamate, histidine, arginine, ornithine, citrulline, and threonine to propionate, acetate and hydrogen. Propionate was formed via the oxidative decarboxylation of -ketoglutarate to succinyl-CoA. In addition, the strain degraded glucose, fructose, glycerol, pyruvate, serine, alanine, citrate and malate to acetate, carbon dioxide and hydrogen, and branched-chain amino acids to branched-chain fatty acids. With all single substrates solely hydrogen was formed as reduced fermentation product. Mixed cultures of strain Su883 and Methanobacterium thermoautotrophicum H showed a more rapid conversion of substrates and with some substrates a shift from acetate to propionate formation.Strain Su883 is a motile, gram-negative, non-sporeforming, slightly curved rod with a DNA base ratio of 56.5 mol% guanine-plus-cytosine. Selenomonas acidaminovorans Su883 is proposed as type strain for the new species within the genus Selenomonas.