Biodegradation of 2-Chlorobenzoate by Recombinant Burkholderia Cepacia Expressing Vitreoscilla Hemoglobin Under Variable Levels of Oxygen Availability

The influence of bacterial hemoglobin, VHb, on dechlorinationand degradation of 2-chlorobenzoate (2-CBA) by recombinantBurkholderia sp. under variable oxygen availability with an initial dissolved oxygenconcentration of 0.27 mM-0.72 mM was investigated in batch and continuous culture. Abilityto express VHb was provided to recombinant Burkholderia by transformationwith the VHb gene, vgb, on plasmid pSC160. 100% of 0.5 mM CBA was degraded incultures with 85% and 70% of total volume as headspace air in closed reactorsby both wild type and recombinant Burkholderia. The recombinant cultures were able todechlorinate and degrade 100% of the 2-CBA in less than 48 hours at 30 °Ccompared to more than 120 hours for wild type cultures. The rate and extent of CBAdegradation by recombinant cultures with 40% of total volume as headspace air was higher than thoseachieved by wild type cells at the end of the 168 hours of incubation period, 98and 73%, respectively. The chloride released: CBA degraded molar ratio for cultures with 40%of total volume headspace air was nearly stoichiometric (molar ratio = 1.0) for recombinantstrains, whereas it was non-stoichiometric (molar ratio = 0.24)for wild type cells. The results suggest a suicidal meta-pathway for wild type cells and a complete dechlorinationand degradation pathway for recombinant cells under hypoxic conditions.The degradation and dechlorination ability of both types of cells was alsoinvestigated in continuous reactor studies by varying the dilution rate under hypoxicconditions. Regarding potential of the recombinant strain for 2-CBA degradation in eitheropen ecosystems or closed bioreactor bioremediation systems, the stability of the plasmidcontaining vgb in the recombinant cells was also studied; the plasmid was100% stable at 0.025 h^-1 dilution rate (1.7 d hydraulic retention time),even after one month.     

Scale-up of a high cell density continuous culture with Pichia pastoris X-33 for the constitutive expression of rh-chitinase

The feasibility of large-scale production of recombinant human chitinase using a constitutive Pichia pastoris expression system was demonstrated in a 21-L continuous stirred tank reactor. A steady-state recombinant protein concentration of 250 mg/L in the supernatant was sustained for 1 month at a dilution rate of 0.042 h(-1) (equivalent to one volume exchange per day), enabling a volumetric productivity of 144 mg/L d (240 U/L d). The steady-state dry cell weight concentration in this high cell density culture reached 110 g/L. Considering safety and economical aspects, all large-scale cultivations were conducted without molecular oxygen supplementation. Conventional air sparging was used instead. The oxygen demand of the process was determined by off-gas analysis (OUR = 4.8 g O(2) L(-1) h(-1) with k(L)a = 846 h(-1)) and evaluated with regard to further reactor scale-up.     

Kinetics of growth, oxygen uptake, and substrate utilization of wild type and genetically engineeredBurkholderia sp

The gene (vgb) encoding the hemoglobin (VHb) ofVitreoscilla sp. was cloned intoBurkholderia sp. and the effect of VHb on the growth characteristics of genetically engineeredBurkholderia (YV1) were compared with wild typeBurkholderia (R34) using continuous flow reactors (chemostat) at various dilution rates under aerobic conditions. Batch oxygen uptake rate showed that YV1 has much higher oxygen uptake rate than R34 (i.e. 0.63 mg O₂/g biomass/min vs. 1.43 mg O₂/g biomass/min for R34 and YV1 respectively at a dilution rate of 1.2 day⁻¹). Monod parameters, maximum growth rate (μ_max) and half saturation coefficient (K_s) were found to be 7.03 day⁻¹ and 691 mg/L for R34 respectively, compared to 5.49 day⁻¹ and 404 mg/L for YV1 respectively. At low dilution rates (<2.5 day⁻¹), when the substrate is present in low concentrations, the growth yield was much higher in YV1 (0.52) than in R34 (0.37). Although substrate utilization rates were similar between R34 and YV1, the latter showed much higher oxygen uptake rate than did R34 at all dilution rates. When the stability of VHb was tested on agar plates containing 40 μg/L of kanamycin and 100 μg/L of ampicillin,vgb gene containing VHb plasmid in YV1 was stable over 82 days. When survivability under oxygen limited conditions was tested, R34 survived only for 11 days whereas YV1 survived over 25 days in liquid media; in agar plate experiments, R34 did not survive more than 40 days whereas more than 75% of YV1 survived over 110 days.     

