Lipid Class and Fatty Acid Composition of the Protozoan Parasite of Oysters, Perkinsus marinus Cultivated in Two Different Media

The meront stage of the oyster protozoan parasite, Perkinsus marinus, cultivated in two media with different fatty acid profiles was analyzed for its fatty acid and lipid class composition. The composition of fatty acids in the prezoosporangium stage of the parasite as well as that of the host oyster were investigated. Although the lipid class composition of meronts was dominated by phospholipids and triacylglycerol, there was no triaclgycerol detected in either culture medium. Despite the difference in fatty acid composition of the two media, the fatty acid composition of meronts in each medium was dominated by 14:0, 16:0, 18:0, 18:1(n-9), 20: (n-9), 18:2(n-6) and 20:4(n-6), a profile that differed from its host. The quantities of total lipids and fatty acids in meronts increased as the number of meronts increased and far exceeded the initial amounts in the media and in the initial cell inoculum. The meronts harvested 25 d post-inoculation, had about 3 to 6 times higher total lipids and 4 to 13 times higher fatty acids than the amounts contained in the media. The fatty acid profiles of both prezoosporangia and oysters resembled each other and consisted primarily of 16:0, 20:4(n-6), 20:5(n-3), 22:2delta7,15, and 22:6(n-3). These results indicate that during meront proliferation, the parasite synthesizes certain fatty acids and lipid classes. For development from meront to prezoosporangium, the parasite may rely on its host for lipid resources.     

The effect of fluoride intake on the total lipid,cholesterol and vitamin E levels in a sera and liver of guinea pigs on high fat diet

Guinea pigs were raised on a diet containing 18 percent fat and were provided 25 or 0 ppm fluoride in the drinking water. Animals were sacrificed at the end of 0, 3, 6, 9 and 13 weeks on the dietary regimen and blood serum was analyzed for fluoride, total lipid, cholesterol and alpha tocopherol. The serum total lipid levels, cholesterol and alpha tocopherol levels were increased in the high fluoride group between 9 and 13 weeks. An increase in total lipid and alpha tocopherol levels was also observed in the livers of these animals. The increase in liver content of alpha tocopherol was proportional to the increase in total lipid content whereas the corresponding increase in serum alpha tocopherol content was significant even when the increase in total lipid was taken into account. The data suggest a specific effect of fluoride on the serum alpha tocopherol levels of the high fluoride animals.     

Effects of triclosan on growth, viability and fatty acid synthesis of the oyster protozoan parasite Perkinsus marinus

Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica, has severely impacted oyster populations from the Mid-Atlantic region to the Gulf of Mexico coast of North America for more than 30 yr. Although a chemotherapeutic treatment to reduce or eliminate P. marinus from infected oysters would be useful for research and hatchery operations, an effective and practical drug treatment does not currently exist. In this study, the antimicrobial drug triclosan 5-chloro-2-(2,4 dichlorophenoxy) phenol, a specific inhibitor of Fab1 (enoyl-acyl-carrier-protein reductase), an enzyme in the Type II class of fatty acid synthetases, was tested for its effects on viability, proliferation and fatty acid synthesis of in vitro-cultured P. marinus meronts. Treatment of P. marinus meront cell cultures with concentrations of > or = 2 microM triclosan at 28 degrees C (a temperature favorable for parasite proliferation) for up to 6 d stopped proliferation of the parasite. Treatment at > or = 5 microM at 28 degrees C greatly reduced the viability and fatty acid synthesis of meront cells. Oyster hemocytes treated with > or = 20 microM triclosan exhibited no significant (p < 0.05) reduction in viability relative to controls for up to 24 h at 13 degrees C. P. marinus meronts exposed to > or = 2 microM triclosan for 24 h at 13 degrees C exhibited significantly (p < 0.05) lower viability relative to controls. Exposure of P. marinus meronts to triclosan concentrations of > or = 20 microM resulted in > 50% mortality of P. marinus cells after 24 h. These results suggest that triclosan may be effective in treating P. marinus-infected oysters.     

