The extracellular productions of beta-xylanase, beta-xylosidase, beta-glucosidase, beta-mannanase, arabinosidase, alpha-glucuronidase, alpha-galactosidase and Fpase from Bacillus pumilus CBMAI 0008 were investigated with three different xylan sources as substrate. The enzymatic profiles on birchwood, Eucalyptus grandis and oat were studied at alkaline and acidic pH conditions. B. pumilus CBMAI 0008 grown on the three carbon sources produced mainly beta-xylanase. At pH 10, the levels of xylanase were 328, 160 and 136 U/ml, for birch, oat and E. grandis, respectively. beta-Mannanase production was induced on E. grandis (5 U/ml) and arabinofuranosidase on oat (5 U/ml). Although small quantities of alpha-glucuronidase had been produced at pH 10, activity at pH 4.8 was 1.5 U/ml, higher than observed for Aspergillus sp. in literature reports. Preliminary assays carried out on E. grandis kraft pulp from an industrial paper mill (RIPASA S.A. Celulose e Papel, Limeira, SP, Brazil) showed a reduction of 0.3% of chlorine use in the pulp treated with the enzymes, resulting in increased brightness, compared to conventional bleaching. The enzymes were more efficient if applied before the initial bleaching sequence, in a non-pre-oxygenated pulp.
A strain YZN-001 was isolated from swine manure effluent and was identified as Pseudomonas stutzeri. It can utilise not only nitrate and nitrite, but also ammonium. The strain had the capability to fully remove as much as 275.08 mg L(-1) NO(3)(-)-N and 171.40 mg L(-1) NO(2)(-)-N under aerobic conditions. Furthermore, At 30°C, the utilization of ammonium is approximately 95% by 18 h with a similar level removed by 72 h and 2 weeks at 10 and 4°C, respectively. Triplicate sets of tightly sealed serum bottles were used to test the heterotrophic nitrifying ability of P. stutzeri YZN-001. The results showing that 39% of removed NH(4)(+)-N was completely oxidised to nitrogen gas by 18 h. Indicating that the strain has heterotrophic nitrification and aerobic denitrification abilities, with the notable ability to remove ammonium at low temperatures, demonstrating a potential using the strain for future application in waste water treatment.
Effects of operation parameters on hydrogen production rate, substrate utilization efficiency and hydrogen bioconversion yield were investigated in a photobioreactor packed with sodium alginate/polyvinyl alcohol-124/carrageenan granules containing Rhodopseudomonas palustris CQK 01. Results demonstrate that an increase in influent glucose concentration and flow rate enhanced glucose transport from the bulk liquid into the granules, resulting in high hydrogen production. The bacteria mainly utilized the photo-heterotrophic pathway under optimal light illumination and produced hydrogen at low influent substrate loading, while they produced organic acids at high influent loading. The optimal temperature and pH of the influent medium for hydrogen production were independent of the illumination wavelength. The maximal hydrogen production rate of 2.61mmol/L/h was achieved under illumination at 590nm and 6000lx, a substrate concentration of 60mmol/L, a temperature of 30 degrees C and a pH7 of the influent medium.
Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P(s)) of 7.784 mL and production yield of (P(r)) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R(2) 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H(2) evolution rate and higher butyrate concentration in the fermentate.
In the present study, the photo-hydrogen production performances by Rhodopseudomonas palustris CQK 01 growing from the inoculated cells with ultra-sonication pretreatment (R. palustris CQK 01-USP) were experimentally investigated in batch culture and compared with that without pretreatment (R. palustris CQK 01-NP). It was found that the ultra-sonication pretreatment modified membrane morphology and broke up part of the cells, resulting in improvement of membrane permeability and bacterial activities and hence, helping the improvement of hydrogen production. The hydrogen production rate, hydrogen yield and energy conversion efficiency with R. palustris CQK 01-USP were increased to be nearly 2 times higher than that with R. palustris CQK 01-NP. The parametric study showed that under the conditions of initial glucose concentration 50 mmol/l, inoculum size 12%, illumination wavelength 590 nm, the photobioreactor with R. palustris CQK 01-USP obtained the optimal hydrogen production rate 0.54 mmol/l/h, hydrogen yield 1.2 mol-H2/mol-glucose and energy conversion efficiency 9.03%.