Biodegradation of 2-Chlorobenzoic Acid by Enterobacter cloacae: Growth Kinetics and Effect of Growth Conditions

Enterobacter cloacae was originally isolated from soil irrigated with wastewater on the basis of its ability to grow with linear alkylbenzene sulfonate (LAS) as the sole source for carbon and energy. The isolated bacterium was grown in batch cultures using a 2-chlorobenzoic acid (2-CBA)-containing minimal salt medium (MSM). 2-CBA was found to be the sole source for carbon and energy. 2-CBA inhibited the growth rate with a maximum concentration of 10 mM, after which no growth occurred. The Haldane model was used to predict the specific growth rate concentration data. 2-CBA degradation by starved E. cloaca cells was faster than that of nonstarved cells. The maximum growth rates on 2-CBA (2 mM) for starved and nonstarved cells reached only 0.34 and 0.28 h^?1, respectively. Glucose, lactose, sucrose, maltose, succinic acid, and mannitol as additional carbon sources at a fixed concentration (0.2%) caused the degradation rate of 2-CBA to proceed faster at ranges between 1.08- and 1.5-fold higher than that of the control. In contrast, using only fructose and sorbitol as the carbon sources showed catabolic repression of the degradation activity of 2-CBA by E. cloaca cells, although their cell mass was improved. All nitrogen sources supplied caused an increase in cell mass, whereas only lysine, alanine, glutamine, casein, and yeast extract caused a decrease in the degradation rate of 2-CBA, with a range between 12% and 28%. The activity of C120 could be detected in a crude extract of E. cloacae cells, indicating that the chloroaromatic ring fission occurs through the ortho pathways, not through the meta pathways. The data showed that different initial cell (inocula) densities did not affect the induction time for 2-CBA degradation. However, doubling the initial cell densities reduced the time required for reaching the complete degradation. 2-CBA degradation was optimally achieved at a 37°C incubation temperature and a pH of 7.5.     

Construction and characterization of two recombinant bacteria that grow on ortho- and para-substituted chlorobiphenyls

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISPOHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.     

High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system

A continuous fermentation process has been developed in Pichia pastoris (P. pastoris) with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in order to produce large quantities of recombinant human chitinase (rh-chitinase) for preclinical studies as a potential high-dose antifungal drug. Expression levels of about 200 to 400 mg/L have been demonstrated in fed-batch fermentations using strains with either the traditional methanol-inducible or the constitutive GAP promoter. Proteolytic degradation of the enzyme was typically seen in fed-batch fermentations. Continuous production of the enzyme by P. pastoris with the GAP promoter was demonstrated in a 1.5-L working volume fermentor using either glucose or glycerol as the carbon source. The fermentation could be extended for >1 month with a steady-state protein concentration of approximately 300 mg/L. Cell densities were >400 g/L wet cell weight (WCW) (approximately 100 g/L dry cell weight [DCW]) at a dilution rate (D) of 0.83 day(-1) or 1.2 volume exchanges per day (VVD). No proteolytic degradation of the enzyme was seen in the continuous fermentation mode.     

Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia sp. strain DNT for enhancement of 2,4-dinitrotoluene degradation

The gene (vgb) encoding the hemoglobin (VHb) of Vitreoscilla sp. was cloned into a broad host range vector and stably transformed into Burkholderia (formerly Pseudomonas) sp. strain DNT, which is able to degrade and metabolize 2,4-dinitrotoluene (DNT). Vgb was stably maintained and expressed in functional form in this recombinant strain (YV1). When growth of YV1, in both tryptic soy broth and minimal salts broth containing DNT and yeast extract, was compared with that of the untransformed strain, YV1 grew significantly better on a cell mass basis (A(600)) and reached slightly higher maximum viable cell numbers. YV1 also had roughly twice the respiration as strain DNT on a cell mass basis, and in DNT-containing medium, YV1 degraded DNT faster than the untransformed strain. YV1 cells pregrown in medium containing DNT plus succinate showed the fastest degradation: 100% of the initial 200 ppm DNT was removed from the medium within 3 days.     

