Thermophilic sulphate and sulphite reduction in lab-scale gas-lift reactors using H(2) and CO(2) as energy and carbon source

Feasibility of thermophilic (55 degrees C) sulphate and sulphite reduction with H(2) and CO(2) gas-mixtures was studied in gas-lift reactors, which contained pumice particles as carrier material. Particular attention was paid to biomass retention and the competition between hydrogenotrophic sulphate-reducers and other hydrogenotrophic thermophiles. A model medium with defined mineral nutrients was used.The results of the experiments clearly demonstrate that sulphate conversion rates up to 7.5 g SO(4) (2-)/L per day can be achieved. With sulphite, a reduction rate of 3.7 g S/L per day was obtained, which equals a sulphate conversion rate of 11.1 g SO(4) (2-)/L per day. Under the applied conditions, a strong competition for hydrogen between hydrogenotrophic sulphate-reducers, tentatively designated as Desulfotomaculum sp., and hydrogenotrophic methanogens was observed. The outcome of the competition could not be predicted. Growth of the mixed culture was totally inhibited at an H(2)S concentration of 250 mg/L. Poor attachment of sulphate-reducing bacteria was observed in all experiments. The biomass concentration did not exceed 1.2 g/L, despite the presence of 50 g/L of pumice. The reason for this phenomenon remains to be understood. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 807-814, 1997.     

High-rate ferric sulfate generation by a Leptospirillum ferriphilum-dominated biofilm and the role of jarosite in biomass retention in a fluidized-bed reactor

The aims of this work were to develop a high-rate fluidized-bed bioprocess for ferric sulfate production, to characterize biomass retention, and to determine the phylogeny of the enrichment culture. After 7 months of continuous enrichment and air aeration at 37 degrees C, the iron oxidation rate of 8.2 g Fe(2+) L(-1)h(-1) (4.5.10(-12) g Fe(2+) cell(-1) h(-1)) was obtained at a hydraulic retention time (HRT) of 0.6 h. However, oxygen supply became the rate-limiting factor. With gas mixture (99.5% O(2)/0.5% CO(2) (vol/vol)) aeration and HRT of 0.2 h, the iron oxidation rate was 26.4 g Fe(2+) L(-1)h(-1) (1.0.10(-11) g Fe(2+) cell(-1) h(-1)). Leptospirillum sp. was predominant in the mesophilic fluidized-bed reactor (FBR) enrichment culture as determined by fluorescent in situ hybridization, while Acidithiobacillus ferrooxidans was not detected. Denaturing gradient gel electrophoresis (DGGE) of the amplified partial 16S rDNA showed only three bands, indicating a simple microbial community. DGGE fragment excision and sequencing showed that the populations were related to L. ferriphilum (100% similarity in sequence) and possibly to the genus Ferroplasma (96% similarity to F. acidiphilum). Jarosite precipitates accumulated on the top of the activated carbon biomass carrier material, increasing the rate of iron oxidation. The activated carbon carrier material, jarosite precipitates, and reactor liquid contained 59% (or 3.71.10(9) cells g(-1)), 31% (or 3.12.10(10) cells g(-1)) and 10% (or 1.24.10(8) cells mL(-1)) of the total FBR microbes, respectively, demonstrating that the jarosite precipitates played an important role in the FBR biomass retention.     

