固定化光合细菌利用有机物产氢的研究

应用固定化细胞技术包埋荚膜红假单胞菌(Rhodopseudomonas capsulata)菌株386．研究在光照下利用有机物产氢的特性。实验观察到,光照培养120小时,悬浮培养物的产氢量为68．2ml·比产氢速率为104．1ml H2/g(生物量)·h;用琼脂包埋后．其产氢能力得到改善,产氢量和比产氢速率分别达到128．4ml和l 9s．8mlH2/g·h。该菌株除可利用苹果酸外,还可利用葡萄糖、乳酸、丙酸等基质高效地产氢。基质浓度只有控制在适当水平时,才具有较高的基质转化产氢效率。此外．菌体生物量、菌龄、培养液pH、光照强度、光照／黑暗时间比以及温度对产氢过程均有不同程度的影响。     

Enhancement of monoclonal antibody production by immobilized hybridoma cell culture with hyperosmolar medium

To determine the effect of hyperosmotic stress on the monoclonal antibody (MAb) production by calcium-alginate-immobilized S3H5/gamma2bA2 hybridoma cells, the osmolalities of medium in the MAb production stage were varied through the addition of NaCI. The specific MAb productivity (q(MAb)) of immobilized cells exposed to abrupt hyperosmotic stress (398 mOsm/kg) was increased by 55% when compared with that of immobilized cells in the control culture (286 mOsm/kg). Furthermore, this enhancement of q(MAb) was not transient. Abrupt increase in osmolality, however, inhibited cell growth, resulting in no increase in volumetric MAb productivity (r(MAb)). On the other hand, gradual increase in osmolality allowed further cell growth while maintaining the enhanced q(MAb) immobilized cells. The q(MAb) immobilized cells at 395 mOsm/kg was 0.661 +/- 0.019 mug/10(6) cells/h, which is almost identical to that of immobilized cells exposed to abrupt osmotic stress. Accordingly, the r(MAb) was increased by ca. 40% when compared with that in the control immobilized cell culture. This enhancement in i(MAb) of immobilized S3H5/gamma2bA2 hybridoma cells by applying gradual osmotic stress suggests the potential of using hyperosmolar medium in other perfusion culture systems for improved MAb production. (c) 1995 John Wiley & Sons, Inc.     

Hydrogen sulfide removal by immobilized Thiobacillus novellas on SiO2 in a fluidized bed reactor

The removal of hydrogen sulfide (H2S) from aqueous media was investigated using Thiobacillus novellas cells immobilized on a SiO2 carrier (biosand). The optimal growth conditions for the bacterial strain were 30 degrees C and initial pH of 7.0. The main product of hydrogen sulfide oxidation by T. novellus was identified as the sulfate ion. A removal efficiency of 98% was maintained in the three-phase fluidized-bed reactor, whereas the efficiency was reduced to 90% for the two-phase fluidized-bed reactor and 68% for the two-phase reactor without cells. The maximum gas removal capacity for the system was 254 g H2S/m3/h when the inlet H2S loading was 300 g/m3/h (1,500 ppm). Stable operation of the immobilized reactor was possible for 20 days with the inlet H2S concentration held to 1,100 ppm. The fluidized bed bioreactor appeared to be an effective means for controlling hydrogen sulfide emissions.     