Response surface methodology was used to optimize extraction parameters for Morchella esculenta SO-01 exopolysaccharide (EPS) produced during submerged culture. The optimum conditions for EPS extraction were predicted to be, concentration at 84.07 degrees C, precipitation for 22.19 h and pH 8.44, and EPS production was estimated at 5.45 g/L. The actual yield of EPS under these conditions was 5.32 g/L. The antioxidant capacity of the EPS was measured in vivo after filling the stomach with different doses of EPS and results showed that the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of blood, spleen, liver, heart, and kidney were increased by 125%, 46.11%, 23.33%, 12.19%, 41.29%, and 63.24%, 63.12%, 166.54%, 98.01%, 57.68%, respectively, and that malonaldehyde (MDA) of blood, spleen, liver, heart, and kidney were decreased by 21.80%, 67.84%, 28.48%, 56.15%, 41.62%. The results provide a reference for large-scale extraction of EPS by M. esculenta SO-01 in industrial fermentation.
High costs and issues such as a high cell concentrations in effluents are encountered when utilizing microalgae for wastewater treatment. The present study analyzed nitrogen and phosphate removal under autotrophic, heterotrophic, mixotrophic and micro-aerobic conditions by Chlorella sorokiniana GXNN 01 immobilized in calcium alginate. The immobilized cells grew as well as free-living cells under micro-aerobic conditions and better than free-living cells under the other conditions. The immobilized cells had a higher ammonium removal rate (21.84%, 43.59% and 41.46%) than free living cells (14.35%, 38.57% and 40.59%) under autotrophic, heterotrophic, and micro-aerobic conditions, and higher phosphate removal rate (87.49%, 88.65% and 84.84%) than free living cells (20.21%, 42.27% and 53.52%) under heterotrophic, mixotrophic and micro-aerobic conditions, respectively. The data indicate that immobilized Chlorella sorokiniana GXNN 01 is a suitable species for use in wastewater treatment.
The performance of the entrapped-cell photobioreactor during H(2) production was assessed by using glucose as substrate in a continuous operation mode. The maximal hydrogen production rate and light conversion efficiency, 2.61mmol/L/h and 82.3%, were obtained at a HRT of 11.4h, an substrate loading rate of 4.2mmol/h and an illumination of 590nm and 6000lux, the corresponding hydrogen yield and total energy efficiency were 0.62mmol H(2)/(mmol glucose) and 4.8%, respectively. The results indicate the H(2) production system illuminated at 590nm wavelength engaged in energy storage for H(2) production due to more ATP synthesized in primary reaction center, and was of higher energy recovery capacity. Furthermore, the total energy efficiency was far lower than the corresponding light conversion efficiency due to intermediates production.
A novel gene, EG encoding enzymes involved in carboxymethyl cellulose (CMC) degradation was isolated, sequenced from the filamentous fungus Rhizopus stolonifer var. reflexus TP-02, and expressed in Escherichia coli BL21. The results showed that the gene amplified from the cDNA of the strain could be classified as the family of endoglucanase. During the fermentation process, the maximum endoglucanase activity (i.e. 0.715 IU/ml) of the recombinant bacteria was obtained at 36 h. The SDS-PAGE analysis on purified samples showed that a band with apparent molecular weight of about 40 kDa was detected after staining with Coomassie brilliant blue.
Cellulase production using corn cob residue from xylose manufacture as substrate was carried out by Trichoderma reesei ZU-02. It was found that on the same cellulose basis, the cellulase activity and yield produced on corn cob residue were comparable with that on purified cellulose. Under batch process, the optimum concentration of substrate was 40 g/l and the optimum C/N ratio was 8.0. In 500 ml flasks, cellulase activity reached 5.25 IU/ml (213.4 IU/g cellulose) after seven days' cultivation. In a 30 m(3) stirred fermenter for large scale production, cellulase and cellobiase activity were 5.48 IU/ml (222.8 IU/g cellulase) and 0.25 IU/ml (10.2 IU/g cellulose), respectively, after four days' submerged fermentation. The produced cellulase could effectively hydrolyze the corn cob residue, and the yield of enzymatic hydrolysis reached 90.4% on 10% corn cob residue (w/v) when the cellulase dosage was 20 IU/g substrate.