Biological sulfuric acid transformation: Reactor design and process optimization

As an alternative to the current disposal technologies for waste sulfuric acid, a new combination of recycling processes was developed. The strong acid (H(2)SO(4)) is biologically converted with the weak acid (CH(3)COOH) into two volatile weak acids (H(2)S, H(2)CO(3)) by sulfate-reducing bacteria. The transformation is possible without prior neutralization of the sulfuric acid. The microbially mediated transformation can be followed by physiochemical processes for the further conversion of the H(2)S.The reduction of sulfate to H(2)S is carried out under carbon-limited conditions at pH 7.5 to 8.5. A fixed-bed biofilm column reactor is used in conjunction with a separate gas-stripping column which was installed in the recycle stream. Sulfate, total sulfide, and the carbon substrate (in most cases acetate) were determined quantitatively. H(2)S and CO(2) are continually removed by stripping with N(2). Optimal removal is achieved under pH conditions which are adjusted to values below the pK(a)-values of the acids. The H(2)S concentration in the stripped gas was 2% to 8% (v/v) if H(2)SO(4) and CH(3)COOH are fed to the recycle stream just before the stripping column.Microbiol conversion rates of 65 g of sulfate reduced per liter of bioreactor volume per day are achieved and bacterial conversion efficiencies for sulfate of more than 95% can be maintained if the concentration of undissociated H(2)S is kept below 40 to 50 mg/L. Porous glass spheres, lava beads, and polyurethane pellets are useful matrices for the attachment of the bacterial biomass. Theoretical aspects and the dependence of the overall conversion performance on selected process parameters are illustrated in the Appendix to this article. (c) 1993 John Wiley & Sons, Inc.     

The effect of hydroxylamine on the activity and aggregate structure of autotrophic nitrifying bioreactor cultures

Addition of hydroxylamine (NH2OH) to autotrophic biomass in nitrifying bioreactors affected the activity, physical structure, and microbial ecology of nitrifying aggregates. When NH2OH is added to nitrifying cultures in 6-h batch experiments, the initial NH3-N uptake rates were physiologically accelerated by a factor of 1.4-13. NH2OH addition caused a 20-40% decrease in the median aggregate size, broadened the shape of the aggregate size distribution by up to 230%, and caused some of the microcolonies to appear slightly more dispersed. Longer term NH2OH addition in fed batch bioreactors decreased the median aggregate size, broadened the aggregate size distribution, and decreased NH3-N removal from >90% to values ranging between 75% and 17%. This altered performance is explained by quantitative fluorescence in situ hybridization (FISH) results that show inhibition of nitrifying populations, and by qPCR results showing that the copy numbers of amoA and nxrA genes gradually decreased by up to an order-of-magnitude. Longer term NH2OH addition damaged the active biomass. This research clarifies the effect of NH2OH on nitrification and demonstrates the need to incorporate NH2OH-related dynamics of the nitrifying biomass into mathematical models, accounting for both ecophysiological and structural responses.     

pH regulation of alkaline wastewater with carbon dioxide: a case study of treatment of brewery wastewater in UASB reactor coupled with absorber

Studies were carried out with carbon dioxide absorber (CA) to evaluate the usage of carbon dioxide (CO(2)) in the biogas as an acidifying agent by Up-flow Anaerobic Sludge Blanket (UASB) reactor. Investigation on the 5l absorber revealed that ratio of brewery wastewater (BW) flow rate to biogas flow rate of 4.6-5.2 was optimum for minimum consumption of CO(2) for acidification. The acidified BW after the absorber was treated in UASB reactor with optimum organic loading rate (OLR) of 23.1 kg COD/m(3)/day and hydraulic retention time (HRT) of 2h. UASB reactor exhibited good performance with respect to reduction of chemical oxygen demand (COD) and methane yield. The implications of the present study on the full scale anaerobic reactor of medium scale brewery revealed that sufficient cost savings could be made if CO(2) in the biogas or CO(2) that was being wasted (let out to the atmosphere) can be used instead of sulfuric acid (H(2)SO(4)) for pH control.     

Effect of molybdate on methanogenic and sulfidogenic activity of biomass

The effect of molybdate, a sulfate analog, on the total methanogenic activity (TMA) and total sulfidogenic activity (TSA) of biomass metabolizing synthetic sucrose based substrate containing sulfate was investigated in batch assays. In Phase I of the study, TMA and TSA were assessed twice for four feed changes at a chemical oxygen demand to sulfate (COD/SO(4)(2-)) ratio of 3.5. In Phase II, long-term experiments were conducted for 10-13 feed changes with varying chemical oxygen demand (COD) concentration, sulfate concentration, COD/SO(4)(2-) ratio, molybdate dose and biomass with different growth histories. Assays with 3mM molybdate showed TSA inhibition over 85%. Dose dependency was observed for sulfate concentration, COD/SO(4)(2-) ratio, and biomass history. The minimum concentration that gave over 93% TSA inhibition was 0.25 mM. However, intermediate concentrations of molybdate inhibited methane producing bacteria (MPB) activity. TMA stimulation was observed at 0.75-2.0 mM molybdate.     

