The glucose metabolic frame of conditions induce the overproduction of a co-culture pigment: induction mechanism of the pigment and development of a specific eutrophic–oligotrophic transition cultivation system

Induction mechanism of a potential red pigment (RPc) was investigated in the present paper. A typical competition relationship exists between Penicillium sp. HSD07B and Candida tropicalis during co-culture, and C. tropicalis converts glucose into glycerol, organic acids and other substances, resulting in a stricter glucose limitation and the secretion of RPc. Moreover, a novel eutrophic–oligotrophic transition cultivation system (E-OTCS) was developed to produce red pigment during monoculture of Penicillium sp. HSD07B. However, the monoculture pigment (RPm) is different from RPc in components, and RP3 and RP4 only occur in RPm when glycerol is supplied. In addition, the additions of glycerol and organic acids to glucose exhaustion medium can significantly improve the pigment yield. These facts not only prove the feasibility of producing RPm using E-OTCS, but also reveal that, besides glucose exhaustion, the accumulation of metabolites of glucose including glycerol and organic acids is also an important factor influencing the production of RPc.     

Hydrogen production by the newly isolated Clostridium beijerinckii RZF-1108

A fermentative hydrogen-producing strain, RZF-1108, was isolated from a biohydrogen reactor, and identified as Clostridium beijerinckii on the basis of the 16S rRNA gene analysis and physiobiochemical characteristics. The effects of culture conditions on hydrogen production by C. beijerinckii RZF-1108 were investigated in batch cultures. The hydrogen production and growth of strain RZF-1108 were highly dependent on temperature, initial pH and substrate concentration. Yeast extract was a favorable nitrogen source for hydrogen production and growth of RZF-1108. Hydrogen production corresponded to cell biomass yield in different culture conditions. The maximum hydrogen evolution, yield and production rate of 2209 ml H₂/l medium, 1.97 mol H₂/mol glucose and 104.20 ml H₂/g CDW h⁻¹ were obtained at 9 g/l of glucose, initial pH of 7.0, inoculum volume of 8% and temperature of 35 °C, respectively. These results demonstrate that C. beijerinckii can efficiently produce H₂, and is another model microorganism for biohydrogen investigations.     

Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells

In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2 g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2 l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6 g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (50.6%).     

Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production

A mutant plant (Arabidopsis thaliana), sex1-1 (starch excess 1-1), accumulating high starch content in leaves was created to serve as better biomass feedstock for a H₂-producing strain Clostridium butyricum CGS2, which efficiently utilizes starch for H₂ production but cannot assimilate cellulosic materials. The starch content of the mutant plant increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using sex1-1 mutant plant as feedstock, C. butyricum CGS2 could produce 490.4 ml/l of H₂ with a H₂ production rate of 32.9 ml/h/l. The H₂ production performance appeared to increase with the increase in the concentration of mutant plant from 2.5 to 10 g/l. The highest H₂ to plant biomass yield was nearly 49 ml/g for the mutant plant. This study successfully demonstrated the feasibility of using a starch-rich mutant plant for more effective bioH₂ production with C. butyricum CGS2.     

Kappaphycus alvarezii as a source of bioethanol

The present study describes production of bio-ethanol from fresh red alga, Kappaphycus alvarezii. It was crushed to expel sap - a biofertilizer - while residual biomass was saccharified at 100 °C in 0.9 N H₂SO₄. The hydrolysate was repeatedly treated with additional granules to achieve desired reducing sugar concentration. The best yields for saccharification, inclusive of sugar loss in residue, were 26.2% and 30.6% (w/w) at laboratory (250 g) and bench (16 kg) scales, respectively. The hydrolysate was neutralized with lime and the filtrate was desalted by electrodialysis. Saccharomyces cerevisiae (NCIM 3523) was used for ethanol production from this non-traditional bio-resource. Fermentation at laboratory and bench scales converted ca. 80% of reducing sugar into ethanol in near quantitative selectivity. A petrol vehicle was successfully run with E10 gasoline made from the seaweed-based ethanol. Co-production of ethanol and bio-fertilizer from this seaweed may emerge as a promising alternative to land-based bio-ethanol.     

Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49

In order to increase the hydrogen yield from glucose, hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria Ethanoligenens harbinense B49 was investigated. The soluble metabolites from dark-fermentation mainly were ethanol and acetate, which could be further utilized for photo-hydrogen production. Hydrogen production by B49 was noticeably affected by the glucose and phosphate buffer concentration. The maximum hydrogen yield (1.83 mol H₂/mol glucose) was obtained at 9 g/l glucose. In addition, we found that the ratio of acetate/ethanol (A/E) increased with increasing phosphate buffer concentration, which is favorable to further photo-hydrogen production. The total hydrogen yield during dark- and photo-fermentation reached its maximum value (6.32 mol H₂/mol glucose) using 9 g/l glucose, 30 mmol/l phosphate buffers and immobilized R. faecalis RLD-53. Results demonstrated that the combination of dark- and photo- fermentation was an effective and efficient process to improve hydrogen yield from a single substrate.     

