Cell Dry Wt Research Articles

Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate) production by a new isolate of Bacillus megaterium

This research aimed at increasing the cell density and production of the homopolymer polyhydroxybutyrate (PHB) by Bacillus megaterium BA-019, using renewable and inexpensive bioresources as a substrate. A higher cell density and a greater PHB production level were obtained by using sugarcane molasses and urea as carbon and nitrogen sources, respectively. The limitation of nitrogen at a C/N molar ratio of 25 resulted in enhanced cell growth and PHB production in batch cultures. Fed-batch cultivation with the feeding nutrient composed of MSM with sugarcane molasses, urea and trace elements, and controlled by a pH-stat feeding control, lead to a significantly enhanced cell concentration and PHB production. The optimal feeding medium in this system required a higher total sugar concentration (400 g/l) and a C/N molar ratio of 10 mol/mol. Under these conditions the highest attained cell mass (72.6 g/l DW) and PHB content (42% of cell dry wt.) were achieved in a short cultivation time (24 h), leading to improved PHB productivity (1.27 g/l/h). However, dissolved oxygen was limiting and thus the system is likely to be suboptimal and capable of even further improvements to the PHB production rate.

Enantioselective oxidation of racemic lactic acid to d-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM

d-Lactic acid and pyruvic acid are two important building block intermediates. Production of d-lactic acid and pyruvic acid from racemic lactic acid by biotransformation is economically interesting. Biocatalyst prepared from 9 g dry cell wt l −1 of Pseudomonas stutzeri SDM could catalyze 45.00 g l −1 dl-lactic acid into 25.23 g l −1 d-lactic acid and 19.70 g l −1 pyruvic acid in 10 h. Using a simple ion exchange process, d-lactic acid and pyruvic acid were effectively separated from the biotransformation system. Co-production of d-lactic acid and pyruvic acid by enantioselective oxidation of racemic lactic acid is technically feasible.

Degradation of 3-chlorobenzoate and phenol singly and in mixture by a mixed culture of two ortho-pathway-following Pseudomonas strains

The compatibility and efficiency of two ortho-cleavage pathway-following pseudomonads viz. the 3-chlorobenzoate (3-CBA)-degrader, Pseudomonas aeruginosa 3mT (3mT) and the phenol-degrader, P. stutzeri SPC-2 (SPC-2) in a mixed culture for the degradation of these substrates singly and simultaneously in mixtures was studied. Another phenol-degrading strain, Pseudomonas sp. SoPC-5 (SoPC-5) that utilizes a meta-cleavage mode also was tried in co-culture with 3mT. The former combination was found to be a better degrader of both the substrates when present alone. But, with inoculum levels of 0.15 mg cell dry wt each of 3mT/SPC-2 or 3mT/SoPC-5 growth with 2 mM each of 3-CBA and phenol was slow with a lag of 24 h and degradation being incomplete. However, with higher inocula in the ratios 1:1, 1:2, and 2:1, i.e., 0.3 + 0.3, 0.3 + 0.6, and 0.6 + 0.3 mg cell dry wt of 3mT and SPC-2, respectively complete degradation of both the substrates occurred. Degradation of 3-CBA was complete with the release of stoichiometric amounts of chloride (Cl(-)) when concentrations of phenol/3-CBA were varied as 2:2, 2:4, and 4:2 mM, i.e., even when the concentration of the more toxic co-substrate 3-CBA was higher than phenol effective simultaneous degradation occurred at the inoculums ratio of 1:1 (0.3 mg dry cell wt. of each strain). These studies clearly indicated the better suitability of ortho-cleavage-utilizing strains as partners in a mixed culture than those follow different modes.

Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant Escherichia coli

The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF at 48 g dry cell wt l(-1) during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as approximately 64 g dry cell wt l(-1), 223 mg hG-CSF g(-1) dry cell wt and 775 mg hG-CSF l(-1) h(-1), respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose. Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show valuable decrease in activity in purified form.

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l(-1)) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l(-1) to 180 mg l(-1), approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)(-1) h(-1). A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.

High-cell-density cultivation of Schizochytrium sp. in an ammonium/pH-auxostat fed-batch system

Thraustochytrids, in particular Schizochytrium spp., are used for the production of the valuable polyunsaturated fatty acid, docosahexaenoic acid (DHA; 22:6 n-3). Growth of Schizochytrium sp. G13/2S in a defined medium was initially made in shake-flask cultures to determine the optimum concentrations of glucose (100-200 g l(-1)) and ammonia ( approximately 300 mg l(-1)) that could be used by this microorganism. In subsequent fermenter cultures, a pH-auxostat method was used to maintain NH(3) from 200-300 mg l(-1). During the first 49 h of fermentation, 150 g glucose l(-1) produced 63 g cell dry wt l(-1). Although growth was not limited by the supply of nitrogen, total fatty acids were at 25% cell dry wt which is more than half the final lipid content of commercially-grown Schizochytrium biomass which uses N-limited medium in the final stages for maximum lipid accumulation. This strategy is therefore useful for the cultivation of Schizochytrium to a high cell density up to the point when lipid accumulation can be triggered by N exhaustion.