Microbial hydrogen production with immobilized sewage sludge

Municipal sewage sludge was immobilized to produce hydrogen gas under anaerobic conditions. Cell immobilization was essentially achieved by gel entrapment approaches, which were physically or chemically modified by addition of activated carbon (AC), polyurethane (PU), and acrylic latex plus silicone (ALSC). The performance of hydrogen fermentation with a variety of immobilized-cell systems was assessed to identify the optimal type of immobilized cells for practical uses. With sucrose as the limiting carbon source, hydrogen production was more efficient with the immobilized-cell system than with the suspended-cell system, and in both cases the predominant soluble metabolites were butyric acid and acetic acid. Addition of activated carbon into alginate gel (denoted as CA/AC cells) enhanced the hydrogen production rate (v(H2)) and substrate-based yield (Y((H2)/sucrose)) by 70% and 52%, respectively, over the conventional alginate-immobilized cells. Further supplementation of polyurethane or acrylic latex/silicone increased the mechanical strength and operation stability of the immobilized cells but caused a decrease in the hydrogen production rate. Kinetic studies show that the dependence of specific hydrogen production rates on the concentration of limiting substrate (sucrose) can be described by Michaelis-Menten model with good agreement. The kinetic analysis suggests that CA/AC cells may contain higher concentration of active biocatalysts for hydrogen production, while PU and ALSC cells had better affinity to the substrate. Acclimation of the immobilized cells led to a remarkable enhancement in v(H2) with a 25-fold increase for CA/AC and ca. 10- to 15-fold increases for PU and ALSC cells. However, the ALSC cells were found to have better durability than PU and CA/AC cells as they allowed stable hydrogen production for over 24 repeated runs.     

Gastrointestinal and microbial responses to sulfate-supplemented drinking water in mice

There is increasing evidence that hydrogen sulfide (H2S), produced by intestinal sulfate-reducing bacteria (SRB), may be involved in the etiopathogenesis of chronic diseases such as ulcerative colitis and colorectal cancer. The activity of SRB, and thus H2S production, is likely determined by the availability of sulfur-containing compounds in the intestine. However, little is known about the impact of dietary or inorganic sulfate on intestinal sulfate and SRB-derived H2S concentrations. In this study, the effects of short-term (7 day) and long-term (1 year) inorganic sulfate supplementation of the drinking water on gastrointestinal (GI) sulfate and H2S concentrations (and thus activity of resident SRBs), and the density of large intestinal sulfomucin-containing goblet cells, were examined in C3H/HeJBir mice. Additionally, a PCR-denaturing gradient gel electrophoresis (DGGE)-based molecular ecology technique was used to examine the impact of sulfate-amended drinking water on microbial community structure throughout the GI tract. Average H2S concentrations ranged from 0.1 mM (stomach) to 1 mM (cecum). A sulfate reduction assay demonstrated in situ production of H2S throughout the GI tract, confirming the presence of SRB. However, H2S generation and concentrations were greatest in the cecum and colon. Sulfate supplementation of drinking water did not significantly increase intestinal sulfate or H2S concentrations, suggesting that inorganic sulfate is not an important modulator of intestinal H2S concentrations, although it altered the bacterial profiles of the stomach and distal colon of 1-year-old mice. This change in colonic bacterial profiles may reflect a corresponding increase in the density of sulfomucin-containing goblet cells in sulfate-supplemented compared with control mice.     

Enzymatic synthesis of formic acid from H2 and CO2 and production of hydrogen from formic acid

Whole cells of Alcaligenes eutrophus (as well as isolated P. oxalaticus formate dehydrogenase and A. eutrophus hydrogenase coupled via NAD(+) or methyl viologen) have been shown to produce H(2) from formic acid. Immobilization of the cells in kappacarrageenan gel greatly enhances their stability at room temperature. The rate of hydrogen production catalyzed by immobilized A. eutrophus has been studied as a function of the concentrations of the cells and formate and also pH. An inhibition by high concentrations of formate has been found. Immobilized cells were also capable of synthesizingformate from H(2) and bicarbonate. Yields of formate up to 30% have been obtained. The catalytic efficiency of immobilized A. eutrophus cells was compared with that of palladium adsorbed on activated carbon.     