Staphylococcus sp. KW-07 contains nahH gene encoding catechol 2,3-dioxygenase for phenanthrene degradation and a test in soil microcosm

We isolated three species of phenanthrene-degrading bacteria from oil-contaminated soils and marine sediment, and assessed the potential use of these bacteria for bioremediation of soils contaminated by polycyclic aromatic hydrocarbons (PAHs). Based on 16S rDNA sequences, these bacteria were Staphylococcus sp. KW-07 and Pseudomonas sp. CH-11 from soil, and Ochrobactrum sp. CH-19 from the marine sediment. By PCR amplification, catechol 2,3-dioxygenase genes (nahH genes) mediating PAH degradation in the chromosome of Staphylococcus sp. KW-07 and Ochrobactrum sp. CH-19, and in plasmid DNA of Pseudomonas sp. CH-11 were detected. All isolates had a similar optimal growth temperature (25 °C) and optimal growth pH (7.0) in a minimal salt medium (MSM) with 0.1% (w/v) phenanthrene as the sole source of carbon and energy. Pseudomonas sp. CH-11 and Staphylococcus sp. KW-07 degraded 90% of added phenanthrene in 3 days and Ochrobactrum sp. CH-19 degraded 90% of the phenanthrene in 7 days under laboratory batch culture conditions. However, Staphylococcus sp. KW-07 was the most effective among the three strains in degradation of phenanthrene in soil. After inoculation of 1 × 10¹¹ cells of Staphylococcus sp. KW-07, over 90% degradation of 0.1% phenanthrene (0.1 g/100 g soil) was achieved after 1 month at 25 °C. The results collectively suggest that the Staphylococcus sp. KW-07 strain isolated may be useful in bioremediation of PAH-contaminated soils.     

Use of glycerol for producing 1,3-dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor

1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6 g/l yeast extract, 4.7 g/l glycerol, 42.1 g/l mannitol, 0.5 g/l K₂HPO₄, 0.5 g/l KH₂PO₄, 0.1 g/l MgSO₄·7H₂O, and 2.0 g/l CaCO₃ with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3 ± 7.8 g/l of maximal DHA concentration with 89.8 ± 2.4% of conversion rate of glycerol to DHA was achieved after 72 h of biotransformation using 10 g/l resting cells at 30 °C, pH 5.0 and 1.5 vvm of aeration rate.     

Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1

A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H₂ production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H₂ production rate and yield of 57.7 ml/h/L and 2.03 mol H₂/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H₂ fermentation.     

Enhancement of ethanol production from glycerol in a Klebsiella pneumoniae mutant strain by the inactivation of lactate dehydrogenase

Previously, using γ-irradiation treatment, we isolated a mutant strain of Klebsiella pneumoniae (named GEM167) that showed high-level ethanol production from glycerol. In the present study, in an effort to enhance ethanol production, we used a deletion of the lactate dehydrogenase gene to engineer a mutant strain incapable of lactate synthesis. In the ΔldhA mutant of GEM167, the production of ethanol was significantly increased from 21.5 g/l to 28.9 g/l and from 0.93 g/(l h) to 1.2 g/(l h). Introduction of the Zymomonas mobilis pdc and adhII genes encoding pyruvate decarboxylase and aldehyde dehydrogenase, respectively, further improved the ethanol production level from glycerol to 31.0 g/l; this is the highest level reported to date.     

A novel assay for rapid in vivo determination of phenotypic stability of recombinant ethanol-producing microorganisms

A rapid empirical assay is presented for assessing the phenotypic stability of continuous cultures of recombinant bacteria containing transposed pdc and adh genes for ethanol production. The method measures spectrophotometrically the rate of colour formation when cells oxidize added ethanol to acetaldehyde in the presence of Schiff’s reagent. During chemostat cultures of the recombinant ethanologen Escherichia coli KO11 on 20 g/l glucose, assay activities were stable and high at ca 8 × 10⁻⁴ ΔOD₅₄₀/(s.OD₅₅₀), reflecting the high, stable ethanol yield (ca 95%). On 20 g/l and 50 g/l xylose, ethanol yields declined rapidly to about 60% and this was closely mirrored by the assay activities which fell to ca 1.5 ΔOD₅₄₀/(s.OD₅₅₀), only slightly higher than those measured for the parent strain. Typically taking only about an hour to perform, the assay provides a faster means of gauging the phenotypic stability of ethanol production than is possible by conventional methods.     