Two-stage glycerol feeding for enhancement of recombinant hG-CSF production in a fed-batch culture of Pichia pastoris

Recombinant hG-CSF was expressed in Pichia pastoris under the control of the AOX1 promoter. In this study, the glycerol feeding rate was adjusted to achieve the maximum attainable specific growth rate before induction. Using a two-stage glycerol feeding method, the specific growth rate was changed from a maximum value of 0.21 h(-1) (at the beginning of feeding) to 0.15 h(-1) prior to induction. With this approach, the final dry cell wt and rhG-CSF yield achieved was close to 120 g l(-1) and 320 mg l(-1), respectively. Our study found that the two-stage feeding method allowed the overall productivity of rhG-CSF to increase 2.9 times that of the conventional fed-batch method.

UDP-glucose:(6-methoxy)podophyllotoxin 7- O-glucosyltransferase from suspension cultures of Linum nodiflorum

Cell cultures of Linum species store 6-methoxypodophyllotoxin (MPTOX), podophyllotoxin (PTOX) and related lignans as O-glucosides. UDP-glucose:(M)PTOX 7- O-glucosyltransferase has been detected and characterised in protein preparations of suspension-cultured cells of Linum nodiflorum L. (Linaceae). The maximal lignan glucoside contents in the cells are preceded by a rapid increase of the specific glucosyltransferase activity on day six of the culture period. MPTOX glucoside is the major lignan with up to 1.18 mg g −1 of the cell dry wt which is more than 30-fold of the PTOX glucoside content. Of the three aryltetralin lignans tested as substrates, PTOX and MPTOX display comparable apparent K m values of 4.7 and 5.4 μM, respectively. 5′-Demethoxy-6-methoxypodophyllotoxin is converted with the highest velocity of 25.2 pkat mg −1 while also possessing a higher K m of 14.7 μM. Two-substrate test series indicate that all three compounds compete for the active site of a single protein. The structurally similar lignan β-peltatin acts as competitive inhibitor as well. However, the 6- O-glucosidation is most likely catalysed by a separate enzyme. The (M)PTOX 7- O-glucosyltransferase works best at a pH around 9 and a temperature around 35 °C. A 15–30% increase of the reaction rate is effected by the addition of 0.9 mM Mn 2+.

Osteoinduction in human fat-derived stem cells by recombinant human bone morphogenetic protein-2 produced in Escherichia coli

Bioactive recombinant human bone morphogenetic protein-2 (rhBMP-2) was obtained using Escherichia coli pET-25b expression system: 55 mg purified rhBMP-2 were achieved per g cell dry wt, with up to 95% purity. In murine C2C12 cell line, rhBMP-2 induced an increase in the transcription of Smads and of osteogenic markers Runx2/Cbfa1 and Osterix, measured by semi-quantitative RT-PCR. Bioassays performed in human fat-derived stem cells showed an increased activity of the early osteogenic marker, alkaline phosphatase, and the absence of cytotoxicity.

Biosynthesis of intracellular 5-aminolevulinic acid by a newly identified halotolerant Rhodobacter sphaeroides

Of 23 strains of halotolerant (up to 12% w/v NaCl) photosynthetic bacteria isolated from various sources, one isolate, SH5, accumulated intracellular 5-aminolevulinic acid (ALA) at 0.45 microg/g dry cell wt (DCW) growing aerobically in the dark. The strain was identified as Rhodobacter sphaeroides using 16S rDNA sequencing. Biosynthesis of ALA was enhanced to 14 microg/g DCW using modified glutamate/glucose (50 mM) medium with the addition of 10 mM levulinic acid after 24 h cultivation. Addition of 30 microM Fe(2+) to this medium increased the yield to 226 microg/g DCW.