A bacterial strain able to rapidly degrade tetrabromobisphenol A (TBBPA), JXS-2-02, was isolated from anaerobic sludge that had been successfully enrichment by adding TBBPA for 240days. JXS-2-02 can use TBBPA as the sole carbon and energy source for growth in mineral salt medium. Based on morphology, biochemical characteristics and 16S rDNA sequence analysis, JXS-2-02 was identified as Comamonas sp.. Under the optimal conditions (pH 7.0, a temperature of 30°C and an inoculum of 1% OD(600)=0.6), more than 86% of the initial TBBPA (0.5mgL(-1)) was degraded after 10d. A TBBPA biodegradation pathway was proposed on the basis of the metabolite production patterns and the identification of the metabolites by GC-MS analysis. This study is the first report to isolate a single TBBPA-degrading bacterial strain under anaerobic conditions for TBBPA debromination and detoxification.
The effect of coconut water (CW) on biomass and docosahexaenoic acid (DHA, C22:6 n3) formation by Schizochytrium mangrovei Sk-02 was studied in a yeast extract-diluted sea water medium. Optimal CW-level was ca. 33% (v/v), resulting in a biomass level of 28 g/l with a DHA-content of 20% (w/w) or 6 g DHA/l, almost 50% higher than in non-supplemented cultures at the same initial sugar level. Study on the growth-promoting effects of coconut water suggested that it could be (partially) mimicked by addition of trace elements; the fatty acids present in CW did not appear to be incorporated or effect fatty acid formation by the organism. CW-addition was also effective in media with other nitrogen sources such as casitone, peptone and tryptone. Its inclusion (at 50% v/v) increased biomass levels two-to-three-fold with concomitant increases in the DHA-level.
A novel extracellular hydrolase of ∼45kDa molecular mass was purified from Kluyveromyces marxianus IFO 0288 cultures and characterized as serine protease. The K(m)-value of protease (designated protease-KM-IFO-0288-A), which was found active in media containing elevated [NaCl] but lacking EDTAK(2), decreased with increasing [Ca(2+)]. The protease maintained considerable activity at the range of 10-60°C and pH 6.00-10.25, with optimum k(cat)/K(m)-value at 35.5°C and pH 7.75. It was strongly affected by specific irreversible inhibitors of serine proteases while was unaffected by inhibitors of cysteine proteases. Significant rate constants, activation energies, and proton inventories were estimated from the profiles of Michaelis-Menten parameters, versus pH, temperature and deuterium atom fraction, in the hydrolysis of Suc-AAPF-pNA showing that protease-KM-IFO-0288-A performs catalysis via a charge-relay system. The properties of protease-KM-IFO-0288-A suggest that K. marxianus represents a valuable source of extracellular protease of biotechnological interest which, given its GRAS status, could find several important applications.
An organic solvent stable, alkaline serine protease (Bsubap-I) with molecular mass of 33.1 kDa, purified from Bacillus subtilis DM-04 showed optimum activity at temperature and pH range of 37-45 degrees C and 10.0-10.5, respectively. The enzyme activity of Bsubap-I was significantly enhanced in presence of Fe(2+). The thermal resistance and stability and of Bsubap-I in presence of surfactants, detergents, and organic solvents, and its dehairing activity supported its candidature for application in laundry detergent formulations, ultrafiltration membrane cleaning, peptide synthesis and in leather industry. The broad substrate specificity and differential antibacterial property of Bsubap-I suggested the natural ecological role of this enzyme for the producing bacterium.
Bacillus megaterium RB-05 was grown on glucose and on "tossa-daisee" (Corchorus olitorius)-derived jute, and production and composition of extracellular polysaccharide (EPS) were monitored. An EPS yield of 0.065 ± 0.013 and of 0.297 g ± 0.054 g(-1) substrate after 72 h was obtained for glucose and jute, respectively. EPS production in the presence of jute paralleled bacterial cellulase activity. High performance liquid chromatography (HPLC), matrix assisted LASER desorption/ionization-time of flight (MALDI-ToF) mass spectroscopy, and fourier transform infrared (FT-IR) spectroscopy demonstrated that the EPS synthesized in jute culture (JC) differed from that synthesized in glucose mineral salts medium (GMSM). While fucose was only a minor constituent (4.9 wt.%) of EPS from GMSM, it a major component (41.9 wt.%) of EPS synthesized in JC. This study establishes jute as an effective fermentation substrate for EPS production by a cellulase-producing bacterium.
