Cell mass production of Acinetobacter calcoaceticus in palm oil-limited chemostat culture

Chemostat culture of Acinetobacter calcoaceticus KB-2 was done under palm oil-limiting conditions for cell production, and variation of cell compositions and yield coefficients were investigated in connection with the specific growth rates. At the concentration of 0.6% palm oil, the productivity of cells and yield coefficient were 4.76 g cells/l/h and 1.18 g cells/g palm oil, respectively, at a practical dilution rate of 0.85 h⁻¹. About 80% of the palm oil was assimilated by the strain, and the maintenance coefficient was 0.035 g palm oil/g cells/h. Although the carbohydrate content remained essentially constant when the growth rate was varied, the lipid, protein, and nucleic acid contents were increased slightly at higher growth rates. Although the protein content increased only 3%, the protein yield coefficient (Y_p) increased about 1.5 times over the range of specific growth rates between 0.1 and 0.7 h⁻¹. The increase in Y_p was due to the higher protein content of the biomass and to higher values of the cell yield coefficient.     

Characterization of energy conversion of Synechococcus sp. PCC7942 under photoautotrophic conditions based on metabolic flux and chlorophyll fluorescence analysis

Energy conversion efficiency of photoautotrophic microalgae plays an important role in the utilization of light energy for cell growth and production of metabolites. To understand the utilization of light energy, Synechococcus sp. PCC7942 was cultivated at different incident light intensities of 15.8, 47.3, and 94.6 μmol/m²/sec in continuous culture. The influence of light on the carbon and energy metabolism of microalgae was investigated by combining metabolic flux analysis (MFA) and chlorophyll fluorescence analysis (CFA). Results showed that the yields of biomass based on ATP (Y _ATP) and total light energy (Y _E) both declined with increasing light, and the maximal values of Y _ATP and Y _E were estimated to be 4.73 g/mol-ATP, and 17.10 × 10^?3 g/kJ respectively, at the examined conditions. The overall efficiency of energy conversion against total absorbed energy changed with the varying irradiances. However, the actual conversion efficiency of total energy based on CFA was almost constant, regardless of the different irradiances used in the present study.     

Metal biosorption by two cyanobacterial mats in relation to pH, biomass concentration, pretreatment and reuse

The pH-dependent metal sorption by Oscillatoria- and Phormidium-dominated mats was effectively expressed by the Hill function. The estimated Hill functions can fruitfully predict the amount of metal sorbed at a particular initial pH. Pretreatment of biomass with 0.1 mmol L⁻¹ HCl was more effective than pretreatment with CaCl₂, HNO₃, NaOH, and SDS in enhancing metal sorption ability of the biomass. Desorption of metal ions in the presence of 100 mmol L⁻¹ HCl from metal-loaded mat biomass was completed within 1 h. After six cycles of metal sorption/desorption, sorption decreased by 6-15%. Only 6% and 11% of the biomass derived from the Oscillatoria sp.- and Phormidium sp.-dominated mats was lost during the cycling. The cyanobacterial mats seem to have better potential than several biomass types for use in metal sorption from wastewaters as they are ubiquitous, self-immobilized, and have good reusability.     

Biotransformation of oleic acid into (E)-10-hydroxy-8-octadecenoic acid and (E)-7,10-dihydroxy-8-octadecenoic acid by Pseudomonas sp. 42A2 in an immobilized system

Oleic acid was transformed into 10-hydroxy-8E-octadecenoic acid and 7,10-dihydroxy-8E-octadecenoic acid using Pseudomonas sp. 42A2 NCIMB 40045 in different immobilised supports. Celite R633 gave highest conversions in 48 h: the cellular yield (Y _p/x) g products g^–1 of cellular protein in the immobilised culture was 5 compared with the free-cell culture Y _p/x of 3.8. Conversion of oleic acid in the immobilised cell culture was 50% of the carbon source supplied.     