Influence of photoperiod on pigmentation and metabolic efficiency of the marine aerobic anoxygenic photosynthetic bacterium Erythrobacter longus Strain NJ3Y

The marine photoheterotroph Erythrobacter longus Strain NJ3Y was grown in continuous culture under variable light:dark (L:D) regimes of 24:0, 12:12 and 0:24 h to examine the influence of photoperiod on pigmentation and metabolic efficiency. Average carbon mass balances for each light regime closed to within 5 %, giving confidence in the measured variables as well as calculations of physiological parameters. Significant differences in photosynthetic pigmentation were observed for the experimental light treatments, with highest and lowest pigment concentrations measured in the 0:24 and 24:0 h L:D regimes, respectively, indicating that the photosynthetic pigments of E. longus Strain NJ3Y are preferentially synthesized in the dark. These differences are consistent with the strategy that photosynthetic pigments are produced during periods of darkness, for use in energy harvesting during subsequent light periods. In contrast, concentrations of the photoprotective pigment caloxanthin sulfate were highest for cells grown under a 12:12 h L:D cycle, indicating that this carotenoid is synthesized in the light and the dark. It is concluded that the 12:12 h L:D regime provides the maximum opportunity for E. longus Strain NJ3Y to derive a metabolic advantage from light availability (i.e. by enabling it to both synthesize and utilize its light harvesting apparatus). The corresponding carbon-specific glucose uptake rate was similar to 30% higher than that measured for the 0:24 and 24:0 h L:D regimes. It is hypothesized that light-stimulated rates of dissolved organic carbon uptake by bacteriochlorophyll a containing bacteria confer a slight competitive advantage over bacteria that lack this photosynthetic machinery. Lowest glucose-specific biomass yields (Y-x/s, g dry cell wt g(-1) glucose) were observed for the 12:12 h L:D treatment, suggesting that under the culture conditions used in this study, photosynthesis does not contribute significantly to the growth of E. longus Strain NJ3Y.

Biodesulfurization of dibenzothiophene by growing cells of Gordonia sp. in batch cultures

A new isolate, identified as Gordonia sp. ZD-7 by 16S rDNA sequence analysis, grew in n-hexadecane containing dibenzothiophene (DBT) which was degraded from 2.8 mM to 0.2 mM within 48 h. Biodesulfurization could be repeatedly performed for more than 190 h, with average desulfurization rates of 5 mmol DBT kg cells (dry wt)(-1) h(-1).

Chromate reduction by immobilized palladized sulfate‐reducing bacteria

Resting cells of Desulfovibrio vulgaris NCIMB 8303 and Desulfovibrio desulfuricans NCIMB 8307 were used for the hydrogenase-mediated reduction of Pd(II) to Pd(0). The resulting hybrid palladium bionanocatalyst (Bio-Pd(0)) was used in the reduction of Cr(VI) to the less environmentally problematic Cr(III) species. The reduction of Cr(VI) by free and agar-immobilized Bio-Pd(0) was evaluated. Investigations using catalyst suspensions showed that Cr(VI) reduction was similar ( approximately 170 nmol Cr(VI)/h/mg Bio-Pd(0)) when Bio-Pd(0) was produced using D. vulgaris or D. desulfuricans. Continuous-flow studies using D. vulgaris Bio-Pd(0) with agar as the immobilization matrix investigated the effect of Bio-Pd(0) loading, inlet Cr(VI) concentration, and flow rate on the efficiency of Cr(VI) reduction. Reduction of Cr(VI) was highest at a D. vulgaris Bio-Pd(0) loading of 7.5 mg Bio-Pd(0)/mL agar (3:1 dry cell wt: Pd(0)), an input [Cr(VI)] of 100 microM, and a flow rate of 1.75 mL/h (approx. 3.5 column volumes/h). A mathematical interpretation predicted the activity of the immobilized Bio-Pd(0) for a given set of conditions within 5% of the value found by experiment. Considering the system as an 'artificial enzyme' analog and application of applied enzyme kinetics gave an apparent K(m) value (K(m app)) of 430 microM Cr(VI) and a determined value of flow-through reactor activity which differed by 11% from that predicted mathematically.

Effect of operating parameters on molinate biodegradation

The effect of operating conditions during molinate degradation by the defined mixed bacterial culture DC, previously described as able to mineralize molinate, was evaluated in a batch reactor. Parameters such as the rate of molinate degradation, the dissolved organic carbon (DOC) consumption and the accumulation of molinate degradation products were monitored along the culture growth. The effect of conditions such as temperature, pH, aeration rate, salinity, and presence of additional carbon and/or nitrogen sources, was tested independently. Degradation of molinate in river water was also evaluated. Culture DC was able to grow and to mineralize molinate at all the conditions assayed. Temperature was the factor with the strongest influence on bacterial growth and molinate mineralization. The lowest and the highest rate values of growth (0.010 and 0.110 h −1) and of molinate degradation (0.027 and 0.180 g molinate g −1 cell dry wt h −1) were obtained at 15 and 35 °C, respectively. In cultures with approximately 187 mg l −1 of molinate, 2-oxo-molinate was the major molinate degradation product accumulated in the medium, in concentrations below 0.133 mg l −1. Degradation of molinate was also evaluated in a continuous stirred tank reactor (CSTR). Operating the CSTR at a hydraulic retention time (HRT) of 83 h, fed with medium containing molinate concentrations ranging from 1 to 3 mM, culture DC degraded the herbicide with specific degradation rates similar to those obtained in the batch systems.