Mass transfer limitation of sulfate in methanogenic aggregates

The role of mass transfer limitation of sulfate as a factor governing the competition between sulfate reducing and methane producing bacteria in methanogenic aggregates was theoretically evaluated by the calculation of steady-state sulfate microprofiles using a reference set of parameters obtained from the literature. The shooting method was used as a numerical technique for solving the mathematical model. The effect of the parameters on mass transport limitation was tested by varying each reference value of the parameters with a factor of 3. Sulfate limitation within granules prevailed at moderate (0.1 kg m(-3)) and low sulfate concentrations in the bulk liquid, at high maximum sulfate utilization rates (3.73 x 10(-5) kg SO(4) (2-) kg(-1) VSS S(-1) or biomass concentrations (40 KG VSS m(-3)), and in large aggregates (radius of 7.5 10(-4) m). The effective diffusion coefficient of sulfate and the affinity constant were less determinative for the penetration depth of sulfate within a granule. (c) 1994 John Wiley & Sons, Inc.     

Application of molecular techniques to evaluate the methanogenic archaea and anaerobic bacteria in the presence of oxygen with different COD:sulfate ratios in a UASB reactor

In this paper, the microbial characteristics of the granular sludge in the presence of oxygen (3.0+/-0.7mgO(2)l(-1)) were analyzed using molecular biology techniques. The granules were provided by an upflow anaerobic sludge blanket (UASB) operated over 469 days and fed with synthetic substrate. Ethanol and sulfate were added to obtain different COD/SO(4)(2-) ratios (3.0, 2.0, and 1.6). The results of fluorescent in situ hybridization (FISH) analyses showed that archaeal cells, detected by the ARC915 probe, accounted for 77%, 84%, and 75% in the COD/SO(4)(2-) ratios (3.0, 2.0, and 1.6, respectively). Methanosaeta sp. was the predominant acetoclastic archaea observed by optical microscopy and FISH analyses, and confirmed by sequencing of the excised bands of the DGGE gel with a similarity of 96%. The sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris (similarity of 99%) was verified by sequencing of the DGGE band. Others identified microorganism were similar to Shewanella sp. and Desulfitobacterium hafniense, with similarities of 95% and 99%, respectively. These results confirmed that the presence of oxygen did not severely affect the metabolism of microorganisms that are commonly considered strictly anaerobic. We obtained mean efficiencies of organic matter conversion and sulfate reducing higher than 74%.     

Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor

The microalga incorporated photobioreactor is a highly efficient biological system for converting CO2 into biomass. Using microalgal photobioreactor as CO2 mitigation system is a practical approach for elimination of waste gas from the CO2 emission. In this study, the marine microalga Chlorella sp. was cultured in a photobioreactor to assess biomass, lipid productivity and CO2 reduction. We also determined the effects of cell density and CO2 concentration on the growth of Chlorella sp. During an 8-day interval cultures in the semicontinuous cultivation, the specific growth rate and biomass of Chlorella sp. cultures in the conditions aerated 2-15% CO2 were 0.58-0.66 d(-1) and 0.76-0.87 gL(-1), respectively. At CO2 concentrations of 2%, 5%, 10% and 15%, the rate of CO2 reduction was 0.261, 0.316, 0.466 and 0.573 gh(-1), and efficiency of CO2 removal was 58%, 27%, 20% and 16%, respectively. The efficiency of CO2 removal was similar in the single photobioreactor and in the six-parallel photobioreactor. However, CO2 reduction, production of biomass, and production of lipid were six times greater in the six-parallel photobioreactor than those in the single photobioreactor. In conclusion, inhibition of microalgal growth cultured in the system with high CO2 (10-15%) aeration could be overcome via a high-density culture of microalgal inoculum that was adapted to 2% CO2. Moreover, biological reduction of CO2 in the established system could be parallely increased using the photobioreactor consisting of multiple units.     