Effects of calcium salts of fatty acids and calcium salt of methionine hydroxy analogue on plasma prostaglandin F2alpha metabolite and milk fatty acid profiles in late lactation Holstein-Friesian cows

Effects of a dietary lipid supplement containing calcium salts of fatty acids and methionine hydroxy analogue on plasma prostaglandin F2alpha (PGF2alpha) metabolite (PGFM) and milk fatty acid profiles were examined in 40 late lactation, nonpregnant, Holstein-Friesian cows for a period of 70 days. Effects on milk production, milk composition, and blood metabolites were also examined. Cows were paired on the basis of lactation number (first lactation, n = 8; second lactation, n = 32) and randomly assigned from within pairs to one of two dietary treatments: unsupplemented control (C) or 400 g per cow per day of the lipid supplement (S). Cows receiving the supplement had higher (P < 0.05) total milk production, total fat production (kg), and total lactose production (kg). Plasma cholesterol was significantly higher (P < 0.01) after 30 days of treatment in cows receiving the supplement. Cows receiving the supplement had lower (P < 0.01) concentrations of short chain milk fatty acids (C4:0 to C14:1) and higher concentrations of long chain fatty acids (C18:1 and C18:2; P < 0.01) than control animals. Oxytocin-induced prostaglandin release on Day 16 postovulation was increased (P < 0.01) in cows receiving the supplement. In conclusion, supplementation with calcium salts of fatty acids and methionine hydroxy analogue significantly increased milk yield and plasma PGFM.     

An investigation of the mechanisms for sterol synthesis and dietary sterol bioconversion in the heterotrophic protists Oxyrrhis marina and Gyrodinium dominans

The ability of the marine heterotrophic protists Oxyrrhis marina and Gyrodinium dominans to synthesize sterols de novo and modify dietary sterols was investigated using ¹³C-labeled substrates. De novo sterol synthesis of O. marina was determined by incorporation of ¹³C acetate into the culture medium. For G. dominans which has low tolerance of acetate, a protozoan prey Perkinsus marinus that cannot synthesize sterols, was cultured with ¹³C acetate then fed to G. dominans. Both heterotrophs utilized dietary ¹³C to synthesize fatty acids de novo, but not sterols. The ability of O. marina and G. dominans to alkylate, saturate, and desaturate dietary sterols was tested using P. marinus incorporated with ¹³C-labeled cholesterol as prey. O. marina did not modify the dietary ¹³C-cholesterol, but G. dominans produced 5 labeled sterols (brassicasterol, C28:1, and unknown C28, C29 and C30 sterols) indicating that G. dominans has the ability to desaturate and alkylate dietary cholesterol. The ability of O. marina and G. dominans to dealkylate dietary sterols was tested by feeding them gelatin acacia microspheres (GAMs) containing ¹³C-labeled brassicasterol. Neither heterotroph dealkylated brassicasterol to make cholesterol, but G. dominans alkylated and saturated brassicasterol to make 2 sterols (C29:1 and C30:0). The lack of dealkylation of brassicasterol by both protist species suggests problems with the substrate and/or delivery system since previous studies suggest that dealkylation of brassicasterol occurs when either species is fed algae containing this sterol.     

Perkinsus marinus, a protozoan parasite of the Eastern oyster (Crassostrea virginica): effects of temperature on the uptake and metabolism of fluorescent lipid analogs and lipase activities