毕赤酵母中表达透明颤菌血红蛋白提高重组脂肪酶的表达

解脂耶氏酵母胞外脂肪酶Lip2(YlLip2)是一种具有广泛应用前景的工业酶.为了改善高密度发酵生产Y1Lip2过程中的溶氧限制,提高Y1Lip2的表达量,将YlLip2基因lip2和透明颤菌血红蛋白(VHb)基因vgb分别置于AOXl启动子和PsADH2启动子的调控之下,进行YlLip2和VHb在毕赤酵母中的共表达.PsADH2启动子来源于树干毕赤酵母Pichia stipitis,在低氧条件下能被激活.SDS-PAGE和CO-差式光谱分析表明,Y1Lip2和VHb在重组菌中成功实现了共表达.在氧限制性条件下,VHb表达的细胞(VHb+,GS 115/9Klip2-pZPVT)与对照细胞(VHb-,GS 115/9Klip2)相比,摇瓶和10 L发酵罐中YlLip2表达量分别提高了25％和83％.此外,在低氧条件下,VHb+细胞在10 L发酵罐中的生物量也比VHb-细胞高.文中也获得了一株表达了VHb的并携带有多个lip2基因拷贝的克隆子GS 115/9Klip2-pZP VTlip2 49#,在低氧条件下,该克隆子在10L发酵罐中的最高脂肪酶水解活力达33 900 U/mL.因此,在毕赤酵母中用PsADH2启动子表达VHb,同时增加lip2基因的拷贝数是提高YlLip2表达量的一种有效策略.     

Trichloroethylene mineralization in a fixed-film bioreactor using a pure culture expressing constitutively toluene ortho -monooxygenase

An aerobic, single-pass, fixed-film bioreactor was designed for the continuous degradation and mineralization of gas-phase trichloroethylene (TCE). A pure culture of Burkholderia cepacia PR1(23)(TOM(23C)), a Tn5transposon mutant of B. cepacia G4 that constitutively expresses the TCE-degrading enzyme, toluene ortho-monooxygenase (TOM), was immobilized on sintered glass (SIRANtrade mark carriers) and activated carbon. The inert open-pore structures of the sintered glass and the strongly, TCE-absorbing activated carbon provide a large surface area for biofilm development (2-8 mg total cellular protein/mL carrier with glucose minimal medium that lacks chloride ions). At gas-phase TCE concentrations ranging from 0.04 to 2.42 mg/L of air and 0.1 L/min of air flow, initial maximum TCE degradation rates of 0.007-0.715 nmol/(min mg protein) (equivalent to 8.6-392.3 mg TCE/L of reactor/day) were obtained. Using chloride ion generation as the indicator of TCE mineralization, the bioreactor with activated carbon mineralized an average of 6.9-10.3 mg TCE/L of reactor/day at 0.242 mg/L TCE concentration with 0.1 L/min of air flow for 38-40 days. Although these rates of TCE degradation and mineralization are two- to 200-fold higher than reported values, TOM was inactivated in the sintered-glass bioreactor at a rate that increased with increasing TCE concentration (e.g., in approximately 2 days at 0.242 mg/L and <1 day at 2.42 mg/L), although the biofilter could be operated for longer periods at lower TCE concentrations. Using an oxygen probe and phenol as the substrate, the activity of TOM in the effluent cells of the bioreactor was monitored; the loss of TOM activity of the effluent cells corroborated the decrease in the TCE degradation and mineralization rates in the bioreactor. Repeated starving of the cells was found to restore TOM activity in the bioreactor with activated carbon and extended TCE mineralization by approximately 34%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 674-685, 1997.     