The present study investigated to find out the suitability of the CO2 sequestered algal biomass of Chlorella sorokiniana as substrate for the hydrogen production by Enterobacter cloacae IIT-BT 08. The maximum biomass productivity in continuous mode of operation in autotrophic condition was enhanced from 0.05gL-1h-1 in air to 0.11gL-1h-1 in 5% air-CO2 (v/v) gas mixture at an optimum dilution rate of 0.05h-1. Decrease in steady state biomass and productivity was less sensitive at higher dilution and found fitting with the model proposed by Eppley and Dyer (1965). Pretreated algal biomass of 10gL-1 with 2% (v/v) HCl-heat was found most suitable for hydrogen production yielding 9±2mol H2 (kg COD reduced)-1 and was found fitting with modified Gompertz equation. Further, hydrogen energy recovery in dark fermentation was significantly enhanced compared to earlier report of hydrogen production by biophotolysis of algae.
Acarbose, a potent α-glucosidase inhibitor, is as an oral anti-diabetic drug for treatment of the type two, noninsulin-dependent diabetes. Actinoplanes utahensis ZJB-08196, an osmosis-resistant actinomycete, had a broad osmolality optimum between 309 mOsm kg(-1) and 719 mOsm kg(-1). Utilizing this unique feature, an fed-batch culture process under preferential osmolality was constructed through intermittently feeding broths with feed medium consisting of 14.0 g l(-1) maltose, 6.0 g l(-1) glucose and 9.0 g l(-1) soybean meal, at 48 h, 72 h, 96 h and 120 h. This intermittent fed-batch culture produced a peak acarbose titer of 4878 mg l(-1), increased by 15.9% over the batch culture.
A fungal strain, KSU-09, isolated from the roots of date palm (Phoenix dactylifera), was identified as Amylomyces rouxii based on sequence analysis of the internal transcribed spacer (ITS) region of its rRNA genes. Mycelia-free water extracts obtained from mycelium suspended in water for 72h facilitated the production of stable, predominantly monodispersed and spherical silver nanoparticles (AgNPs) in the size range of 5-27nm upon addition of 1mM silver nitrate, as determined by the XRD, AFM and TEM. The AgNPs exhibited antimicrobial activity against Shigella dysenteriae type I, Staphylococcus aureus, Citrobacter sp., Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Fusarium oxysporum. Thus, the strain KSU-09 could be used for simple, non-hazardous and efficient synthesis of antimicrobial AgNPs.
Some acetals can be produced from renewable resources (bioalcohols) and seem to be good candidates for different applications such as oxygenated diesel additives. In the present case the production of 1,1 diethoxy butane from bioethanol and butanal is presented. Butanal can be obtained from biobutanol following a partial oxidation or a dehydrogenation process. In this paper innovative process development about the synthesis of the mentioned acetal including catalytic reactive distillation experimental and simulation results will be presented and discussed. Katapak SP modules containing Amberlyst 47 resin were used as structured catalytic packings. This reactive system allowed reaching higher conversions than the equilibrium ones at the same temperatures. All the experimental data gathered allowed to tune a simulation model for the reactive distillation operation which showed a fairly good behavior in order to perform initial 1,1 diethoxy butane production process design studies.
This study examines the performance of bench-scale Upflow Anaerobic Sludge Blanket (UASB) reactor in treating simulated wastewaters containing 1,1,2,2-tetrachloroethane (TeCA). Reactors R1 (control) and R2 (containing TeCA) were operated at Hydraulic Retention Times (HRTs) of 36, 30, 24, 18 and 12 h. The removal of TeCA decreased from 99.85% to 98.40% as the HRT was lowered down from 36 to 12 h. The maximum TeCA dechlorination rate and the half velocity coefficient were 0.2 d(-1) and 18.58 mg TeCA/l, respectively. The Organic Loading Rate (OLR) was varied from 1.5 to 3.1 kg/m(3)/d to yield various substrate:co-substrate ratios and the overall removal of TeCA throughout this study was more than 99%. The average effluent TeCA concentration at optimum HRT (24 h) and substrate:co-substrate ratio (100:1) was less than 0.1 mg/l. The granules within the reactor were 0.1-3.2 mm in size with a heterogeneous bacterial population.
