万古霉素发酵废渣固态发酵生产单细胞蛋白的研究

以万古霉素发酵废渣为唯一培养基,对固态发酵生产单细胞蛋白(SCP)进行了研究.经过筛选得到了一株白地霉HCCB 2267,它能以万古霉素发酵废渣为唯一底物生长.通过菌株驯化和培养条件优化,固态发酵产生的SCP可迭9.42×108个/g干重,其粗蛋白含量达47.24%,氨基酸总量从30.60%提高到46.28%.其优化的培养条件是:培养基起始含水量60%,起始pH 5.0～6.0,接种量15%,培养温度28～30℃,培养时间72h等.此外,筛选到的菌株对废渣中残存的菌丝体有降解作用.     

Fed-batch and continuous culture ofPhaffia rhodozyma (Xanthophyllomyces dendrorhous)

The basidiomycetous yeastPhaffia rhodozyma was grown in batch, fed-batch and continuous culture, and some parameters governing growth and total carotenoid production were determined.     

Pulse velocities in cylindrical,tapered and curved anisotropic elastic arteries

Modified Moens-Korteweg formulae have been developed for the pulse velocities in anisotropic elastic arteries of varying geometries. In particular, cylindrical straight, tapered and curved tubes have been considered. Numerical results indicate that the cylindrical tube formula can be applied in all cases and that the circumferential elastic modulus is the dominant elastic parameter.     

L-异亮氨酸产生菌XQ-4在连续培养中的动力学特性

以黄色短杆菌（Brevibacterium flavum)ATCC14067诱变选育获得的L-异亮氨酸高产菌XQ－4（AHV^rSuc^gSG^rEth^rα-AB^rIleHx^r）在连续培养中进行动力学特性研究,以葡萄糖为限制性底物时,XQ－4菌株的生长符合Monod方程,其最大比生长速率μmax=0.265h^-1,饱和常数Ka=0.789g/L.XQ-4菌株L-异亮氨酸发酵时菌体最大实际转化率Yx=0.499g/g,产物最大实际转化率Yp=0.379g/g。     

Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (K_m 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO₄³⁻-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F⁻, PO₄³⁻) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.     

Deacetylation of cephalosporins in batch and continuous reactors

We wish to report here the principal observations and conclusions concerning the deacetylation of three substrates, 7-ACA¹), cephalosporin C and 2-methoxyethyl acetate by the immobilized esterase from B. subtilis (Konecny and Voser, 1977) in three types of reactors: A stirred tank and recirculation reactor, both operated in the batch mode under pH-stat conditions, and a continuous packed bed, operated with excess buffer in the feed. The results, formulated in terms of simple empirical equations, are discussed in relation to the choice of carriers, reactor efficiency, scale up and other factors governing the choice of reactor. Certain general considerations apply to all reactions which generate acid and involve a catalyst whose activity and life are adversely effected by low pH.     

Studies on the stability of solvent production by Clostridium acetobutylicum in continuous culture

Various methods of continuous flow culture of Clostridium acetobutylicum NCIB 8052 were investigated, with the aim of obtaining prolonged production of acetone and butanol. In ammonia-limited chemostat culture, maximal concentrations of solvents were obtained at pH 5–5 at a relatively high biomass concentration of 1.3–2.0 g/1 dry weight maintained at a dilution rate of 0.06/h. Similar dependence of solvent production on the sustenance of a relatively high cell density was observed in magnesium- or phosphate-limited chemostat cultures. Solvent production was always transient, however, with a shift to production of only acetic and butyric acids being observed after 4–16 volume changes. Longer term solvent production was obtainable under conditions of glucose limitation but the solvent yield was low. Cultivation in a pH-auxostat permitted solvent production in reasonably high yield over at least 70 volume changes with no signs of culture degeneration. Although none of the continuous flow cultures achieved a true steady state, we conclude that turbidostat or pH-auxostat culture are the methods of choice for continuous solvent production by Cl. acetobutylicum NCIB 8052.     

Enrichment of linear alkylbenzenesulphonate (LAS) degrading bacteria in continuous culture

Enrichment experiments were carried out in continuous-flow units using a mineral medium with commercial linear alkylbenzenesulphonate (LAS) as the limiting carbon- and energy-source. The mixed bacterial culture originating from the waste water of a detergent plant consisted of five strains belonging to the genus Pseudomonas and two strains each of the genera Achromobacter and Acinetobacter. The cultivation conditions corresponding to dilution rates of 0.025-0.1 h^-1 and LAS concentrations of 20–50 mg/1 were examined. During the experiments the composition of mixed cultures and the kinetics of LAS biodegradation were followed. Continuous-flow enrichment experiments resulted in the selection of six bacterial cultures with different compositions of individual species and capability to utilize LAS. From the original seven strains at lower dilution rates (0.025 and 0.05 h^-1) six were selected, excluding Pseudomonas sp. 3, while at the highest dilution rate (0.1/h^-1) five strains were selected after eliminating Pseudomonas sp. 5 and Achromobacter sp. 1. All enriched mixed cultures were more efficient in primary than in ultimate LAS degradation. Two of the culture strains were able to achieve primary LAS degradation ( Pseudomonas sp. 1 in mineral medium with LAS as the sole carbon- and energy-source and Acinetobacter sp. 3 in medium supplemented by yeast extract and nutrient broth).
None of the strains could degrade LAS completely, which indicates that many types of interactions based on combined metabolic attack as well as those based on provision of specific nutrients, may exist between culture members during the complete LAS bio-oxidation.     