Energy conservation in Bacillus megaterium

1. The respiratory chain energy conservation systems of Bacillus megaterium strains D440 and M have been investigated following growth in batch and continuous culture. Respiratory membranes from these strains contained cytochromes b, aa ₃ , o and b, c, a, o, respectively; both readily oxidised NADH but neither showed any pyridine nucleotide transhydrogenase activity.
2. Whole cells of both strains exhibited endogenous →H^-/O ratios of approximately 4; when loaded with specific substrates the resultant →H⁺/O ratios indicated that proton translocating loops 1 and 2 were present in strain D440 and that loops 2 and 3 were present in strain M.
3. In situ respiratory activities were measured as a function of dilution rate during growth in continuous culture. True molar growth yields with respect to oxygen (Y _{O 2}) of approximately 50 g cells·mole oxygen^-1 were obtained for most of the nutrient limitations employed. Average values for Y _ATP of 12.7 and 10.8 g cells·mole ATP equivalents^-1 were subsequently calculated for strains D440 and M respectively.
4. Energy requirements for maintenance purposes were low in energy-limited cultures but were substantially increased when growth was limited by nitrogen source (NH ₄ ⁺ ). Under the latter conditions there is probably a partial uncoupling of energy-conserving and energy-utilising processes leading to energy wastage.

     

Chromium-resistant bacteria and cyanobacteria: impact on Cr(VI) reduction potential and plant growth

Two chromium-resistant bacterial strains, Bacillus cereus S-6 and Ochrobactrum intermedium CrT-1, and two cyanobacterial strains, Oscillatoria sp. and Synechocystis sp., were used in this study. At initial chromate concentrations of 300 and 600 μg K₂CrO₄ mL⁻¹, and an inoculum size of 9.6×10⁷ cells mL⁻¹, B. cereus S-6 completely reduced Cr(VI), while O. intermedium CrT-1 reduced Cr(VI) by 98% and 70%, respectively after 96 h. At 100 μg K₂CrO₄ mL⁻¹, Synechocystis sp. MK(S) and Oscillatoria sp. BJ2 reduced 62.1% and 39.9% of Cr(VI), respectively, at 30°C and pH 8. Application of hexavalent chromate salts adversely affected wheat seedling growth and anatomical characters. However, bacterial inoculation alleviated the toxic effects, as reflected by significant improvements in growth as well as anatomical parameters. Cyanobacterial strains also led to some enhancement of various growth parameters in wheat seedlings.

     

Structure and composition of hydrocarbons and fatty acids from a marine blue-green alga,Synechococcus sp.

The major hydrocarbons of Synechococcus sp., a marine blue-green alga isolated from the Gulf of Mexico, were identified as 1-nonadecene and 1,14-nonadecadiene. The content of 1-nonadecene increased with culture age from about 0.5 mg/g dry weight in young cells to about 2.3 mg/g dry weight in old cells, while the content of 1,14-nonadecadiene remained constant with culture age at about 1 mg/g dry weight. Both [1-¹⁴C]acetate and [2-¹⁴C]acetate were incorporated to equal extents into the hydrocarbons. [1-¹⁴C]Stearate was incorporated into the hydrocarbons, but [³H]arachidate was not. The fatty acids of Synechococcus sp. were typical of blue-green algae, consisting of C_16:0, C_16:1, C_16:1, C_16:0, C_18:0, C_18:1, C_18:2 and C_18:3 fatty acids were detected. The hexadecenoic acid was shown to be 9-C_16:1 while the octadecenoic acid was a mixture of 93% 11-C_18:1 and 7% 9-C_18:1. The fatty acid content increased during the first 4 days of growth and then decreased slightly.     

Performance assessment of two patent strains ofZymomonas mobilis in batch and continuous fermentations