Microalgal biomass production and on-site bioremediation of carbon dioxide, nitrogen oxide and sulfur dioxide from flue gas using Chlorella sp. cultures

The growth and on-site bioremediation potential of an isolated thermal- and CO?-tolerant mutant strain, Chlorella sp. MTF-7, were investigated. The Chlorella sp. MTF-7 cultures were directly aerated with the flue gas generated from coke oven of a steel plant. The biomass concentration, growth rate and lipid content of Chlorella sp. MTF-7 cultured in an outdoor 50-L photobioreactor for 6 days was 2.87 g L?1 (with an initial culture biomass concentration of 0.75 g L?1), 0.52 g L?1 d?1 and 25.2%, respectively. By the operation with intermittent flue gas aeration in a double-set photobioreactor system, average efficiency of CO? removal from the flue gas could reach to 60%, and NO and SO? removal efficiency was maintained at approximately 70% and 50%, respectively. Our results demonstrate that flue gas from coke oven could be directly introduced into Chlorella sp. MTF-7 cultures to potentially produce algal biomass and efficiently capture CO?, NO and SO? from flue gas.     

Effect of aeration and carbon dioxide on cell morphology of Candida utilis

The effect of CO2 availability on cell size, shape, and aggregation in continuous cultures of Candida utilis was studied in minimal medium with glucose or ethanol as the sole carbon and energy source. Enrichment with CO2 was achieved (i) by using the substrate with more C atoms, (ii) by using pure oxygen and thus decreasing aeration intensity at the same dissolved-oxygen concentration, or (iii) by adding CO2 to the aeration gas. The cells were always of yeast shape, and no filaments were formed. In cultures with a biomass concentration above 6 g (dry weight) per liter, no cell aggregates were observed. In cultures with a lower biomass, the daughter cells failed to separate from the parent cells and formed aggregates with thickened walls. The average cell number per aggregate was found to be higher, and the average protoplast volume lower, under conditions of probable CO2 limitation. Simultaneously, the ratio of total dry weight to wet weight of protoplasts was considerably higher, indicating an increased share of wall or extracellular material. The possible effect of the observed morphological changes for maintaining a suitable concentration gradient of CO2 around the cell is discussed.     

Effect of aeration and carbon dioxide on cell morphology of Candida utilis.

The effect of CO2 availability on cell size, shape, and aggregation in continuous cultures of Candida utilis was studied in minimal medium with glucose or ethanol as the sole carbon and energy source. Enrichment with CO2 was achieved (i) by using the substrate with more C atoms, (ii) by using pure oxygen and thus decreasing aeration intensity at the same dissolved-oxygen concentration, or (iii) by adding CO2 to the aeration gas. The cells were always of yeast shape, and no filaments were formed. In cultures with a biomass concentration above 6 g (dry weight) per liter, no cell aggregates were observed. In cultures with a lower biomass, the daughter cells failed to separate from the parent cells and formed aggregates with thickened walls. The average cell number per aggregate was found to be higher, and the average protoplast volume lower, under conditions of probable CO2 limitation. Simultaneously, the ratio of total dry weight to wet weight of protoplasts was considerably higher, indicating an increased share of wall or extracellular material. The possible effect of the observed morphological changes for maintaining a suitable concentration gradient of CO2 around the cell is discussed.     