The effects of temperature on the uptake and metabolism of fluorescent labeled palmitic acid (FLC16) and phosphatidylcholine (FLPC) and lipase activities in the oyster protozoan parasite, Perkinsus marinus, meront stage were tested at 10, 18, and 28 degrees C. Temperature significantly affected not only the uptake, assimilation, and metabolism of both FLC16 and FLPC in P. marinus, but also its triacylglycerol (TAG) lipase activities. The incorporation of both FLC16 and FLPC increased with temperature and paralleled the increase in the amount of total fatty acids in P. marinus meront cultures. The incorporation of FLC16 was higher than FLPC at all temperatures. The percentage of FLC16 metabolized to TAG was significantly higher at higher temperatures. Trace amounts of incorporated FLC16 were detected in monoacylglycerol (MAG) and PC at 18 and 28 degrees C. P. marinus meronts metabolized FLPC to TAG, diacylglycerol (DAG), monoacylglycerol (MAG), free fatty acids (FFA), phosphatidylethanolamine (PE), and cardiolipin (CL). The conversion of FLPC to TAG and PE was highest at 28 degrees C. The relative proportions of individual fatty acids and total saturated, monounsaturated and polyunsaturated fatty acids changed with temperatures. While total saturated fatty acids (SAFAs) increased with temperature, total monounsaturated fatty acids (MUFAs) decreased with temperature. Total polyunsaturated fatty acids (PUFAs) increased from 28 to 18 degrees C. The findings of increase of total SAFAs and decrease of total MUFAs with the increase of temperatures and upward shift of total PUFAs from 28 to 18 degrees C suggest that, as in other organisms, P. marinus is capable of adapting to changes in environmental temperatures by modifying its lipid metabolism. Generally, higher lipase activities were noted at higher cultivation temperatures. Both TAG lipase and phospholipase activities were detected in P. marinus cells and their extra cellular products (ECP), but phospholipase activities in both the cell pellets and ECP were very low. Also, lipase activities were much lower in ECP than in the cells. The observations of low metabolism, bioconversion of incorporated fluorescent lipid analogs and lipase activities at low temperatures are consistent with the low in vitro growth rate and low infectivity of P. marinus at low temperatures.     

Uptake and interconversion of fluorescent lipid analogs in the protozoan parasite, Perkinsus marinus, of the oyster, Crassostrea virginica

Uptake, distribution, and interconversion of fluorescent lipid analogs (phosphatidylcholine, PC; cholesteryl ester, CHE; phosphatidylethanolamine, PE; palmitic acid, C16; sphingomyelin, SM) by the two life stages, meront and prezoosporangium, of the oyster protozoan parasite, Perkinsus marinus, were investigated. Class composition of these two life stages and lipid contents in meront cells were also examined. Both meronts and prezoosporangia incorporated and modified fluorescent lipids from the medium, but their metabolic modes differ to some extent. Results revealed that among the tested analogs, neutral lipid components (CHE and C16) were incorporated to a greater degree than the phospholipids (PC, PE, and SM). HPLC analysis of meront lipids showed that while the majority of the incorporated PC, CHE, and PE remained as parent compounds, most of the incorporated C16 was in triacylglycerol (TAG) and SM was in ceramide and free fatty acids. The cellular distribution of fluorescent labels varied with lipid analogs and the extent of their metabolism by the parasite. Fluorescence distribution was primarily in cytoplasmic lipid droplets of both life stages after 24 h incubation with PC. After 24 h incubation with SM, fluorescence appeared in the membrane and cytosol. Total lipid contents in meront cultures increased during proliferation and TAG accounted for most of the increased total lipids. Since total lipid content per meront cell did not increase until the day of culture termination, the lipid increase in the meront culture was mainly a result of increased cell numbers. Both life stages contain relatively high levels of phospholipids, 53.8% in 8-day-old meronts and 39.4% in prezoosporangia. PC was the predominant phospholipid.     

Effects of plasma from bivalve mollusk species on the in vitro proliferation of the protistan parasite Perkinsus marinus

The in vitro culture of the Eastern oyster parasite Perkinsus marinus has provided a unique opportunity to examine its susceptibility to putative recognition and effector defense mechanisms operative in refractory bivalve species. In this study, we report the effect of supplementing the culture medium with plasma from: (1) uninfected to heavily infected Eastern oysters; (2) oyster species considered to be disease-resistant; and (3) bivalve mollusk species that are naturally exposed to the parasite but show no signs of disease. We also examined in vitro the interaction between hemocytes from Crassostrea virginica and C. gigas and P. marinus trophozoites. Our results revealed a significant decrease (32%) in proliferation of P. marinus in the presence of plasma from heavily infected C. virginica oysters. The inhibitory effects were less pronounced with plasma from moderately infected and uninfected oysters. In contrast, plasma from C. rivularis and C. gigas enhanced P. marinus proliferation. Proliferation was significantly reduced in media supplemented with plasma from Mytilus edulis, Mercenaria mercenaria, and Anadara ovalis. The highest inhibitory activity was apparent in M. edulis, for which 5% plasma-supplemented medium reduced growth by 35% relative to the controls. M. edulis active component(s) was heat-stable, yet pronase-sensitive. The significantly higher uptake of live P. marinus trophozoites by hemocytes from C. virginica, relative to those from C. gigas, suggests a certain level of specificity in the recognition/endocytosis of the parasite by its natural bivalve host species.     