In this study, two statistical experimental designs, Plackett-Burman design (PBD) and response surface methodology (RSM), were employed to enhance the expression of 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC_LA-4), which was subsequently used for the construction of catechol biosensor. Ten important factors were evaluated by PBD, and four significant parameters were then optimized by RSM. Under the favorable fermentation conditions, the maximal specific activity of BphC_LA-4 was about 0.58U/mg with catechol as substrate. Meanwhile, homology modeling and molecular docking were utilized to help understand the interaction between BphC_LA-4 and catecholic substrates, which illustrated that BphC_LA-4 presented lower binding affinity towards 4-methylcatechol in comparison with 3-methylcatechol and catechol. Interestingly, the BphC_LA-4 enzyme electrode prepared by SiO2 sol-gel showed good response to all these three catecholic compounds. The differences of selectivity to 4-methylcatechol between free and immobilized enzyme implied that the introduction of electro-catalysis might have an effect on the enzyme-catalysis process.
An (S)-specific carbonyl reductase (SCRII) was purified to homogeneity from Candida parapsilosis by following an anti-Prelog reducing activity of 2-hydroxyacetophenone. Peptide mass fingerprinting analysis shows SCRII belongs to short-chain dehydrogenase/reductase family. Its coding gene was cloned and overexpressed in Escherichia coli. The recombinant SCRII displays the similar enzymatic characterization and catalytic properties to SCR. It catalyzes the enantioselective reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with excellent optical purity of 100% in higher yield than SCR. Based on the sequence-structure alignment, several single-point mutations inside or adjacent to the substrate-binding loop or active site were designed. With respect to recombinant native SCRII, the A220 and E228 mutations almost lost enantioselectivity towards 2-hydroxyacetophenone reduction. The catalytic efficiencies (kcat/Km) for the A220 or E228 variants are <7% that of the unmutated enzyme. This work provides an excellent catalyst for enantiopure alcohol preparation and the lethal mutations of A220 and E228 suggest their importance in substrate-binding and/or catalysis.
Production of 1,2-propanediol (1,2-PDO) from glycerol hydrogenolysis was carried out in bio-ethanol solvent over small amount of Rh-promoted Cu/solid-base catalysts prepared via layered double hydroxide precursors. It was found that glycerol hydrogenolysis proceeded easily on Rh-Cu/solid-base catalysts than separated Rh and Cu/solid-base. The conversion of glycerol and selectivity to 1,2-PDO over Rh(0.02)Cu(0.4)/Mg(5.6)Al(1.98)O(8.57) reached 91.0% and 98.7%, respectively, at 2.0 MPa H(2), 180 °C. And this catalyst was stable in five consecutive hydrogenolysis tests in ethanol.
This study investigated the effect of co-substrate amendments on EDB biodegradation under aerobic conditions. Microcosms were established using contaminated soil and groundwater samples and maintained under in situ conditions to determine EDB degradation rates, and the diversity and abundance of EDB degrading indigenous bacteria. After 100days of incubation, between 25% and 56% of the initial EDB was degraded in the microcosms, with added jet fuel providing highest degradation rates (2.97±0.49yr(-1)). In all microcosms, the quantity of dehalogenase genes did not change significantly, while the number of BTEX monooxygenase and phenol hydroxylase genes increased with jet fuel amendments. These results indicate that EDB was not degraded by prior dehalogenation, but rather by cometabolism with adapted indigenous microorganisms. This is also reflected in the history of the plume, which originated from an aviation gasoline pipeline leak. This study suggests that biostimulation of EDB is possible at aerobic groundwater sites.
(R)-2-Hydroxybutyric acid ((R)-2-HBA) is an important building block for azinothricin family of antitumour antibiotics and biodegradable poly(2-hydroxybutyric acid). However, optically active (R)-2-HBA could not be produced through microbial fermentation or chemical synthesis. Several biocatalytic methods have been reported for the production of (R)-2-HBA. Those processes used expensive and scarce substrates and would not be suitable for practical application. In this work, Gluconobacter oxydans DSM 2003 was confirmed to have the ability to produce (R)-2-HBA from 1,2-butanediol, a non-toxic and inexpensive compound that had a great potential for biotechnological processes. Under the optimal conditions, the biocatalytic process produced (R)-2-HBA at a high concentration (18.5 g l(-1)) and a high enantiomeric excess (99.7%). The biocatalysis process introduced in this study may provide a technically and economically interesting route for production of (R)-2-HBA.