大气CO2浓度升高对绿豆叶片光合作用及叶绿素荧光参数的影响

利用FACE系统在大田条件下通过盆栽试验研究了大气CO2浓度升高[CO2浓度平均为(550+60) μmol·mo1-1]对绿豆叶片光合生理和叶绿素荧光参数的影响.结果表明:与对照[ CO2浓度平均为(389+40) μmol·mol-1左右]相比,大气CO2浓度升高使花荚期绿豆叶片净光合速率(Pn)和胞间CO2浓度(Ci)分别升高11.7％和9.8％,气孔导度(Gs)和蒸腾速率(Tr)分别下降32.0％和24.6％,水分利用效率(WUE)提高83.5％;在蕾期,CO2浓度升高对绿豆叶片叶绿素初始荧光(Fo)、最大荧光(Fm)、可变荧光(Fv)、Fv/Fm和Fv/Fo没有显著影响;在鼓粒期,CO2浓度升高使绿豆叶片Fo增加19.1％,Fm和Fv分别下降9.0％和14.3％,Fv/Fo和Fv/Fm分别下降25.8％和6.2％.表明大气CO2浓度升高可能使绿豆生长后期光系统Ⅱ反应中心结构受到破坏,叶片的光合能力下降.     

Energetic aspects of CO oxidation in desulfovibrio desulfuricans

In cell suspension of Desulfovibrio desulfuricans B-1388, oxidation of CO as the only energy source is associated with reduction of SO42-. After a 2-h incubation of cells in 8% CO, 81% of the gas is converted. Oxidation of 1 mole CO results in formation of 0.23 mole H2S. Intracellular ATP content increases from 2.5 (control) to 8.3 nmoles/mg (during CO conversion). Dinitrophenol inhibits sulfate reduction and CO oxidation. CO dehydrogenase was detected in cytoplasmic and membrane cell fractions (59 and 34%, respectively).     

The effect of precursor structures on the action of glucosaminyl 3-O-sulfotransferase-1 and the biosynthesis of anticoagulant heparan sulfate

To understand how 2-O-sulfation of uronic acid residues influences the biosynthesis of anticoagulant heparan sulfate, the cDNA encoding glucosaminyl 3-O-sulfotransferase-1 (3-OST-1) was introduced into wild-type Chinese hamster ovary cells and mutant pgsF-17 cells, which are defective in 2-O-sulfation. 3-OST-1-transduced cells gained the ability to bind to antithrombin. Structural analysis of the heparan sulfate chains showed that 3-OST-1 generates sequences containing GlcUA-GlcN(SO(3))3(SO(3)) and GlcUA-GlcN(SO(3))3(SO(3))6(SO(3)) in both wild-type and mutant cells. In addition, IdoUA-GlcN(SO(3))3(SO(3)) and IdoUA-GlcN(SO(3))3(SO(3))6(SO(3)) accumulate in the mutant chain. These disaccharides were also observed by tagging [6-(3)H]GlcN-labeled pgsF-17 heparan sulfate in vitro with [(35)S]PAPs and purified 3-OST-1. Heparan sulfate derived from the transduced mutant also had approximately 2-fold higher affinity for antithrombin than heparan sulfate derived from the transduced wild-type cells, and it inactivated factor Xa more efficiently. This study demonstrates for the first time that (i) 3-O-sulfation by 3-OST-1 can occur independently of the 2-O-sulfation of uronic acids, (ii) 2-O-sulfation usually occurs before 3-O-sulfation, (iii) 2-O-sulfation blocks the action of 3-OST-1 at glucosamine residues located to the reducing side of IdoUA units, and (iv) that alternative antithrombin-binding structures can be made in the absence of 2-O-sulfation.     

Thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor

Sulfate reduction outcompeted methanogenesis at 65 degrees C and pH 7.5 in methanol and sulfate-fed expanded granular sludge bed reactors operated at hydraulic retention times (HRT) of 14 and 3.5 h, both under methanol-limiting and methanol-overloading conditions. After 100 and 50 days for the reactors operated at 14 and 3.5 h, respectively, sulfide production accounted for 80% of the methanol-COD consumed by the sludge. The specific methanogenic activity on methanol of the sludge from a reactor operated at HRTs of down to 3.5 h for a period of 4 months gradually decreased from 0. 83 gCOD. gVSS(-1). day(-1) at the start to a value of less than 0.05 gCOD. gVSS(-1). day(-1), showing that the relative number of methanogens decreased and eventually became very low. By contrast, the increase of the specific sulfidogenic activity of sludge from 0. 22 gCOD. gVSS(-1). day(-1) to a final value of 1.05 gCOD. gVSS(-1). day(-1) showed that sulfate reducing bacteria were enriched. Methanol degradation by a methanogenic culture obtained from a reactor by serial dilution of the sludge was inhibited in the presence of vancomycin, indicating that methanogenesis directly from methanol was not important. H(2)/CO(2) and formate, but not acetate, were degraded to methane in the presence of vancomycin. These results indicated that methanol degradation to methane occurs via the intermediates H(2)/CO(2) and formate. The high and low specific methanogenic activity of sludge on H(2)/CO(2) and formate, respectively, indicated that the former substrate probably acts as the main electron donor for the methanogens during methanol degradation. As sulfate reduction in the sludge was also strongly supported by hydrogen, competition between sulfate reducing bacteria and methanogens in the sludge seemed to be mainly for this substrate. Sulfate elimination rates of up to 15 gSO(4)(2-)/L per day were achieved in the reactors. Biomass retention limited the sulfate elimination rate.     

Sulfide oxidation at halo-alkaline conditions in a fed-batch bioreactor

A biotechnological process is described to remove hydrogen sulfide (H(2)S) from high-pressure natural gas and sour gases produced in the petrochemical industry. The process operates at halo-alkaline conditions and combines an aerobic sulfide-oxidizing reactor with an anaerobic sulfate (SO(4) (2-)) and thiosulfate (S(2)O(3) (2-)) reducing reactor. The feasibility of biological H(2)S oxidation at pH around 10 and total sodium concentration of 2 mol L(-1) was studied in gas-lift bioreactors, using halo-alkaliphilic sulfur-oxidizing bacteria (HA-SOB). Reactor operation at different oxygen to sulfide (O(2):H(2)S) supply ratios resulted in a stable low redox potential that was directly related with the polysulfide (S(x) (2-)) and total sulfide concentration in the bioreactor. Selectivity for SO(4) (2-) formation decreased with increasing S(x) (2-) and total sulfide concentrations. At total sulfide concentrations above 0.25 mmol L(-1), selectivity for SO(4) (2-) formation approached zero and the end products of H(2)S oxidation were elemental sulfur (S(0)) and S(2)O(3) (2-). Maximum selectivity for S(0) formation (83.3+/-0.7%) during stable reactor operation was obtained at a molar O(2):H(2)S supply ratio of 0.65. Under these conditions, intermediary S(x) (2-) plays a major role in the process. Instead of dissolved sulfide (HS(-)), S(x) (2-) seemed to be the most important electron donor for HA-SOB under S(0) producing conditions. In addition, abiotic oxidation of S(x) (2-) was the main cause of undesirable formation of S(2)O(3) (2-). The observed biomass growth yield under SO(4) (2-) producing conditions was 0.86 g N mol(-1) H(2)S. When selectivity for SO(4) (2-) formation was below 5%, almost no biomass growth was observed.     

Chloride-stimulated sulfate efflux in Ehrlich ascites tumor cells: evidence for 1:1 coupling

The kinetics of Cl-SO4-(2) exchange in Ehrlich ascites tumor cells was investigated in an attempt to determine the stoichiometry of this process. When tumor cells, equilibrated in Cl--free, 25 mM SO4-(2) medium are placed in SO4-(2)-free, 25 mm Cl-medium, both the net amount and rate of Cl-uptake far exceeds SO4-(2) loss.. Addition of the anion transport inhibitor SITS (4-acetamido-4,-isothiocyano-stilbene-2,2'-disulfonic acid) greatly reduces sulfate efflux (97%), but has no measurable effect on chloride uptake. Addition of furosemide, a Cl-transport inhibitor, reduces chloride uptake 94% but is without effect on sulfate efflux. These findings suggest that a chloride permeability pathway exists distinct from that utilized by SO4-(2). SITS, when added to furosemide treated cells, further reduces chloride uptake as well as inhibiting sulfate efflux, and under these experimental conditions, a linear relationship exists between SITS-sensitive, net chloride uptake and sulfate loss. The slope of this line is 1.05 (correlation coefficient = 0.996) which suggests the stoichiometry of Cl-SO4-(2) exchange is 1:1. Assuming a 1:1 stoichiometry, measurement of the initial chloride influx and initial sulfate efflux indicate that 92% of net chloride uptake is independent of sulfate efflux. Taken altogether, these results support the contention that the tumor cell possesses a permeability pathway which facilitates the exchange of one sulfate for one chloride. Under conditions where anion transport is not inhibited, this coupling is obscured by a second and quantitatively more important pathway for chloride uptake. This pathway is SITS-insensitive, although partially inhibited by furosemide.     