Summary The performance ofZymomonas mobilis strains ATCC 31821 and ATCC 31823 was assessed in batch and continuous culture. In batch culture using a medium containing 250 g/l glucose, identical maximum specific growth rates of 0.16/h were found, though final biomass concentration and growth yield were significantly lower for ATCC 31 823 than for ATCC 31 821. Final ethanol concentrations in this medium were about 110 g/l vor both organisms. In continuous culture at increasing dilution rates using a medium containing 100 g/l glucose, no significant differences were seen between the two strains with respect to the fermentation parameters studied. For ATCC 31 821, maximum rates of glucose uptake (Q_s) and ethanol produktion (Q_p) of 8.7 g glu/g/h and 4.4 g eth/g/h, respectively, were found. Both strains showed a similar performance at a fixed dilution rate of 0.1/h, where maximum ethanol concentrations of about 68 g/l were reached at a feed glucose concentration of about 139 g/l. At this dilution rate the maximum values of Q_s and Q_p were about 5.8 g glu/g/h and 2.8 g eth/g/h, respectively. Test tube experiments showed that growth, measured as optical density, decreased with increasing concentrations of exogenous ethanol with complete inhibition of growth at ethanol concentrations >8% (v/v). As evidenced by the results presented here, we have been unable to practice the invention as described in U.S. Patent 4,403,034 (Rogers and Tribe 1983).Nomenclature D Dilution rate, 1/h - _max maximum specific growth rate, 1/h - S_R Initial substrate concentration, g glucose/1 - S Residual substrate concentration, g glucose/1 - S₀ Effluent substrate concentration, g glucose/1 - X Blomass concentration; g cells/l - OD₆₂₀ Optical density at 620 nm, dimensionless - [P] Product concentration, g ethanol/1 - Y_x/s Growth yield, g cells/g glucose used - Y_p/s Product yield, g ethanol/g glucose used - %, Yield Percentage yield, Y_p/sx100/Y _p ^s/max =Y_p/sx100/0.51 - Q_s Specific rate of glucose uptake, g glucose/g cells/h - Q_p Specific rate of ethanol formation, g ethanol/g cells/h - m_e Maintenance energy coefficient, g glucose/g cells/h - VP Volumetric productivity, g ethanol/l/h - t Fermentation time, h     

Growth, Respiration, and Polypeptide Patterns of Bradyrhizobium sp. (Arachis) Strain 3G4b20 from Succinate- or Oxygen-Limited Continuous Cultures

Succinate- or oxygen-limited continuous cultures were used to study the influences of different concentrations of dissolved oxygen and ammonia on the growth, respiration, and polypeptide patterns of Bradyrhizobium sp. (Arachis) strain 3G4b20. During succinate-limited growth, molar growth yields on succinate (Y_succ) ranged from 38.9 to 44.4 g (dry weight) of cells mol of succinate⁻¹ and were not greatly influenced by changes in dilution rates or changes in the oxygen concentrations that we tested. Succinate, malate, and fumarate induced the highest rates of oxygen uptake in all of the steady states in which the supply rates of (NH₄)₂SO₄ ranged between 322 and 976 μmol h⁻¹. However, the amino acids aspartate, asparagine, and glutamate could also be used as respiratory substrates, especially when the (NH₄)₂SO₄ supply rate was decreased to 29 μmol h⁻¹. Glutamine-dependent respiration was seen only when the (NH₄)₂SO₄ supply rate was 29 μmol h⁻¹ and thus appears to be under tight ammonia control. Nitrogenase activity was detected only when the culture was switched from a succinate-limited steady state to an oxygen-limited steady state. Comparison of major silver-stained proteins from three steady states by two-dimensional gel electrophoresis revealed that nearly 60% were affected by oxygen and 24% were affected by ammonia. These data are consistent with reports that oxygen has a major regulatory role over developmental processes in Rhizobium sp. and Bradyrhizobium sp.     

A study of mixed continuous cultures of sulfate-reducing and methane-producing bacteria

Ecological relationships between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten have been studied by continuous culture studies using the chemostat technique. The maximum specific growth rate (μ _max) and saturation constant (K _s) were, respectively, 0.36 hr⁻¹ and 0.047 mM for lactate-limited growth ofDesulfovibrio desulfuricans and 0,011 hr⁻¹ and 0.17 mM for acetate-limited growth ofMethanobacterium sp. Calculated values for the true molar growth yieldsY _G) and maintenance coefficients (m) were 30.6 g bacterial mass/mole of lactate and 0.53 g substrate/g dry wt hr forD. desulfuricans and 37.8 g bacterial mass/mole of acetate and 0.54 g substrate/g dry wt hr forMethanobacterium. No growth ofMethanobacterium was observed at apS²⁻ value (the hydrogen sulfide potential) of more than 11 and there was no effect on the growth atpS²⁻ values above 13. In mixed continuous culture experiments the concentration of acetate decreased in the secondstage growth vessel, whereas that of methane increased stoichiometrically. If the substrate concentration in the reservoirs (S _r) was increased from 0.1 to 0.5 mg/ml, the population ofDesulfovibrio increased and that ofMethanobacterium was washed out of the culture vessel, since the concentration of hydrogen sulfide reached apS²⁻ value of 10.5. From the mixed continuous culture experiments a commensalism between the two species can be described, i.e., the acetate-fermentingMethanobacterium benefits from the acetate released byDesulfovibrio which is, in turn, not affected in the presence of the former.     