Presence and synthesis of cholesterol in stable staphylococcal L-forms.

The sterol which was present in two strains of a stable staphylococcal L-form was analyzed by gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry. The retention time of the sterol on gas-liquid chromatography was the same as that of authentic cholesterol. Analysis of the sterol by mass spectrometry showed a molecular ion at an m/e of 386 and the same patterns of major ions above an m/e of 145 as those of authentic cholesterol. As a result, the sterol in staphylococcal L-form was identified as cholesterol. A parent strain and its L-forms were cultured in medium containing [14C]acetate, and the synthesis of cholesterol was examined. In the L-forms, 0.52% of the total lipid radioactivity was found in cholesterol fraction, whereas no significant radioactivity was detected in the cholesterol fraction of the parent strain, indicating that staphylococcal L-forms have acquired the capacity to synthesize cholesterol.     

Efficient lipid delivery to hybridoma culture by use of cyclodextrin in a novel granulated dry-form medium technology

Lipids are critical nutrients for high density eukaryotic cell cultivation, but inclusion of lipid components into dry-form media has been technically challenging. Lipid supplements are usually supplied for separate addition after powder reconstitution and filtration, which increases manipulation and risk in a biopharmaceutical manufacturing facility. Advanced Granulation Technology (AGT) is a novel dry-form media format designed for large-scale biopharmaceutical manufacture. All components of a complex formulation are homogeneously incorporated into a single granulated medium that simply requires water addition to yield a complete reconstituted 1X medium. We investigated whether cyclodextrin technology could be combined with the AGT process to deliver usable lipid in a dry medium format. The test lipids were cholesterol and several fatty acids supplied either as an aseptic supplement to liquid media or as part of a complete AGT formulation. The test system utilized a cholesterol auxotroph, NS0, and a protein-free basal formulation (CD Hybridoma Medium), with or without supplemental lipid. Post-filtration recovery of cholesterol from AGT-processed materials compared favorably with media supplemented with liquid lipid concentrates. Cell growth and viability and expression of recombinant protein were equivalent in all test media.     

Clonal growth of primary cultures of human hyaline chondrocytes in a defined medium

Rapid clonal growth of primary cultures of human costal chondrocytes in a defined medium has been achieved. The basal nutrient medium used for such growth is MCDB 104. It is prepared without linoleic acid and supplemented with 1 microgram/ml insulin, 100 ng/ml fibroblast growth factor, 1.0 x 10(-7) M dexamethasone, and 5 micrograms/ml mixed lipids, presented to the cells in the form of liposomes. The lipid supplement contains soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. No expression of cartilage-like differentiation occurs in the defined medium. However, colonies grown for several days in the defined medium and then grown for an additional period of time in medium F12 supplemented with fetal bovine serum and chicken embryo extract synthesize large amounts of refractile matrix that is stained intensely by acidified alcian green, thus demonstrating that the cells growing in the defined medium are capable of expressing cartilage matrix in a permissive environment. Good clonal growth and expression of differentiation can also be obtained by inoculating primary cultures of human chondrocytes directly into the F12-serum-embryo extract medium.     