Culture of photomixotrophic soybean and pine in a modified fermentor using a novel impeller

Photomixotrophic suspensions of Glycine max (soybean) and Pinus elliottii (slash pine) have been successfully cultured in a hybrid stirred tank photobioreactor using a novel cell-lift impeller. A cell-lift impeller exhibited cell viabilities over 90% and an average cell aggregate size of 1.0 mm or less. Flat-bladed turbines produced equivalent biomass to the cell-lift impeller, but cell viability was reduced (85%) and cell aggregate size increased (3-5 mm diameter). Maximum fresh weights of 82 g L(-1) (soybean) and 52 g L(-1) (slash pine) were achieved in 15 days using continuous lighting (90-100 muE m(-2) s(-1)) and supplemental 2% CO(2) inlet gas. Maximum biomass was achieved using an impeller speed of 60 rpm with air-flow rate of 0.2 vvm for the cell-lift impeller and the pair of flat bladed turbines. The lag and early exponential phases were characterized by (1) rapid hydrolysis of sucrose followed by preferential use of glucose and (2) a reduction in chlorophyll levels. Carbon dioxide (2%-5%) was an essential nutrient for photomixotrophic cell culture in the bioreactors.     

Growth and exopolysaccharide production during free and immobilized cell chemostat culture of Lactobacillus rhamnosus RW-9595M

AIMS: Biomass and exopolysaccharide (EPS) production were studied during chemostat cultures in whey permeate medium with Lactobacillus rhamnosus RW-9595M-free cells and cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: A continuous culture with free cells was conducted for 9 days at dilution rates (D) between 0.3 and 0.8 h(-1) in yeast extract (YE)/mineral supplemented whey permeate. Maximum EPS production (1808 mg l(-1)) and volumetric productivity (542.6 mg l(-1) h(-1)) were obtained for a low D of 0.3 h(-1). A continuous fermentation in a two-stage bioreactor system, composed of a first stage with immobilized cells and a second stage inoculated with free cells produced in the first reactor, was carried out for 32 days. The influence of YE concentration, temperature and dilution rate, and their interactions on biomass, EPS and lactic acid production was investigated. A statistically significant model was found only for lactic acid production. Marked cell morphological and physiological changes led to the formation of very large cell-containing aggregates and a low mean soluble EPS production (138 mg l(-1)). Aggregate volumetric productivity of the two-stage system varied between 5.7 and 49.5 g l(-1) h(-1) for different fermentation conditions and times. Aggregates contained a very high biomass concentration, estimated at 74% of aggregate dry weight by nitrogen analysis and 4.3 x 10(12) CFU g(-1) by a DNA extraction method and a high nonsoluble polysaccharide content (14.2%). At age 24 days, insoluble EPS concentration and volumetric productivity were 1250 mg l(-1) and 2240 mg l(-1) h(-1) respectively. The physiological changes were shown to be reversible when cells were incubated during three successive batch cultures. CONCLUSIONS: EPS production and volumetric productivity during continuous free-cell chemostat cultures with L. rhamnosus RW-9595M are among the highest values reported for lactobacilli in literature. Immobilization and continuous culture resulted in low soluble EPS production and large morphological and physiological changes of L. rhamnosus RW-9595M, with formation of macroscopical aggregates mainly composed of biomass and nonsoluble EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on continuous EPS production by immobilized LAB. Immobilization and culture time-induced cell aggregation and could be used to produce new synbiotic products with very high viable cell and EPS concentrations.