Stability trends and fragmentation patterns of gaseous yttrium oxide clusters studied by fast atom bombardment tandem mass spectrometry

The fragmentation patterns of yttrium oxide cluster species YO⁺, Y₂O₂⁺, Y₂O₃⁺, Y₃O₄⁺, Y₄O₆⁺, Y₅O₇⁺, Y₆O₈⁺ and Y₇O₁₀⁺ were investigated at collision energies 30–110 and 170 eV by fast atom bombardment tandem mass spectrometry. The collision activated dissociation (CAD) spectra obtained revealed higher thermodynamic stability for the clusters of general formula Y_αO_(3α−1)/2⁺, where a is an odd number (e.g. YO⁺, Y₃O₄⁺, Y₅O₇⁺, Y₇O₁₀⁺) which are also the preferred CAD products for all oxide clusters studied. These most stable oxides are constituted by trivalent yttrium only whereas those containing formally tetravalent yttrium Y_aO_3a/2⁺, (where a is even) e.g. Y₂O₃⁺ and Y₄O₆⁺, are extremely unstable. The clusters Y_aO_(3a−2)/2⁺, (where a is even) containing divalent yttrium, e.g. Y₂O₂⁺ and Y₆O₈⁺, have considerable stability but their CAD products are again the thermodynamic products Y_aO_(3a−1)/2⁺. Electronic structures appear to have overriding significance in determining the thermo- dynamic stabilities of the oxide cluster species.     

Biomass production in mass cultures of marine phytoplankton at varying temperatures

Natural populations of marine phytoplankton obtained from a large outdoor pond were grown on waste water-sea water mixtures in laboratory continuous cultures in the temperature range 5–33 °C. Virtually all of the influent inorganic nitrogen (14.0 mg l^?1) was assimilated at every temperature tested. There was, however, a distinct change in dominant species with temperature: below 19.8 °C Phaeodactylum tricornutum was dominant, at 27 °C Nilzschia sp. was the main species, and as the temperature increased above 27 °C a blue-green alga, Oscillatoria sp., became increasingly dominant. There is some indication that the excellent growth of P. tricornutum below 10 °C was related to a dramatic increase in the nutrient content per cell as the temperature decreased. Thus at low temperatures reduced division rates are compensated for by increased nutrient uptake rates. It follows that there is a transfer of phytoplankton protein from numerous small cells at intermediate temperatures to large cells that are reduced in numbers at lower temperatures but which represent the same total organic matter. The effect of this phenomenon on annual food chain efficiencies in both controlled and natural marine ecosystems is unknown.     

Comparative Amperometric Study of Uptake Hydrogenase and Hydrogen Photoproduction Activities between Heterocystous Cyanobacterium Anabaena cylindrica B629 and Nonheterocystous Cyanobacterium Oscillatoria sp. Strain Miami BG7

Heterocystous filamentous cyanobacterium Anabaena cylindrica B629 and nonheterocystous filamentous cyanobacterium Oscillatoria sp. strain Miami BG7 were cultured in media with N₂ as the sole nitrogen source; and activities of oxygen-dependent hydrogen uptake, photohydrogen production, photooxygen evolution, and respiration were compared amperometrically under the same or similar experimental conditions for both strains. Distinct differences in these activities were observed in both strains. The rates of hydrogen photoproduction and hydrogen accumulation were significantly higher in Oscillatoria sp. strain BG7 than in A. cylindrica B629 at every light intensity tested. The major reason for the difference was attributable to the fact that the heterocystous cyanobacterium had a high rate of oxygen-dependent hydrogen consumption activity and the nonheterocystous cyanobacterium did not. The activity of oxygen photoevolution and respiration also contributed to the difference. Oscillatoria sp. strain BG7 had lower O₂ evolution and higher respiration than did A. cylindrica B629. Thus, the effect of O₂ on hydrogen photoproduction was minimized in Oscillatoria sp. strain BG7.     