Saccharomyces cerevisiae JUC15 was successfully obtained by target reaction-oriented screening, which reduced 2-hydroxy-1-phenylethanone (HPE) to (R)-phenyl-1,2-ethanediol ((R)-PED) of excellent enantiomeric excess (e.e. >99.9%). There was no significant decrease in the yield and optical purity of (R)-PED when the free cells were reused for 40 repeated cycles at 2gL(-1) substrate concentration. The strain used cheap sucrose for cofactor regeneration and worked over a considerably wider range of pH (4-9). The optimum substrate concentration was 8gL(-1), which was higher than any other biocatalysts reported so far. Interesting, when HPE concentration reached 20gL(-1) in reaction system, where 43.2% of the substrate was present in suspended solid form, the reaction still gave enantiopure (R)-PED in 71% yield. Last but not least, the product e.e. kept above 99.9% in all examined conditions. These results suggest the potential of this strain for the industrial production of optically active (R)-PED.
In this study, a highly efficient process for Candida parapsilosis-catalyzed deracemization of racemic 1-phenyl-1,2-ethanediol (PED) was described, based on a resin-based in situ substrate feeding and product removal (ISSFPR) methodology. The resin H103 was selected and used to keep the combined concentration of (R)-PED and (S)-PED at about 15g/l in aqueous phase. In the presence of 143g/l H103 resin, 50g/l of racemic substrate was converted to (S)-PED with 99.3% enantiomeric excess (ee) in 92% yield, while the initial reaction could afford an ee>99% only when the substrate concentration was 15g/l or lower. To our knowledge, this was the first report on the application of a resin-based ISSFPR technique for enhancing the efficiency of microbial stereoinversion.
Hydrogenolysis of biodiesel derived glycerol to 1,2-propanediol (1,2-PDO) has attracted much attention in recent years. In this work, glycerol hydrogenolysis to 1,2-PDO was performed over CuO/MgO catalysts prepared by impregnation and coprecipitation at 180 degrees C and 3.0 MPa H(2). It was found that the Cu(15)/MgO catalyst prepared by coprecipitation had the best activity. The conversion of glycerol and the selectivity of 1,2-PDO over Cu(15)/MgO reached 72.0% and 97.6%, respectively. And the conversion of glycerol was further increased to 82.0% when small amount of NaOH was added in the reaction mixture. Those highly active catalysts were characterized by X-ray diffraction, transmission electron microscopy, N(2)-adsorption and temperature-programmed reduction with H(2). Characterization results revealed that the activity of the prepared catalysts depended strongly on the particle sizes of both Cu and MgO. Catalysts that have smaller sized Cu and MgO particles are more active for glycerol hydrogenolysis.
1,3-Propanediol (1,3-PDO) was produced from glycerol in three parallel Expanded Granular Sludge Blanket (EGSB) reactors inoculated with granular sludge (control reactor-R1), heat-treated granular sludge (R2) and disrupted granular sludge (R3) at hydraulic retention times (HRT) between 3 and 24h. Maximum 1,3-PDO yield (0.52molmol(-1)) and productivity (57gL(-1)d(-1)) were achieved in R1 at HRTs of 12 and 3h, respectively. DGGE profiling of PCR-amplified 16S rRNA gene fragments showed that variations in the HRT had a critical impact in the dominant community of microorganisms. However, no appreciable differences in the bacterial population were observed between R2 and R3 at low HRTs. Production of H2 was observed at the beginning of the operation, but no methane production was observed. This study proves the feasibility of 1,3-PDO production in EGSB reactors and represents a novel strategy to valorise glycerol generated in the biodiesel industry.
Medium compositions favoring butanol and 1,3-propanediol (1,3-PDO) production from glycerol by Clostridium pasteurianum DSM525 were investigated using statistical experimental designs. Medium components affecting butanol and 1,3-PDO production were screened using a fractional factorial experimental design. Among the six tested variables (phosphate buffer, MnSO4·H2O, MgSO4·7H2O, FeSO4·7H2O, (NH4)2SO4, and yeast extract), FeSO4·7H2O, (NH4)2SO4, and yeast extract were found to be significant variables for further optimization of medium using a Box-Behnken design. Optimal butanol (0.98 g/L/h) and 1,3-PDO (1.19 g/L/h) productivities were predicted by the corresponding quadratic model for each product and the models were validated experimentally under optimized conditions. The optimal medium composition for butanol production was significantly different from that for 1,3-PDO production (0.06 vs. 0 g/L for FeSO4·7H2O, 7.35 vs. 0 g/L for (NH4)2SO4, and 5.08 vs. 8.0 g/L for yeast extract), suggesting that the product formation from glycerol by C. pasteurianum DSM525 can be controlled by changing medium compositions.