Two-stage system for hydrogen production by immobilized cyanobacterium Gloeocapsa alpicola CALU 743

Previous studies showed that cell suspensions of unicellular nondiazotrophic cyanobacterium G. alpicola grown under nitrate-limiting conditions intensively produces H2 via fermentation of endogenous glycogen with hydrogen yield more then 90% of theoretical maximum (3.8 mol H2 per mol glucose). H2 production is realized by a Hox hydrogenase on the stages of NAD(P)H generation. Exploiting this property, the two-stage cyclic system for sustained hydrogen production was developed using a photobioreactor (PhBR) with G. alpicola immobilized on glass fiber TR-0.3. Immobilization of the cells on the matrix occurred during growth directly in PhBR operated in continuous mode; the density of culture immobilized achieved 37 g Chl alpha cm(-2). The first stage of the cycle was the photosynthetic incubations of G. alpicola in the flow of the culture medium, which contained limiting concentrations of nitrate for efficient glycogen accumulation and activation of hydrogenase. The second stage was the fermentation of glycogen, with H2 production realized in darkness with continuous Ar sparging and without medium flow. Standardization of optimal parameters for both stages provided a stable cyclic regime of the system: photosynthesis (24 hours)-fermentation (24 hours). The total amount of H2 evolved in one cycle was 957.6 mL L(-1)(matrix), and the overage rate of H2 production during the cycle (48 hours) was about 20 mL h(-1) L(-1)(matrix). Ten consequent cycles was carried out in this regime with reproducible H2 production, although PhBR with the same sample of immobilized culture was operated over a period of more then three months.     

产糖化酶黑曲霉固定化方法比较的研究

采用海藻酸钙凝胶电埋法、以沸石、多孔聚酯等材料为固定化载体的吸附法固定黑曲霉（Aspergillus niger AS3.4309)菌丝细胞,以游离菌丝体作为对照,进行发酵产糖化酶的比较,结果表明：以聚酯泡沫作为固定化载体吸附固定化菌丝细胞产糖化酶活力最高。在产糖化酶的发酵过程中,与游离菌丝体细胞相比,固定化黑曲霉持续产酶时间有一定程度的延长。     

Biosynthesis of gibberellic acid from milk permeate in repeated batch operation by a mutant Fusarium moniliforme cells immobilized on loofa sponge

Gibberellic acid (GA) production from milk permeate was studied by 28 mutants of Fusarium moniliforme, among which mutant gamma-14 was selected as the best producer. Experiments were carried out in shaker flasks and fermentative process was analyzed with free and immobilized cells. Immobilization of mutant gamma-14 cells onto loofa sponge discs was studied with respect to the optimization of the incubation temperature, initial pH, inoculum size (number of discs) and its reusability for GA production. Best yield of GA (2.40 gl(-1)) was recorded by immobilized cells under optimized cultural conditions (4 immobilized discs, 30 degrees C and pH 5). Data obtained during four reusable cycles showed high stability of GA production and reduction in the initiation time of acid production, resulting in higher levels of GA in shorter time duration. Immobilization of mutant gamma-14 cells onto loofa sponge discs, permitted repeated reuse under the specified fermentation conditions for GA production from milk permeate.     

Binding interactons of collagen I, laminin and fibronectin with immobilized Escherichia coli 0157:H7 using a surface plasmon resonance biosensor

Real-time interactions of collagen I, fibronectin, laminin, hyaluronic acid and chondroitin sulfate with immobilized Escherichia coli O157:H7 cells were studied with a surface plasmon resonance biosensor. Results showed that collagen I and laminin bound to the E.coli surface but fibronectin had very low binding while hyaluronic acid and chondroitin sulfate had no detectable interaction. Calcium ion inhibited laminin binding but enhanced collagen I binding. This research provides a model system to study the interactions of bacterial cells with extracellular matrix components. © Rapid Science Ltd. 1998     

Transforming growth factor beta 1 stimulates the production of basic fibroblast growth factor binding proteoglycans in Balb/c3T3 cells.