Characterization of multiple chlorobenzoic acid-degrading organisms from pristine and contaminated systems: mineralization of 2,4-dichlorobenzoic acid

Multiple bacterial strains with CBA metabolic properties were isolated using a simple selective strategy. Phylogenetic analysis of the 16S rRNA gene sequences grouped them into two main clusters consisting of four bacterial phyla and belonging to 17 genera. Whereas growth was more frequent with 2-CBA (～68%), 50% grew on 4-CBA and ～7% utilized 3-CBA. One third of the strains exhibited 2,4-dichlorobenzoic acid (2,4-diCBA) catabolic function and were mainly representatives of α-, β- and γ-Proteobacteria. In batch experiments, growth was concomitant with substrate disappearance and near-stoichiometric release of chloride. Doubling times for 2,4-diCBA degradation doubled those determined for mono-substituted CBAs. Out of the six 2,4-diCBA degraders submitted for enzyme assays, significant induction of catechol 1,2-dioxygenase types I and II activities in cell-free extracts were found in four while protocatechuate 3,4-dioxygenase activity was detected in the remaining two. Activities in CBA-grown cells were 20 orders-of-magnitude higher than those grown on benzoic acid.     

Effects of light intensity and dilution rate on the semicontinuous cultivation of Arthrospira (Spirulina) platensis. A kinetic Monod-type approach

Semicontinuous cultures were carried out at different dilution rates (D) and light intensities (I) to determine the maximum productivity of Arthrospira platensis cultivated in helicoidal photobioreactor up to the achievement of pseudo-steady-state conditions. At I=108 μmol photons m(-2) s(-1), the semicontinuous regime ensured the highest values of maximum cell concentration (X(m)=5772±113 mg L(-1)) and productivity (P(XS)=1319±25 mg L(-1) d(-1)) at the lowest (D=0.1 day(-1)) and the highest (D=0.3 day(-1)) dilution rates, respectively. A kinetic model derived from that of Monod was proposed to determine the relationship between the product of light intensity to dilution rate (ID) and the cell productivity, which were shown to exert a combined influence on this parameter. This result put into evidence that pseudo-steady-state conditions could be modified according to circumstances, conveniently varying one or other of the two independent variables.     

Enhancement of 2,4-dinitrotoluene biodegradation by Burkholderia sp. in sand bioreactors using bacterial hemoglobin technology

Continuous flow sand column bioreactor experiments were conducted to investigate the effect of 2,4-dinitrotoluene (DNT) concentration (i.e. DNT loading rate) and influent dissolved oxygen (DO) concentration on aerobic biodegradation of DNT by wild type (DNT) and recombinant (YV1) Burkholderia sp., the latter containing plasmid pSC160 which carries the gene (vgb) encoding the hemoglobin (VHb) from the bacterium Vitreoscilla. The experiments were conducted in two continuous flow packed bed sand column bioreactors, one growing the wild type strain and the other growing YV1. Under oxygen-rich feed conditions (6.8 mg DO/L in the feed) with an influent DNT concentration of 99.6 mg/L (DNT loading rate approximately = 9.2 mg/m2/day), the effluent DNT concentration from the wild type bioreactor reached 0.7 mg DNT/L in 40 days whereas it was less than 0.2 mg DNT/L for the YV1 bioreactor in about 25 days. When influent DNT concentration was increased to 214 mg/L (DNT loading rate approximately = 20.3 mg/m2/day) while maintaining the same influent DO level of 6.8 mg/L, the effluent DNT concentration increased to about 5 mg/L for the wild type bioreactor whereas it was maintained at less than 0.2 mg/L for the YV1 bioreactor. Additionally, when influent DO was reduced from 6.8 mg/L to 3.1 mg/L while the influent DNT concentration remained at 214 mg/L, the effluent DNT concentration increased to more than 20 mg/L for the wild type bioreactor but up to only 1.7 mg/L for the YV1 bioreactor. A subsequent increase of influent DO back to 6.6 mg/L reduced the effluent DNT concentration to about 5 mg/L for the wild type bioreactor and to 0.10-0.19 mg/L for the YV1 bioreactor. These results confirm the utility of vgb technology to enhance biodegradation of aromatic compounds under hypoxic conditions and also that this enhancement can be maintained over extended periods of time as evidenced by plasmid stability in Burkholderia YV1.     

Toluene bioconversion to p-hydroxybenzoate by fed-batch cultures of recombinant Pseudomonas putida.