δ-(D-α-Aminoadipoyl)-Cleaving Amidase of Ochrobactrum anthropi

Ochrobactrum anthropi cleaved the delta-(D-alpha-aminoadipoyl)-side chain from delta-(D-alpha-aminoadipoyl)-7-amino-4-methylcoumarin, a beta-lactamase-resistant cephalosporin C analogue. In whole cell conversions up to 1 nkat g(-1) dry cell wt were achieved. O. anthropi possesses also gamma-D-glutamyltranspeptidase activity, 8 nkat g(-1) dry cell wt, the likely cause of delta-(D-alpha-aminoadipoyl)-cleavage.

High-level Expression of a Synthetic Gene Encoding a Sweet Protein, Monellin, in Escherichia coli

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE.

Expression of Bacterial Hemoglobin in the Yeast, Pichia pastoris, with a Low O2-Induced Promoter

The PsADH2-promoter of Pichia stipitis alcohol dehydrogenase II (ADH II) gene was employed to control the expression of Vitreoscilla hemoglobin (VHb) gene in Pichia pastoris. As in P. stipitis, the promoter was also induced microaerobically in P. pastoris. The expression level of VHb in P. pastoris at low O2 tension (<5% air saturation) was 16 nmol/g dry cell wt, i.e. about 24-fold higher than that at 60% air saturation. The expressed VHb enhanced growth of P. pastoris under microaerobic conditions. The application of O2-regulated promoter in P. pastoris revealed that induction of high-level expression of heterologous protein is feasible without addition of supplementary compounds.

Suspension culture of gametophytes of transgenic kelp in a photobioreactor.

Transgenic Laminaria japonica gametophytes producing a recombinant tissue-type plasminogen activator (rtPA) protein, which is an effective third-generation thrombolytic agent for acute myocardial infarction (AMI), were cultured in an illuminated bubble column bioreactor. A maximum final dry cell weight of 1120 mg l(-1) was obtained in batch culture with an initial dry cell weight of 126 mg l(-1) and with aeration rate of 1.2 l air min(-1 )l(-1) culture, nitrate at 1.5 mM: and phosphate at 0.17 mM: . The yield of rtPA was 56 microg g(-1) dry cell wt. This is the first report regarding cultivation of a transgenic macroalga in a bioreactor.

Induced accumulation of oleanolic acid and ursolic acid in cell suspension cultures of Uncaria tomentosa

Increasing sucrose from 20 to 50 g l(-1) in Uncaria tomentosa cell suspension cultures enhanced ursolic acid and oleanolic acid production from 129 +/- 61 to 553 +/- 193 microg g(-1) cell dry wt. The maximal concentration of both triterpenes (1680 +/- 39 microg g(-1) cell dry wt) was 8 days after elicitation by jasmonic acid, while yeast extract or citrus pectin treatments produced 1189 +/- 20 or 1120 +/- 26 microg g(-1) cell dry wt, respectively. The ratio of ursolic acid:oleanolic acid was constant at 70:30.

Reduction of Cr(VI) by immobilized cells of Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776

Hexavalent chromium, a carcinogen and mutagen, can be reduced to Cr(III) by Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776. This study examined Cr(VI) reduction by immobilized cells of the two strains in a common solution matrix using various entrapment matrices. Chitosan and PVA-borate beads did not retain integrity and supported low or no reduction of Cr(VI) by the cells. A commercial preparation (Lentikats) was stable but also did not support Cr(VI) reduction. K-carrageenan beads were stable in batch suspensions but gel integrity was lost after only 5 h in a flow-through system in the presence of 100 microM Cr(VI). The best immobilization matrices were agar and agarose, where the initial rates of reduction of Cr(VI) (from 500 microM solution) for D. vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776 were 127 (agar) and 130 (agarose), and 15 (agar) and 12 (agarose) nmol h(-1) mg dry cell wt(-1), respectively. The higher removal of Cr(VI) by D. vulgaris was also seen in 14-mL packed-bed flow-through columns, where, at a flow rate of 2.4 mL h(-1), the percentage removal of Cr(VI) was approximately 95% and 60% for D. vulgaris and Microbacterium sp., respectively (agar-immobilized cells). The Cr(VI) reducing activities of D. vulgaris and Microbacterium sp. were lost after 159 and 140 h, respectively. Examination of the beads for structural integrity within the columns in situ using magnetic resonance imaging after 24 and 100 h of continuous operation against Cr(VI) (with negligible Cr retained within the columns) showed that agar beads were more stable with time. The most appropriate system for development of a continuous bioprocess is thus the use of D. vulgaris NCIMB 8303 immobilized in an agar gel matrix.