Compatible Solutes in the Thermophilic Bacteria Rhodothermus marinus and "Thermus thermophilus"

(sup13)C nuclear magnetic resonance spectroscopy and (sup1)H nuclear magnetic resonance spectroscopy were used to identify and quantify the organic solutes of several strains of halophilic or halotolerant thermophilic bacteria. Two strains of Rhodothermus marinus and four strains of "Thermus thermophilus" grown in complex medium containing NaCl were examined. 2-O-Mannosylglycerate was a major compatible solute in all strains: the Thermus strains accumulated the (beta)-anomer only, whereas both anomers were found in R. marinus. 2-O-(beta)-mannosylglycerate and 2-O-(alpha)-mannosylglycerate were the major compatible solutes in R. marinus. The former was the predominant solute in cells grown in 2.0 and 4.0% NaCl-containing medium, while the latter was the predominant compatible solute at higher salinities. Glutamate, trehalose, and glucose were also present as minor components. The intracellular K(sup+) concentration, as determined by (sup39)K nuclear magnetic resonance spectroscopy, in R. marinus increased with salinity and was sufficient to balance the negative charges of the mannosylglycerate. In addition to 2-O-(beta)-mannosylglycerate, trehalose was a major compatible solute of "T. thermophilus." 2-O-(beta)-Mannosylglycerate was the main solute in medium containing 1.0 or 2.0% NaCl, while trehalose predominated in cells grown in medium supplemented with 3.0 or 4.0% NaCl. Glycine betaine, in lower concentrations, was also detected in two "T. thermophilus" strains. This is the first report of mannosylglycerate as a compatible solute in bacteria.     

Preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with p-bromo-phenacyl bromide and alpha-tocopherol inhibitors

PrTX-I, a non-catalytic and myotoxic Lys49-PLA(2) from Bothrops pirajai venom has been crystallized alone and in complex with bromophenacyl bromide (BPB), alpha tocopherol and alpha tocopherol acetate inhibitors. These crystals have shown to diffract X-rays between 2.34 and 1.65 A resolution. All complexes crystals are isomorphous and belong to the space group P2(1) whereas native PrTX-I crystals belong to the P3(1)21.     

The effects of phorbol ester on anabolism of lipids of cultured human skin fibroblasts

We have investigated the independent effects of phorbol ester (phorbol 12-myristate 13-acetate) on anabolism of the major lipid components in cultured diploid human skin fibroblasts. When we incubated these cells with [3H]acetate in serum-free medium for 18 h in the presence of 16 nM phorbol ester, [3H]acetate incorporation and the cellular content of cholesterol ester increased, and free cholesterol decreased. Enhancement of [3H]acetate incorporation into cholesterol ester was also observed when the cells were incubated with phorbol ester for 5 h in medium containing lipoprotein-deficient serum. Incorporation of [3H]galactose into glycosphingolipids increased many fold upon exposure of the cells either to fetal calf serum or separately to phorbol ester. Therefore, phorbol ester independently affects cholesterol and glycosphingolipid metabolism in a way that may be similar to that reported for serum low-density lipoproteins and unknown other factors in fetal calf serum. We have observed these effects of phorbol ester in the intact living cell. These findings should provide useful means for the study of metabolism of the plasma membrane lipid components in fibroblasts.     

Inhibition of lipid synthesis in isolated rat hepatocytes by serum lipoproteins

The incorporation of labeled acetate into lipids was studied in rat hepatocytes isolated after treatment of liver with collagenase and hyaluronidase. About 60% of the lipid radioactivity was in free cholesterol and 13% was in triglycerides. Acetate incorporation was markedly inhibited when human serum lipoproteins were present in the incubation medium. Very low, high, and low density lipoproteins, at concentrations of 1.0 mg/ml, inhibited acetate incorporation by 70, 55, and 35%, respectively. Chylomicrons, at similar concentrations, did not inhibit acetate incorporation. The distribution of radioactivity into lipid classes was unchanged by the addition of lipoproteins. Lipoproteins did not produce a nonspecific toxic effect on hepatocytes, since their addition did not alter the rate of leucine incorporation into protein. The addition of the delipidated protein from low density lipoprotein or of lecithin in amounts comparable to those present in inhibitory concentrations of lipoproteins failed to diminish acetate incorporation. Artificial cholesterol-lecithin emulsions containing small amounts of free cholesterol did not inhibit lipid synthesis. Although the mechanism for the inhibition of acetate incorporation by lipoproteins is unclear, such effects may play some physiological role in the control of lipid biosynthesis in the liver.     