A microbial oxidation process for the production of p-hydroxybenzoate (HBA) from toluene is reported. The oxidation reaction was studied in fed-batch fermentations using a recombinant Pseudomonas putida grown on glutamate as the sole carbon and energy source with salicylate and IPTG induction of tmoABCDE, and pchCF and phbz pathway genes, respectively. An average volumetric HBA productivity of 13.4 mg HBA x L(-1) x h(-1) was obtained under rapid growth conditions (glutamate excess), giving an HBA titer of 132 mg x L(-1) after 9.8 h of fermentation. This corresponded to an average specific HBA productivity of 7.2 microg HBA (mg total protein)(-1) x h(-1). In contrast, maximum HBA titers of 35 mg HBA x L(-1) were achieved in 27 h in comparative studies employing glutumate limited fed-batch cultures. A specific productivity of 4.1 microg HBA (mg total protein)(-1) x h(-1) and volumetric productivity of 1.3 mg HBA x L(-1) x h(-1) were calculated for the growth-rate restricted cultures. The differences in HBA production between the two cultures could be correlated to the levels of specific toluene-4-monooxygenase (T4MO) polypeptides. T4MO catalyzes the rate-limiting step in the pathway. Using experimental data, the half-life value of TmoA was calculated to be approximately 28 h. Assuming linear, monomolecular decay of TmoA, a specific degradation constant of 0.025 x h(-1) was calculated, which placed the stability of recombinant TmoA in the range of relatively stable proteins, even in the absence of co-expression of tmoF, the terminal oxidoreductase subunit of T4MO.     

Kinetic parameters of continuous cultures of Bacillus thermoleovorans sp. A2 degrading phenol at 65 degrees C

In this paper, we report on the kinetics of phenol degradation and cell growth in continuous cultures of suspended cells of Bacillus thermoleovorans sp. A2 at 65 degrees C. A high yield coefficient of Y(x/s)=0.84 g cell dry weight g(-1) phenol was measured at a dilution rate of 0.5 h(-1). At the same dilution rate the coefficient for maintenance metabolism (m(s)) was determined to be 0.045 g phenol g(-1) cell dry weight h(-1). The maximal growth rate (wash-out) determined at a phenol inlet concentration of 188 mg l(-1) was 0.9 h(-1). Up to 7 g phenol l(-1) per day were degraded in a continuously operated 2-l stirred tank reactor with suspended cells (feed concentration 660 mg l(-1)). Additionally, yield coefficients for oxygen and ammonium are reported.     

Kinetics of growth and phosphate uptake in pure culture studies of Acinetobacter species

Acinetobacter has been found to be the major species responsible for mediating biological phosphate removal. The growth kinetics and phosphate uptake were investigated for an isolated Acinetobacter strain growing in a defined medium. The phosphate uptake is dependent on growth rate, temperature, and pH. Polyphosphate granules occurred in a balanced growth stage. The maximum phosphorus content in cells was 4.8% at the dilution rate of 12 day(-1). The specific phosphate uptake rate was found to be a quadratic polynomial function of the dilution rate. Increased calcium (up to 36 mg/L) and magnesium (up to 15 mg/L), and the addition of yeast extract (100 mg/L), primary effluent (20%), and fluoride (10 mg/L) did not affect phosphate uptake. Anaerobic conditioning (N(2) stripping), low pH (CO(2) stripping), and addition of sodium acetate under anaerobic conditions failed to stimulate immediate phosphate release. Nevertheless, After 21-24 h, the phosphate release was ca. 3, 5, and 15 mg P/g cell, respectively, for N(2) purging, the addition of acetate, and CO(2) purging. For two-stage completely stirred reactor operation, there was negligible phosphate overplus at the second reactor when phosphate was added, when the first reactor was subjected to phosphate limitation. When both phosphate and carbon limited the growth in the first reactor, there was slight phosphate accumulation under endogenous respiration conditions in the second reactor.     

Construction and Characterization of Two Recombinant Bacteria That Grow on ortho- and para-Substituted Chlorobiphenyls

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISP_OHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.     