Basic fibroblast growth factor (bFGF) binds to cell surface receptors and to heparin sulfate proteoglycans. Heparan sulfate binding may limit bFGF degradation and be an obligatory step for bFGF cell interaction. Transforming growth factor-beta 1 (TGF-beta 1) is a potent regulator of proteoglycan production and composition. The possibility that TGF-beta 1 synergistically regulates bFGF activity by altering bFGF-proteoglycan interactions was investigated. TGF-beta 1 increased 125I-bFGF binding to the extracellular matrix (ECM) of Balb/c3T3 cells 2-4-fold by increasing the number of bFGF binding sites. Increased bFGF binding correlated with a 2-5-fold increase in the production of sulfated proteoglycans, including heparan sulfate proteoglycans. TGF-beta 1 selectively stimulated production of high molecular mass proteoglycans (190-300 kDa) in conditioned medium and stimulated all proteoglycans in ECM. 125I-bFGF bound to TGF-beta 1 induced proteoglycans immobilized onto cationic nylon filters. Furthermore, ECM isolated from TGF-beta 1-treated cells incorporated more mitogenically active bFGF than native ECM. The mitogenic potential of the ECM was significantly reduced by treatment with heparinase. These results suggest that the ability of TGF-beta 1 to stimulate binding of bFGF to ECM, increase ECM heparan sulfate proteoglycan, and potentiate the mitogenic activity of bFGF are linked. Thus one aspect of TGF-beta 1/bFGF synergy may involve modulation of the ECM.     

A comparative study of the formation of 2-keto-D-gluconic acid by free and immobilized cells of Pseudomonas putida

The effect of glucose, oxygen and 2-keto-D-gluconic acid (2KG) concentrations on the 2KG production by free and immobilized cells of Pseudomonas putida was studied. The effect of these factors was found to be similar in case of both free and immobilized cells, but the rate of the 2KG production by the free cells was a little higher as compared to the immobilized cells.     

Continuous production of glucose-6-phosphate by immobilized Achromobacter butyri cells

Summary Whole cells of Achromobacter butyri OUT 8004 having polyphosphate glucokinase activity were immobilized in polyacrylamide gel. The immobilized cells were activated by organic solvents, especially acetone. The immobilization resulted in increased stability of polyphosphate glucokinase. Continuous high yield production of G-6-P from glucose and metaphosphate was performed with an immobilized cell column, which had a half-life of approximately 20 days.Abbreviations G-6-P glucose-6-phosphate - G-1-P glucose-1-phosphate - Cation-S stearyl trimethyl ammonium chloride - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)-aminomethane; p-NPP, p-nitrophenyl phosphate - S.V. space velocity     

Cooperation of syndecan-2 and syndecan-4 among cell surface heparan sulfate proteoglycans in the actin cytoskeletal organization of Lewis lung carcinoma cells

Syndecan-2 cooperates with integrin alpha 5 beta 1 in cell adhesion to a fibronectin substratum and regulates actin cytoskeletal organization in an expression level-dependent manner; Lewis lung carcinoma-derived P29 cells with high expression form stress fibers, whereas the same tumor-derived low expressers, LM66-H11 cells, form cortex actin [Munesue, S., Kusano, Y., Oguri, K., Itano, N., Yoshitomi, Y., Nakanishi, H., Yamashina, I., and Okayama, M. (2002) BIOCHEM: J. 363, 201-209]. In this study we examined the participation of other cell surface heparan sulfate proteoglycans in this signaling. The two clones expressed syndecan-1, -2 and -4, and glypican-1 at similar levels except for syndecan-2. Treatment of cells with phosphatidylinositol-specific phospholipase C or immobilized anti-syndecan-1 antibodies demonstrated that neither glypican-1 nor syndecan-1 was involved in this signaling, indicating that individual cell surface heparan sulfate proteoglycans have functional specificity. Stimulation with immobilized anti-syndecan-2 or -4 antibodies induced stress fiber formation in P29 cells but not in LM66-H11 cells, despite the similar levels of syndecan-4 expression, suggesting that stress fiber formation required a threshold expression level of syndecan-2 acting downstream of syndecan-4. This was confirmed by cells in which syndecan-2 expression was artificially suppressed by antisense mRNA oligonucleotide treatment or elevated by cDNA transfection. This is the first report demonstrating that syndecan-2 and -4 cooperate in situ in actin cytoskeletal organization.     