Enhanced ethanol production from sugarcane juice by galactose adaptation of a newly isolated thermotolerant strain of Pichia kudriavzevii

The thermotolerant yeast strain isolated from sugarcane juice through enrichment technique was identified as a strain of Pichiakudriavzevii (Issatchenkiaorientalis) through molecular characterization. The P. kudriavzevii cells adapted to galactose medium produced about 30% more ethanol from sugarcane juice than the non-adapted cells. The recycled cells could be used for four successive cycles without a significant drop in ethanol production. Fermentation in a laboratory fermenter with galactose adapted P. kudriavzevii cells at 40 °C resulted in an ethanol concentration and productivity of 71.9 g L⁻¹ and 4.0 g L⁻¹ h⁻¹, respectively from sugarcane juice composed of about 14% (w/v) sucrose, 2% (w/v) glucose and 1% (w/v) fructose. In addition to ethanol, 3.30 g L⁻¹ arabitol and 4.19 g L⁻¹ glycerol were also produced, whereas sorbitol and xylitol were not formed during fermentation. Use of galactose adapted P. kudriavzevii cells for ethanol production from sugarcane juice holds potential for scale-up studies.     

Overproduction of a potential red pigment by a specific self-immobilization biomembrane-surface liquid culture of Penicillium novae-zeelandiae

A specific self-immobilization biomembrane-surface liquid culture (SIBSLC) was developed to overproduce a potential penicillium red pigment. Statistic analysis shows that both glucose concentration and membrane diameter are important factors influencing the yield of red pigment. After the optimization using central composite experimental design, the maximum yield of red pigment in shake flask reaches 4.25?g/l. The growth of strain HSD07B consists of three phases, and the pigment secreted in the decelerated phase, is originated from the interior of biomembrane where glucose exhaustion occurs. In addition, the batch and continuous SIBSLC were conducted for production of the pigment, and the latter was more competitive in consideration of the fact that it not only increased 61.5?% of pigment productivity, but also simplified the production process. Moreover, the pigment produced by SIBSLC is potentially acceptable for food applications although it is distinguished from the co-cultured red pigment we reported previously in components.     

Evaluation of colour removal in synthetic saline wastewater containing azo dyes using an immobilized halotolerant cell system

Studies were carried out to evaluate the colour removal capacity of a moderately halotolerant bacterium, Bacillus firmus, in synthetic saline wastewater medium (SSWM) under static condition. The bacterial strain effectively decolourized Polar red B (an azo dye) in a wide range of sodium chloride (1-6%, w/v), dye (5-100 mg/L) and SDS (0.1-5.0 mg/L) concentrations and at pH range of 6-10 after 24 h of incubation. Cell immobilization studies indicated that colour removal was significantly higher (p < 0.05) in immobilized halotolerant cell systems than with free cells of B. firmus especially at salt concentrations higher than 4%. Results suggest the potential of using the immobilized halotolerant cell system for effective treatment of dye-contaminated saline wastewaters.     

Effect of Fe-Pd bimetallic nanoparticles on Sphingomonas sp. PH-07 and a nano-bio hybrid process for triclosan degradation

In this study, we have evaluated the effect of palladium-iron bimetallic nanoparticles (nFe-Pd) on diphenyl ether (DE) degrading bacterial strain Sphingomonas sp. PH-07 as well as a sequential nano-bio hybrid process with nFe-Pd as catalytic reductant and PH-07 as biocatalyst for degradation of triclosan. Strain PH-07 grew well in the presence of nFe-Pd up to 0.1 g/L in minimal salts medium with DE as carbon source. In aqueous system, TCS (17.3 μM) was completely dechlorinated within 2 h by nFe-Pd (0.1 g/L) with concomitant release of 2-phenoxyphenol (16.8 μM) and chloride ions (46 μM). All possible dichloro- and monochloro-2-phenoxyphenol intermediates were identified by HPLC and GC-MS analyses, and the dechlorination pathway was proposed. Addition of PH-07 cells into the reactor effectively degraded the 2-phenoxyphenol. Our results reveal that strain PH-07 survives well in the presence of nFe-Pd and nFe-Pd/PH-07 hybrid treatment could be a potential strategy for degradation of TCS.     

Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata

Feasibility of producing (R)-3-hydroxybutyric acid ((R)-3-HB) using wild type Azohydromonas lata and its mutants (derived by UV mutation) was investigated. A. lata mutant (M5) produced 780 mg/l in the culture broth when sucrose was used as the carbon source. M5 was further studied in terms of its specificity with various bioconversion substrates for production of (R)-3-HB. (R)-3-HB concentration produced in the culture broth by M5 mutant was 2.7-fold higher than that of the wild type strain when sucrose (3% w/v) and (R,S)-1,3-butanediol (3% v/v) were used as carbon source and bioconversion substrate, respectively. Bioconversion of resting cells (M5) with glucose (1% v/w), ethylacetoacetate (2% v/v), and (R,S)-1,3-butanediol (3% v/v), resulted in (R)-3-HB concentrations of 6.5 g/l, 7.3 g/l and 8.7 g/l, respectively.     