Cloning and Expression of the Vitreoscilla Hemoglobin Gene in Enterobacter Aerogenes: Effect on Cell Growth and Oxygen Uptake

The hemoglobins found in unicellular organisms show a great deal of chemical reactivity, protecting cells against oxidative stress, and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than their VHb counterparts. Here, the expression of VHb, its effect on the growth and antioxidant enzyme status of cells under different culture conditions was studied by cloning the complete regulatory and coding sequences (vgb) for VHb in Enterobacter aerogenes. Contrary to what has been reported for Escherichia coli, the expression of vgb in E.aerogenes decreased several fold under 10% of atmospheric oxygen (2% oxygen) and its growth was not greatly improved by the presence of VHb. Measured either as viable cells or total cell mass, untransformed E. aerogenes grew better than the recombinant strains. At the late exponential phase, however, the vgb-bearing strain was determined to have a higher cell number and total cell mass than the strain bearing only the plasmid vector with no vgb insert. The VHb expressing strain also had an oxygen uptake rate several fold higher than its counterparts. Given that oxidative stress may occur upon elevated oxygen exposure and be balanced by the action of antioxi-dative compounds, the level of antioxidative response of E. aerogenes expressing VHb was also studied. The VHb expressing strain had substantially (1.5–2.6-fold) higher catalase activity than strains not expressing VHb. Both VHb+ and VHb- strains, however, showed similar levels of superoxide dismutase activity. The activity of both enzymes was also growth phase dependent. Stationary phase cells of all strains showed 2–5-fold higher activity for these enzymes than cells at the exponential phase.     

Cometabolic degradation of mono-chloro benzoic acids by <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. R04 grown on organic carbon sources

The aerobic cometabolism of chlorobenzoic acids (CBAs) by Rhodococcus sp. R04 was accomplished by augmenting the medium with organic carbon sources. In mineral medium supplemented with glucose (MMG), 0.5 mM 2-CBA was incompletely metabolized after the 5-day incubation, while the near-complete disappearance of 0.5 mM 4-CBA was monitored. Over the 5-day incubation period, the concentration of chloride increased to 0.17 mM in bottles containing 4-CBA, glucose and strain R04; whereas in cultivation with 2-CBA the chloride content was about 0.1 mM. After 5-day incubation, 28.5% 4-CBA was remained in mineral medium supplemented with ethanol (MME), and the relatively low values of chloride were released. To our knowledge, it is first report that the feasibility of using ethanol as an added substrate for cometabolic degradation of CBA by aerobic polychlorinated biphenyl (PCB)-degrading bacteria. The specific activities of (chloro)benzoate 1,2-dioxygenase and (chloro)catechol 1,2-dioxygenase activities were detected in cell-free extracts (CFEs) of strain R04. These results suggest that the initial degradation of CBAs occurred most likely prior to chloride release.     

Enhancement of mussel adhesive protein production in Escherichia coli by co-expression of bacterial hemoglobin

Mussel adhesive proteins (MAPs) have been considered as potential underwater and medical bioadhesives. Previously, we reported a functional expression of recombinant MAP hybrid fp-151, which is a fusion protein with six type 1 (fp-1) decapeptide repeats at each type 5 (fp-5) terminus, with practical properties in Escherichia coli. In the present work, we introduced the Vitreoscilla hemoglobin (VHb) co-expression strategy to enhance the production levels of hybrid fp-151 since VHb has been successfully used for efficient oxygen utilization in several expression systems, including E. coli. In both batch-type flask and fed-batch-type bioreactor cultures, we found that co-expression of VHb conferred higher cell growth and hybrid fp-151 production. Its positive effects were significantly increased in high cell density bioreactor cultures as the microaerobic environment was more quickly and severely formed. We obtained a approximately 1.9-fold higher (approximately 1 g/L) production of MAP fp-151 from VHb co-expressing cells in fed-batch bioreactor cultures as compared to that from VHb non-expressing cells. Collectively and regardless of the culture type, VHb co-expression strategy was successful in enhancing the production of recombinant mussel adhesive proteins in the E. coli expression system.     

甲醇抑制时重组毕赤酵母发酵的特性研究

本文对毕赤酵母进行了恒化培养研究。以甲醇为唯一碳源时,在稀释率较低时(D<0.048 h^-1),连续培养系统操作很稳定。但在稀释率高时(D>0.048h^-1),连续培养系统的定态点不止一个,实验不能维持,故采用比生长速率恒定的分批流加培养进行研究。结果表明,毕赤酵母的生长符合Andrew普遍化底物抑制模型。综合考虑水蛭素的生成、底物的消耗,在生产中维持甲醇浓度为限制性浓度(0.5 g/L),且维持比生长速率为0.02 h^-1时,水蛭素Hir65的比生成速率达到最大值0.2 mg/(g·h)且甲醇的比消耗速率为0.04 g/(g·h)。