Hydrogen sulfide is an endogenous potentiator of T cell activation

H(2)S is an endogenous signaling molecule that may act via protein sulfhydrylation to regulate various physiological functions. H(2)S is also a byproduct of dietary sulfate metabolism by gut bacteria. Inflammatory bowel diseases such as ulcerative colitis are associated with an increase in the colonization of the intestine by sulfate reducing bacteria along with an increase in H(2)S production. Consistent with its increased production, H(2)S is implicated as a mediator of ulcerative colitis both in its genesis or maintenance. As T cells are well established mediators of inflammatory bowel disease, we investigated the effect of H(2)S exposure on T cell activation. Using primary mouse T lymphocytes (CD3+), OT-II CD4+ T cells, and the human Jurkat T cell line, we show that physiological levels of H(2)S potentiate TCR-induced activation. Nanomolar levels of H(2)S (50-500 nM) enhance T cell activation assessed by CD69 expression, interleukin-2 expression, and CD25 levels. Exposure of T cells to H(2)S dose-dependently enhances TCR-stimulated proliferation with a maximum at 300 nM (30% increase, p < 0.01). Furthermore, activation increases the capacity of T cells to make H(2)S via increased expression of cystathionine γ-lyase and cystathionine β-synthase. Disrupting this response by silencing these H(2)S producing enzymes impairs T cell activation, and proliferation and can be rescued by the addition of 300 nM H(2)S. Thus, H(2)S represents a novel autocrine immunomodulatory molecule in T cells.     

Improvement of acid phosphatase production by immobilization of Humicola lutea mycelium in polyurethane sponge

Acid phosphatase production by the fungus Humicola lutea 120-5, immobilized in polyurethane sponge, was studied under semicontinuous shake flask fermentation and compared to the enzyme secretion by free cells. The effect of parameters such as the carrier content and the duration of the batch in repeated batch experiments on the phosphatase production half-life was investigated. The best results were obtained with 1.0 g of sponge cubes (about 1.0 cm per side) per culture flask using 72 h runs. In these conditions the half-life of enzyme production by immobilized biocatalyst was 15 sequential cycles (45 days) compared to three cycles (9 days) for the free mycelium. The maximal phosphatase titre registered in free cell fermentation was 2500 U/l (i.e. 100%), while the relative enzyme activity of the optimal immobilized system was over 100% during the whole half-life time of 45 days. Significant improvement (200–215%) in the yield was observed in one-third of this period or 15 days. The supernatant medium obtained at any stage of the repeated batch cultures did not contain free cells and, due to the low pH (3.0–3.5), the whole process was carried out without any bacterial contamination. In comparison with free cell fermentation, the significant improvement of the acid phosphatase production by polyurethane sponge-immobilized H. lutea mycelium as well as its operation stability was confirmed by scanning electron microscopy.     

固定化氧化亚铁硫杆菌培养条件对黄铁矾沉淀的影响

以聚乙烯醇-海藻酸钠复合材料为载体,Ca(NO3)2为交联剂对氧化亚铁硫杆菌进行包埋固定化。该固定化细胞的连续培养技术可以用于处理H2S、SO2,为了减少减少固定化细胞培养过程中带来许多不利效应的黄铁矾沉淀 (NH4Fe3(SO4)2(OH)6）,采取了改变初始pH值和目前普遍采用的9K培养基中的(NH4)2SO4浓度,K2HPO4浓度三种方法。结果显示：在三种方法中,降低(NH4)2SO4浓度是比较可行的一种方法,当(NH4)2SO4从3.0 g/L降低到0.5g/L,Fe2+氧化速率几乎没有受到影响,沉淀形成速率却减少了45%。在固定化细胞连续运行时,降低9K培养基中(NH4)2SO4的含量,当稀释率为0.4 h-1,运行时间为96 h,Fe2+氧化速率高达3.75 g/L.H,结果显示反应柱内沉淀明显减少,同时Fe2+氧化速率并没有明显变化。     