Optimization of medium composition for butyric acid production by Clostridium thermobutyricum using response surface methodology

The optimal medium for butyric acid production by Clostridium thermobutyricum in a shake flask culture was studied using statistical experimental design and analysis. The optimal composition of the fermentation medium for maximum butyric acid yield, as determined on the basis of a three-level four-factor Box-Behnken design (BBD), was obtained by response surface methodology (RSM). The high correlation between the predicted and observed values indicated the validity of the model. A maximum butyric acid yield of 12.05 g/l was obtained at K₂HPO₄ 7.2 g/l, 34.9 g/l glucose, 20 g/l yeast extract, and 15 g/l acetate, which compared well to the predicated production of 12.13 g/l.     

Comparative virulence of Beauveria bassiana isolates against lepidopteran pests of vegetable crops

Forty-three isolates of the entomopathogenic fungus Beauveria bassiana were screened for virulence against second-instar larvae of diamondback moth (Plutella xylostella) (DBM), European corn borer (Ostrinia nubilalis) (ECB), corn earworm (Helicoverpa zea) (CEW), and fall armyworm (Spodoptera frugiperda) (FAW); 30 of these isolates were tested against beet armyworm (Spodoptera exigua) (BAW). Highly virulent isolates were also tested against black cutworm (Agrotis ipsilon) (BCW), and the most virulent isolate was also assayed against imported cabbage worm (Pieris rapae) (ICW) and cabbage looper (Trichoplusia ni) (CL). All lepidopteran species tested were susceptible to B. bassiana. Corn earworm and beet armyworm were most susceptible to fungal infection, and fall armyworm was least susceptible. Limited testing suggested low susceptibility of black cutworm and cabbage looper. B. bassiana isolate 1200 exhibited virulence against all pest species greater than or equal to commercial strain GHA of B. bassiana currently registered in the USA as BotaniGard®. In assays in which larvae were topically sprayed and maintained on the treated substrate for 24 h at 100% relative humidity, 6-day (25 °C) median lethal concentrations (LC₅₀s) of this isolate against CEW, BAW, DBM, FAW, ICW, ECB, CL, and BCW were 4, 5, 7, 11, 12, 98, 125, and 273 conidia/mm², respectively. The respective LC₅₀s of commercial strain GHA against these pest species were 9, 67, 97, 1213, 29, 1668, 541, and 3504 conidia/mm². Use of LC₅₀ versus median lethal concentration ratios (comparing LC₅₀s of each isolate to a “standard” strain) generated similar rankings of isolate virulence. Results from parametric ANOVAs of log LC₅₀ values followed by Tukey HSD multiple comparisons tests and those from Kruskal-Wallis nonparametric analyses followed by sequential Bonferroni tests for means comparisons were nearly identical.     

Disruption of the acid protease gene in Saccharomycopsis fibuligera A11 enhances amylolytic activity and stability as well as trehalose accumulation

The acid protease gene in Saccharomycopsis fibuligera A11 was disrupted by integrating the HPT (hygromycin B phosphotransferase) gene into ORF (Open Reading Frame) of the acid protease gene. The mutant 12 obtained could grow in the medium containing hygromycin. No clear zone formed by the mutant grown on the plate containing milk protein was observed whereas a big clear zone formed by the strain A11 was detected. The acid protease and amylases activities produced by the mutant within 3 days were 0.89 U/ml and 424.7 U/ml, respectively while those produced by the strain A11 were 13.5 U/ml and 259.9 U/ml, respectively. The amylases preparations produced by the mutant 12 and the strain A11 kept 88.8% and 45.5% of amylase activity, respectively after they were incubated at 37 °C for two days. Trehalose amount accumulated in the mutant cells was 28.3% (w/w) while that accumulated in the cells of S. fibuligera A11 was 23.6 (w/w).     

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18^T and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0–7.0) and at temperatures between 15 and 40 °C (optimal at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985^T (95.2–94.8%) and Chromohalobacter salexigens DSM 3043^T (95.0–94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA–DNA hybridization between both strains was 82%, whereas the values between strain M1-18^T and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C_18:1ω7c/C_18:1ω6c, C_16:0, and C_16:1ω7c/C_16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18^T (= CCM 8464 = CECT 8192^T = IBRC-M 10767^T = LMG 27461^T).