Cholesterol requirement for growth of IR983F and P3X63-Ag8-U1 myeloma cells in serum-free medium.

Cholesterol, a major lipid component of the plasma membrane, is thought to have profound effects on the structure and function of cells. Most animal tissues are capable of synthesizing cholesterol de novo from acetate; however, there are relatively few mammalian cells in vitro expressing an absolute requirement for an exogenous source of cholesterol. In this paper, it was shown that both IR983F (983) rat myeloma cells and P3X63-Ag8-U1 (P3U1) mouse myeloma cells which had been cultivated in serum-free medium containing cholesterol for more than 6 months still required cholesterol in vitro for growth in serum-free medium. Optimal growth of 983 and P3U1 occurred in cholesterol concentrations of 15 and 5 micrograms/ml, respectively. Moreover, it was demonstrated that the cholesterol could be replaced by human low density lipoprotein in a concentration of 10 micrograms/ml but not by mevalonic acid lactone. In contrast to the parental myeloma cells, hybridoma cells derived from the mouse myeloma cells which had been cultivated in serum-free medium containing cholesterol for more than 6 months did not require cholesterol.     

Supplementation of Perkinsus marinus cultures with host plasma or tissue homogenate enhances their infectivity

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus-susceptible (Crassostrea virginica) and -tolerant (Crassostrea gigas, Crassostrea ariakensis) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.     

大肠杆菌DH5α耐乙酸突变株的选育及其代谢特性研究

大肠杆菌DH5α是基因工程常用的宿主菌之一,但由于对代谢副产物乙酸十分敏感,影响外源基因的表达效率。为了提高E. coli DH5α乙酸耐受力,采用60Co诱变结合连续培养,逐步提高稀释率和乙酸钠选择压力,于含乙酸钠平板进一步筛选,得到5株对乙酸耐受能力显著增强的突变菌株,具有良好的遗传稳定性,其中DA19显示最强的耐受性能。DA19与DH5α相比,在复合培养基YPS和YPS2G中菌体浓度分别提高17％和5％,最大比生长速率分别提高8％和27％,产乙酸分别减少为6％和59％;在基本培养基中的细胞浓度提高24倍,在含10g/L乙酸钠培养基中达到的细胞浓度与不加乙酸钠DH5α的细胞浓度相当。     

Effects of cholesterol surface transfer on cholesterol and phosphatidylcholine synthesis in cultured rat arterial smooth muscle cells

The purpose of this study was to examine the effects of cholesterol surface transfer between lipid vesicles and rat arterial smooth muscle cells on endogenous synthesis of cholesterol and phosphatidylcholine. Lipid vesicles containing cholesterol and egg phosphatidylcholine in different proportions were used as the extracellular lipid source. The rate of cellular cholesterol and phosphatidylcholine synthesis was determined from the [14C]acetate incorporation into these lipid classes. [3H]Cholesterol in lipid vesicles, with a cholesterol/phospholipid (C/P) mole ratio of 1:1, was rapidly transferred into rat smooth muscle cells, with a half-time of about 3.6 hours in the absence of serum proteins. Incubation of cells for 5 hours with vesicles of a high C/P mole ratio (i.e. 1.5:1) at vesicle-cholesterol concentrations above 100 micrograms/ml resulted in a marked reduction of cellular cholesterol synthesis, whereas the rate of phosphatidylcholine synthesis was increased. Cells incubated with lipid vesicles of C/P 1:2 did not show any change in cellular cholesterol or phosphatidylcholine synthesis. Incubation of cells with egg phosphatidylcholine vesicles at concentrations above 300 micrograms/ml, on the other hand, stimulated endogenous synthesis of cholesterol without affecting cellular phosphatidylcholine synthesis. The main conclusion is that cholesterol surface transfer may influence cellular lipid metabolism in the absence of mediating serum lipoproteins in a model system with cultured cells and lipid vesicles.     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.