泥炭固体发酵生产单细胞蛋白研究

对以泥炭为唯一碳源,固体发酵生产单细胞蛋白(SCP)进行了一系列的研究。选用酵母菌和黑曲霉进行混合发酵培养,考察影响单细胞蛋白生产的各个因素,如菌种接种量,培养基含水量,发酵时间,发酵温度,培养基外加氮源等。通过正交实验设计确定了优化的培养条件。即:菌种接种量为10%,培养基含水量为300%,28℃培养72 h,以蛋白胨为氮源。     

Hydrodynamic effects on cells in agitated tissue culture reactors

Tissue cells are known to be sensitive to mechanical stresses imposed on them by agitation in bioreactors. The amount of agitation provided in a microcarrier or suspension bioreactor should be only enough to provide an effective homogeneity. Three distinct flow regions can be identified in the reactor: bulk turbulent flow, bulk laminar flow, and boundary-layer flows. Possible mechanisms of cell damage are examined by analyzing the motion of microcarriers or free cells relative to the surrounding fluid, to each other, and to moving or stationary solid surfaces. The primary mechanisms of cell damage appear to result from (a) direct interaction between microcarriers and turbulent eddies, (b) collisions between microcarriers in turbulent flow, and (c) collisions against the impeller or other stationary surfaces. If the smallest eddies of turbulent flow are of the same size as the microcarrier beads, they may cause high shear stresses on the cells. Eddies the size of the average interbead spacing may cause bead-bead collisions which damage cells. The severity of the collisions increases when the eddies are also of the same size as the beads. Bead size and the interbead distance are virtually equal in typical microcarrier suspensions. Impeller collisions occur when the beads cannot avoid the impeller leading edge as it advances through the liquid. The implications of the results of this analysis on the design and operation of tissue culture bioreactors are also discussed.     

Pullulanase synthesis in Klebsiella (Aerobacter) aerogenes strains growing in continuous culture

1. Pullulanase synthesis was studied in 16 classified (N.C.I.B.) strains and in an industrial strain (R) of Klebsiella aerogenes grown in chemostats containing maltose as inducer and sole carbon source. 2. Maximum synthesis was associated with carbon-limited growth at a low dilution rate (about 0.2h(-1)). The enzyme remained firmly cell-bound and seemed to be located on the cell surface. 3. Three strains had high activity (R, N.C.I.B. 5938, 8017), twelve were intermediate, and two (N.C.I.B. 8153, 9146) had negligible activity but were inducible with pullulan. 4. Pullulan similarly induced low, but adequate, activity in the other strains in conditions (nutrient limitation other than carbon-limitation) in which pullulanase was otherwise very seriously repressed. Nevertheless, in carbon limitation pullulan induced no more enzyme than did maltose, maltotriose or oligosaccharide mixtures, and ;hyperactivity' never developed on protracted culture. 5. Cyclic AMP relieved the transient repression produced by adding glucose to maltose-limited cultures and a further change to glucose-limited conditions led to constitutive pullulanase synthesis. 6. Amylomaltase and alpha-glucosidase activities were also examined but in less detail. 7. The presence of pullulanase in maltose-limited growth is discussed, but no clear function can be assigned to it at present. The molar growth yields for all the strains were very similar, and no correlation was found between the overgrowth of one strain by another and pullulanase activity. Further, any function as a general branching enzyme in polysaccharide synthesis seems unlikely.     

SCP from pineapple (Ananas sativa Schutt)

Summary Pineapple cannery effluents are available nearly round the year. The effluents are rich in microbially utilizable nutrients and when supplemented with ammonia support good growth of Candida utilis and Hansenula sydowiorum.The latter strain produces 45.0 g/l, 21.9 g/l, 1.5%, and 6.4% of cell biomass, protein, DNA, and RNA respectively in batch fermentations of such a medium. Amino acid profile, digestibility, NPU, and actual feed experiments confirm the applicability of the product. Concomitantly, a 90% reduction in BOD₅ value of the effluent can be effected during this process which otherwise proves to be troublesome.     