Application of immobilized hydrogenase for the detritiation of water

Detritiation of contaminated water is an essential part of nuclear power production. Most promising methods used for this process are based on catalyzed hydrogen isotope exchange reactions. It is proposed herein to replace the platinum catalysts which are currently used in industry with immobilized hydrogenase. Whole bacterial cells of Alcaligenes eutrophus immobilized in calcium alginate or κ-carrageenan gels were found to be efficient catalysts of the reaction of hydrogen–tritium (H–T) exchange in both a batch tank reactor and in a column. The dependence of the reaction rate on the amount of immobilized cells in the system, and on the concentration of the cells in the matrix, indicate that enzymatic H–T exchange is not controlled by diffusion. Immobilized A. eutrophus cells are enzymatically active over a wide range of pH, with a broad maximum from pH 6.0 to 8.0, and are quite resistant to inhibitors of hydrogenases such as O₂ and CO. Upon increasing the temperature from 4 to 37°C, the rate of hydrogenase-catalyzed H–T exchange increases by a factor of 5. From the standpoint of catalytic efficiency, 1 g of PtO₂ is approximately equivalent to 10 g of cells (wet weight). In contrast of platinum-based catalysts, bacterial hydrogenases (1) are potentially inexpensive; (2) can be readily available in bulk quantities; (3) are maximally active in liquid water.     

Removal of heavy metals from aqueous solution using Rhizopus delemar mycelia in free and polyurethane-bound form

This study assesses the ability of mycelia of Rhizopus delemar (both free and immobilized on polyurethane foam) to remove heavy metals from single-ion solutions as well as from a mixture of them. All experiments were conducted using 0.5-5 mm solutions of CuSO4 x 5H2O, CoCl2-6H2O and FeSO4 7H2O. Mycelia immobilized on polyurethane foam cells showed some times increase in uptake compared with that of free cells. Metal ions accumulation from a mixed solution was decreased slightly for cobalt and iron and considerable for copper ions. Heavy metal uptake was examined in the immobilized column experiments and more than 92% heavy metal removal (mg heavy metals removed/mg heavy metals added) from a mixed solution was achieved during the 5 cycles. During these experiments, the dry weight of the immobilized cells was decreased by only 2%. These results showed that immobilized mycelia of Rhizopus delemar can be used repeatedly for removal of heavy metals from aqueous solutions.     

Hydrogen production by photoreactive nanoporous latex coatings of nongrowing Rhodopseudomonas palustris CGA009

Nonuniform light distribution is a fundamental limitation to biological hydrogen production by phototrophic bacteria. Numerous light distribution designs and culture conditions have been developed to reduce self-shading and nonuniform reactivity within bioreactors. In this study, highly concentrated (2.0 x 108 CFU/muL formulation) nongrowing Rhodopseudomonas palustris CGA009 were immobilized in thin, nanoporous, latex coatings. The coatings were used to study hydrogen production in an argon atmosphere as a function of coating composition, thickness, and light intensity. These coatings can be generated aerobically or anaerobically and are more reactive than an equivalent number of suspended or settled cells. Rhodopseudomonas palustris latex coatings remained active after hydrated storage for greater than 3 months in the dark and over 1 year when stored at -80 degrees C. The initial hydrogen production rate of the microphotobioreactors containing 6.25 cm2, 58.4 mum thick Rps. palustris latex coatings illuminated by 34.1 PAR mumol photons m-2 s-1 was 6.3 mmol H2 m-2 h-1 and had a final yield of 0.55 mol H2 m-2 in 120 h. A dispersible latex blend has been developed for direct comparison of the specific activity of settled, suspended, and immobilized Rps. palustris.