Growth yield and energy generation in anaerobically-grown Campylobacter spec.

An anaerobic continuous culture study was made with Campylobacter spec. to determine growth yields under various growth conditions. The growth media contained 0.1% (w/v) yeast extract as carbon source. When grown in an aspartate-limited culture Y _asp ^max was 4.6. Inclusion of formate in the culture medium hardly affected the true growth yield. The number of ATP equivalents generated in the fumaratereductase system was 0.66 and the Y _ATP ^max was 7.0. In the nitrate reduction with formate 1.7 ATP equivalents were generated, and a YNO _3- ^max of 12.2 was observed. The true growth yield obtained with a mixture of lactate and aspartate was lower than that found with aspartate alone.     

Continuous ethanol production from sago starch using immobilized amyloglucosidase and Zymomonas mobilis

To produce ethanol more economically than in a conventional process, it is necessary to attain high productivity and low production cost. To this end, a continuous ethanol production from sago starch using immobilized amylogucosidase (AMG) and Zymomonas mobilis cells was studied. Chitin was used for immobilization of AMG and Z. mobilis cells were immobilized in the form of sodium alginate beads. Ethanol was produced continuously in an simultaneous saccharification and ethanol fermentation (SSF) mode in a pacekd bed reactor. The maximum ethanol productivity based on the void volume, V_v, was 37 g/l/h with ethanol yield, Y_p/s, 0.43 g/g (84% of the theoretical ethanol yield) in this system. The steady-state concentration of ethanol (46 g/l could be maintained in a stable manner over two weeks at the dilution rate of 0.46 h⁻.     

Kinetics of Insoluble Cellulose Fermentation by Continuous Cultures of Ruminococcus albus

Data from analyses of continuous culture fermentation of insoluble cellulose by Ruminococcus albus 7 were used to derive constants for the rate of cellulose hydrolysis and fermentation, growth yield, and maintenance. Cellulose concentration was 1% in the nutrient reservoir, and hydraulic retention times of 0.5, 1.0, 1.5, 1.75, and 2.0 days were used. Concentrations of reducing sugars in the cultures were negligible (less than 1%) compared with the amount of hydrolyzed cellulose, indicating that cellulose hydrolysis was the rate-limiting step of the fermentation. The rate of utilization of cellulose depended on the steady-state concentration of cellulose and was first order with a rate constant (k) of 1.18 day⁻¹. The true microbial growth yield (Y) was 0.11 g g⁻¹, the maintenance coefficient (m) was 0.10 g g⁻¹ h⁻¹, and the maximum Y_ATP was 7.7 g of biomass (dry weight) mol of ATP⁻¹.     

Mucilaginibacter calamicampi sp. nov., a member of the family Sphingobacteriaceae isolated from soil at a field of reeds

A Gram-negative, aerobic, non-flagellated, non-gliding, rod-shaped bacterial strain, designated WR-R1Y^T, was isolated from soil at a field of reeds in South Korea. Strain WR-R1Y^T grew optimally at 30 °C, at pH 7.0–7.5 and in the absence of NaCl. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain WR-R1Y^T fell within the clade comprising Mucilaginibacter species, coherently clustering with the type strain of Mucilaginibacter composti, with which it exhibited the highest 16S rRNA gene sequence similarity value of 97.6 %. Sequence similarities to the type strains of the other Mucilaginibacter species and the other species used in the phylogenetic analysis were 93.1–96.9 % and <91.1 %, respectively. Strain WR-R1Y^T contained MK-7 as the predominant menaquinone and iso-C_15:0, summed feature 3 (C_16:1 ω6c and/or C_16:1 ω7c), C_16:0 and iso-C_17:0 3-OH as the major fatty acids. The major polar lipids were phosphatidylethanolamine and one unidentified aminophospholipid. The DNA G+C content of strain WR-R1Y^T was 43.1 mol% and its mean DNA–DNA relatedness value with M. composti KACC 14956^T was 17 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain WR-R1Y^T is separate from other Mucilaginibacter species. On the basis of the data presented, strain WR-R1Y^T represents a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter calamicampi sp. nov. is proposed. The type strain is WR-R1Y^T (= KCTC 32214^T = CCUG 63418^T).     