Composition and nutritive value of single-cell protein (SCP)

In recent years there has been an intensification of efforts to make use of microbial fermentation for the production of nutrients and feeds containing protein. Therefore we have investigated some yeasts and a bacterium produced on unconventional nutrient substrates such as methanol, crude oil, n-alkanes and whey, comparing them with yeasts produced by normal techniques, using sulphite liquors, molasses and extract of barley-malt as a carbon source.The crude protein content of the dry matter of all the years was between 39 and 68% and that of the bacterium examined was about 82%. As regards the amounts of essential amino acids in SCP, the high concentration of lysine, with 6.6–8.4 g/16 g N in the yeasts, and 5.9 g/16 g N in the bacterium, deserves special mention. By contrast, the amount of amino acids containing sulphur in the yeasts examined is not satisfactory at 1.5–3.3 g/16 g N, whereas the amount in the bacterium was more suitable at 3.6 g/16 g N. Methionine and cystine are thus the first limiting amino acids. Also, the arginine content in some products might be inadequate for the growth of fowl.Besides the protein, the crude fat fraction has to be considered, firstly because of its considerable order of magnitude, and secondly because of its very variable composition.In an animal experiment, a satisfactory to good biological value of the protein of 61–80%, and an NPU of 55–76%, were found. By adding 0.2% DL-methionine to the dry feed, it was possible to improve considerably these criteria. On the basis of their very high lysine content, these materials have a good to very good value as supplements to our basic feeds for pigs.Metabolic trials with pigs yielded among other things, a protein digestibility of 93% for 2 yeasts, and 84% for the bacterium studied. In fattening trials with pigs, an equally good feeding effect was achieved through equal-protein replacement of fish meal and extracted soya bean meal by yeasts and the bacterium.For the feeding of calves, it is advisable at first, to replace only small amounts of the milk protein in the milk-substitute feed with SCP.     

Studies on mixed populations of human intestinal bacteria grown in single-stage and multistage continuous culture systems

Mixed intestinal bacteria were grown for 336 h in two identical single-stage chemostats at low growth rates in a carbohydrate-limited medium. Complex bacterial populations were maintained and anaerobes always outnumbered aerobes. The predominant organisms belonged to the genera Bacteroides, Bifidobacterium, Lactobacillus, Clostridium, Eubacterium, Propionbacterium, Peptococcus, and Peptostreptococcus. Bacteroides species predominated in both fermentors, particularly B. ovatus and B. thetaiotaomicron. A high degree of reproducibility of bacteriological and fermentation product data was obtained in these experiments. When gut contents were inoculated into a five-stage continuous culture system (retention time of 79 or 38 h) containing soya bran, the medium flow rate had little quantitative effect on the formation of acidic fermentation products; however, more oxidized fermentation acids were produced at the higher retention time. Diverse bacterial populations were maintained in every vessel at each flow rate. Bacteroides fragilis group organisms, especially B. ovatus, were numerically the most important. The viability of bacteria decreased through the system, especially at a retention time of 79 h, when the bacteria were growing under severely nutrient-limited conditions.     

Hyperosmotic stress-induced somatic embryogenesis and its continuous culture in Japanese honewort (Cryptotaenia japonica)

Japanese honewort (Cryptotaenia japonica) is consumed as a traditional vegetable and has medicinal applications. In Japan, C. japonica is mainly produced using hydroponic culture systems; however, damping-off is often caused by the adherence of pathogens to its seeds. Therefore, the use of sterile artificial seeds in hydroponic culture is likely to be effective for preventing disease. In this study, we established methods for stress-induced somatic embryogenesis and artificial seed production in Japanese honewort. Shoot apex explants from seedlings were treated with 0.7 M sucrose as a hyperosmotic stress for 3 or 6 weeks, and then transferred to stress-free conditions. Somatic embryos were formed after culture in stress-free conditions for 7 weeks. Stress-treated shoot apex explants that formed somatic embryos were cultured in Murashige and Skoog liquid medium with shaking. After 2 weeks of culture, approximately 800 somatic embryos were formed from each explant. Somatic embryos were formed continuously during 37 weeks under the same culture conditions. Thus, somatic embryogenesis was effectively induced in Japanese honewort via hyperosmotic stress, and embryogenic competence was maintained under stress- and phytohormone-free conditions. The somatic embryos produced by liquid culture were used to produce artificial seeds by enveloping the embryos in whipped alginate gel to avoid hypoxic conditions. The artificial seeds had a high germination rate (72%). This system is suitable for the sterile, highly productive hydroponic culture of Japanese honewort.     

发酵法开发甲醇蛋白的研究

通过大量实验从土壤中分离筛选出1株同化甲醇能力较强的菌株15357,该菌能以甲醇作为碳源进行单细胞蛋白的发酵.甲醇蛋白得率为41.5%,粗蛋白含量为80.9%,各类氨基酸齐全,8种主要氨基酸含量丰富.毒性试验证明是一种安全的饲料添加剂,喂养蛋鸡应用试验表明,可代替鱼粉,产蛋率有明显提高.