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 5.6g/l yeast extract, 4.7 g/l glycerol, 42.1g/l mannitol, 0.5 g/l K(2)HPO(4), 0.5 g/l KH(2)PO(4), 0.1g/l MgSO(4)·7H(2)O, and 2.0 g/l CaCO(3) 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 72h of biotransformation using 10g/l resting cells at 30°C, pH 5.0 and 1.5vvm of aeration rate.
In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B12.
The rapid accumulation of glycerol during biodiesel synthesis has attracted much interest on the potential usage. One of the promising applications is the conversion of glycerol to 1,3-propanediol (PDO), as well 2,3-butanediol (BDO), by using Klebsiella pneumonia. The results of this study indicate that the dissolved oxygen level (DO) is a determining factor in the distribution of PDO and BDO. The batch with a low DO could achieve a much higher PDO/BDO ratio than the high DO batch, with results of 9.9 and 0.2, respectively. The enzyme activity of glycerol dehydratase (GDHt) in the low DO batch was about three times that of the high DO batch, and this might be the cause of the enhanced PDO production. In conclusion, the results of this work show that high DO was beneficial to the production of BDO when using glycerol as the carbon source, while low DO could enhance PDO production.
A central composite design was used to study the effect of glycerol, rate of stirring, air aeration and pH on the synthesis of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae AC 15. Among the four variables, glycerol and rate of stirring significantly affected 1,3-PD productivity, whereas air aeration and pH were not effective. A quadratic polynomial equation was obtained for 1,3-PD productivity by multiple regression analysis using response surface methodology. The validation experimental confirmed with the predicted model. The optimum combinations for 1,3-PD productivity was glycerol, rate of stirring, air aeration, and pH of 50 g/l, 318 rpm, 0.6 vvm, 6.48, respectively. The subsequent fed batch experiments produced 1,3-PD of 70 g/l at a fermentation of 30 h.
1,3-Propanediol (1,3-PD) is a bifunctional organic compound of particular importance in the polymer industry for the synthesis of polyesters, polyethers and polyurethanes. Its biotechnological production from glycerol features inherent problems of nutrient limitation and inhibition(s) by substrate and product. In the present study 1,3-PD batch mathematical model developed using average batch kinetics data and independently obtained inhibition data was used to identify fresh nutrient feeding strategies (off-line on the computer) for enhanced production of 1,3-PD. Experimental implementation of one such model-based fed-batch cultivation strategy involving pseudo-steady state of substrate featured a 1,3-PD concentration of 63.5g/L with a 1,3-PD productivity of 1.35g/L/h which were significantly higher than batch fermentation results. This demonstrated the potential application of developed model for the design of suitable nutrient feeding strategies for high production of 1,3-PD. The methodology can also be easily adopted for other cultivations.
To identify the adsorption characteristics of 1,3-propanediol on beta zeolite, the effects of temperature, zeolite dose, and 1,3-propanediol concentration were studied through batch experiments. The results showed that the pseudo-second order model expressed the kinetic data better. The experimental and theoretical adsorption capacities were 116.2 and 119.0mg/g at 293K, respectively. The adsorption equilibrium data were observed to satisfy the Freundlich isotherm model. Based on the Boyd plot, intraparticle diffusion primarily governed the uptake process. Moreover, thermodynamic parameters, such as changes in standard free energy (ΔG(0)), standard enthalpy (ΔH(0)), and standard entropy, were estimated. The negative values of ΔG(0) and ΔH(0) (-9.4kJ/mol) indicated that the adsorption process was spontaneous, exothermic, and feasible. Finally, the activation energy derived from the Arrhenius equation suggested that the interaction mainly constitute physical adsorption.