Overproduction of poly(β-malic acid) (PMA) from glucose by a novel Aureobasidium sp. P6 strain isolated from mangrove system

After over 100 strains of Aureobasidium spp isolated from mangrove system were screened for their ability to produce poly(β-malic acid) (PMA), it was found that Aureobasidium sp. P6 strain among them could produce high level of Ca²⁺-PMA. Fourteen percent glucose and 6.5 % CaCO₃ in the medium were the most suitable for Ca²⁺-PMA production. Then, 100.7 g/l of Ca²⁺-PMA was produced using Aureobasidium sp. P6 strain within 168 h at flask level. During 10-l batch fermentation, when the medium contained 12.0 % glucose, 98.7 g/l of Ca²⁺-PMA in the culture and 14.7 g/l of cell dry weight were obtained within 156 h, leaving 0.34 % reducing sugar in the fermented medium. When glucose concentration in the fermentation medium was 14.0 %, 118.3 g/l of Ca²⁺-PMA in the culture and 16.4 g/l of cell dry weight were obtained within 168 h, leaving 0.4 % reducing sugar in the fermented medium. After purification of Ca²⁺-PMA from the culture and acid hydrolysis of the pure Ca²⁺-PMA, analysis of HPLC showed that Aureobasidium sp. P6 strain only produced two main components of Ca²⁺-PMA and minor amount of calcium malate and that the hydrolysate of PMA was mainly composed of calcium malate. This is the first time to report that the novel yeast strain Aureobasidium sp. P6 strain isolated from the mangrove systems can produce such high amount of Ca²⁺-PMA.     

An Enzymatic Conversion of Lipoxygenase Products by a Hydroperoxide Lyase in Blue-Green Algae (Oscillatoria sp.)

An enzyme has been isolated from blue-green algae Oscillatoria sp. which utilizes the product, 13-hydroperoxy-9, 11-octadecadienoic acid (13-HPOD), of lipoxygenase for its substrate. This enzyme, termed hydroperoxide lyase, converts the conjugated diene 13-hydroperoxide of linoleic acid to 13-oxotrideca-9, 11-dienoic acid. The structure of the latter has been determined by ultraviolet spectroscopy and mass spectrometry. 9-HPOD is not a substrate for this enzyme. The hydroperoxide lyase from Oscillatoria sp. has a maximum of activity at pH 6.4 and 30°C. The molecular weight of the enzyme was estimated at 56,000. The enzyme was not inhibited by BW 755C, but was inhibited by molecules containing more than one hydroxyl group. Quercetin was found to be the best inhibitor of the enzyme activity. The purified hydroperoxide lyase from Oscillatoria sp. showed an apparent K_m of 7.4 micromolar and a V_max of 35 nanomoles per minute per milligram of protein for 13-HPOD. An enzymatic pathway for the biogenesis of oxodienoic acid from linoleic acid is proposed. This involves the sequential activity of lipoxygenase and hydroperoxide lyase enzymes.     

Cell yields of Vibrio succinogenes growing with formate and fumarate as sole carbon and energy sources in chemostat culture

Vibrio succinogenes which gains all the ATP by anaerobic electron transport phosphorylation, was grown in continuous culture on a defined medium with formate and fumarate as sole energy sources. The growth yield at infinite dilution rate (Y ^max) was obtained by extrapolation from the growth yields measured at various dilution rates. With formate as the growth limiting substrate, Y ^max was found as 14 g dry cells/mol formate. Under these conditions growth was limited by the rate of energy supply, because formate is used only as a catabolic substrate (Bronder et al. 1982). The Y _ATP ^max calculated from the ATP requirement for cell synthesis was 18 g dry cells/mol ATP. This gives an ATP/2e ratio of 0.8. The ATP/2e ratio in vitro had been measured as 1 (Kröger and Winkler 1981). It is concluded that growing V. succinogenes gain at least 80% the stoichiometrically possible amount of ATP, when growth is limited by energy supply.