The impact of anaerobiosis strategy on 1,3-propanediol production during cultivation of Clostridium butyricum VPI 1718 in different size bioreactors was studied. In batch trials with N2 gas infusion, the fermentation was successfully accomplished, regardless of initial glycerol concentration imposed and bioreactor geometry. However, in the absence of N2 continual sparging, significant variations concerning the biochemical response of the strain were observed. Specifically, at 1-L bioreactor, the absence of N2 infusion at high initial glycerol concentration induced lactate dehydrogenase activity and thus lactic acid synthesis, probably due to partial blockage of phosphoroclastic reaction caused by insufficient self-generated anaerobiosis environment. During fed-batch cultivation with continual N2 sparging, the strain produced ∼71 g L(-1) of 1,3-propanediol, whereas under self-generated anaerobiosis, 1,3-propanediol pathway was evidently restricted, as only 30.5 g L(-1) of 1,3-propanediol were finally produced. Apparently, N2 infusion strategy paired with bioreactor geometry can alter the biochemical behavior of the particular strain.
The aim of this study was to evaluate the natural producer Lactobacillus diolivorans as potential production organism of 1,3-propanediol from glycerol. Different cultivation parameters, such as oxygen supply, feeding-strategy, or medium composition have been tested in batch and fed-batch cultivations. The 1,3-propanediol concentration obtained in batch cultivations was 41.7 g/l. This could be increased to 73.7 g/l in a fed-batch co-feeding glucose and glycerol with a molar ratio of 0.1. Yeast extract as part of the MRS cultivation medium could be replaced by nicotinic acid and riboflavin. Furthermore, the addition of Vitamin B(12) to the culture medium increased production by 15% to a final titer of 84.5 g/l 1,3-propanediol.
Cofactor-dependent biotransformations often require consumption of a secondary substrate for cofactor regeneration. Alternatively, two synthetic reactions may be coupled together through cofactor regeneration cycles. Simultaneous production of value-added products from glycerol and xylose was realized in this work through an enzymatic NAD(H) regeneration cycle involving two enzymes. Glycerol dehydrogenase (GDH) catalyzed the production of 1,3-dihydroxyacetone (DHA) from glycerol, while xylose reductase (XR) enabled the reduction of xylose to xylitol using the protons released from glycerol. Both enzymes were immobilized with P(MMA-EDMA-MAA) nanoparticles. Interestingly, the immobilized multi-enzyme system showed much improved productivity and stability as compared to native enzymes, such that the total turnover number (TTN) reached 82 for cofactor regeneration while the yield reached 160g/g-immobilized GDH for DHA production.
We investigated the influence of solvent on the positional selectivity of Novozym 435 which was the immobilized Candida antarctica lipase B (CALB) during the esterification of oleic acid with glycerol for 1,3-diolein preparation previously. Herein, molecular modeling was used to elucidate the underlying mechanism of the solvent effect on the positional selectivity of the enzyme. The results showed that the binding energy of sn-1 hydroxyl of glycerol molecular with CALB became higher, and the binding energy of sn-2 hydroxyl of glycerol molecular with CALB became lower along with the increase of the solvent log P. It was demonstrated that, increasing log P of the solvent, the enzyme selectivity to sn-1 hydroxyl of glycerol molecular grew weaker, and the selectivity to sn-2 hydroxyl of glycerol molecular grew stronger.
We have isolated three lactic acid bacteria (Lactobacillus suebicus CUPV221, Pediococcus parvulus CUPV1 and P. parvulus CUPV22) that produced high levels of 2-substituted (1,3)-beta-D-glucans which increased the viscosity of the growth media. The (1,3)-beta-D-glucan consisted of two main molecular species, with masses of approximately 10(7) and 10(4) Da, whose proportions varied among the strains. The three strains survived exposure to saliva and simulated gastric conditions at pH 5, with P. parvulus CUPV22 surviving at pH 3.1, and L. suebicus CUPV221 surviving at pH 1.8. All strains were resistant to pancreatin and bile salts. P. parvulus CUPV22 exhibited the highest adhesion (10.5%) to Caco-2 cells, which decreased to 1.2% after washing the cells. Finally, P. parvulus CUPV22 and L. suebicus CUPV221 induced the production of inflammation-related cytokines by polarized macrophages, and interestingly, L. suebicus stimulated the production of cytokine IL-10. These results indicate that the three strains have potential utility for the production of functional foods.