Probiotics, lactic acid bacteria and bacilli: interesting supplementation for aquaculture.

Probiotics administration in aquafeed is known to increase feed consumption and absorption due to their capacity to release a wide range of digestive enzymes and nutrients which can participate in digestion process and feed utilization, along with the absorption of diet components led to an increase in host's health and well-being. Furthermore, probiotics improve gut maturation, prevention of intestinal disorders, predigestion of antinutrient factors found in the feed ingredients, gut microbiota, disease resistance against pathogens and metabolism. The beneficial immune effects of probiotics are well established in finfish. However, in comparison, similar studies are less abundant in the shellfish. In this review, the discussions will mainly focus on studies reported the last 2 years. In recent studies, native probiotic bacteria were isolated and fed back to their hosts. Although beneficial effects were demonstrated, some studies showed adverse effects when treated with a high concentration. This adverse effect may be due to the imbalance of the gut microbiota caused by the replenished commensal probiotics. Probiotics revealed greatest effect on the shrimp digestive system particularly in the larval and early post-larval stages, and stimulate the production of endogenous enzymes in shrimp and contribute with improved the enzyme activities in the gut, as well as disease resistance.

Do the intestinal microbiotas differ between paddlefish (Polyodon spathala) and bighead carp (Aristichthys nobilis) reared in the same pond?

AIMS: A study was conducted to compare the intestinal microbial compositions of two fish species with similar feeding strategy; paddlefish (Polyodon spathala) and bighead carp (Aristichthys nobilis) reared in the same pond. METHODS AND RESULTS: Age-0 paddlefish and bighead carp with mean average body lengths of 43·39 ± 2·78 and 19·33 ± 3·68 cm, respectively, were reared with natural prey items in the same pond (20 m(2)). After 30 days of rearing, the intestinal microbiota of the two fish species was assessed by pyrosequencing of 16S rRNA genes. Interestingly, deviations were observed in the microbial communities of the two fish species according to the alpha- and beta-diversity measurements and detrended correspondence analysis (DCA). Shannon diversity (P = 0·015) and Pielou.evenness (P = 0·035) revealed significant lower diversity of the intestinal microbiota of paddlefish. Moreover, different core intestinal microbiota was noticed in the two fish species. Proteobacteria (57·3%), Firmicutes (11·9%), Fusobacteria (8·9%), Planctomycetes (7·3%), Actinobacteria (6·0%) and Verrucomicrobia (3·2%) were detected in bighead carp, while the dominant phyla in paddlefish intestines were Bacteroidetes (37·0%), Fusobacteria (35·1%), Firmicutes (14·8%) and Proteobacteria (12·6%). CONCLUSIONS: Our results revealed that the intestinal microbiota differed between paddlefish and bighead carp reared in the same pond when fed similar nature food. The potential host factors, such as the genetic background, gut histology and physiology are assumed to be involved in the intestinal bacterial compositions. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the similar feeding strategy of paddlefish and bighead carp, this study presents basic knowledge for evaluation of the importance of host factors (genetic background and gut anatomy) on intestinal microbial composition.

Characterisation and identification of enzyme-producing autochthonous bacteria from the gastrointestinal tract of two Indian air-breathing fish.

Characterisation and identification of autochthonous enzyme-producing bacteria isolated from the proximal intestine and distal intestine of two species of Indian air-breathing fish, murrel (Channa punctatus) and stinging catfish (Heteropneustes fossilis), were investigated using conventional culture technique. Population levels of proteolytic strains were highest in the digestive tract of stinging catfish. In both species, the viable counts of amylase-producing bacteria were somewhat higher than cellulase-producing bacteria. Among the gut bacteria isolated, 8 strains (4 from murrel and 4 from stinging catfish) were selected as potent enzyme-producers on the basis of quantitative enzyme assays. All these strains were Gram-positive rods, but only four isolates (CPF4, CPH6, CPH7 and HFH4) were capable of forming endospores. The tested bacteria grew in wide range of temperatures and pH. The strains were further identified by 16S rRNA gene sequence analysis. Two strains, CPF3 (isolated from murrel) and HFH4 (isolated from stinging catfish) showed high similarity to Bacillus sp., strain HFH7 (isolated from the stinging catfish) was most closely related to Bacillus subtilis, while five strains belonged to Bacillus licheniformis. Based on the results of the present study, we suggest that incorporation of autochthonous enzyme-producing bacteria in aquafeeds merits further investigations.

Luminal uptake of Vibrio (Listonella) anguillarum by shed enterocytes--a novel early defence strategy in larval fish.

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.

Effects of dietary antibiotic growth promoter and Saccharomyces cerevisiae fermentation product on production, intestinal bacterial community, and nonspecific immunity of hybrid tilapia (Oreochromis niloticus female x Oreochromis aureus male).

To investigate the effects of a dietary antibiotic growth promoter (florfenicol) and a Saccharomyces cerevisiae fermentation product (DVAQUA) on growth, G:F, daily feed intake, intestinal bacterial community, and nonspecific immunity of hybrid tilapia (Oreochromis niloticus ♀ × Oreochromis aureus ♂), a 16-wk feeding trial was conducted in a recirculating aquaculture system. Four feeding regimens were evaluated: control, dietary florenicol (0.02 g/kg; 16 wk), dietary DVAQUA (0.5 g/kg; 16 wk), and sequential use of florenicol (0.02 g/kg; 8 wk), and DVAQUA (0.5 g/kg; 8 wk). Each regimen had 4 replicate tanks (0.5 × 0.5 × 0.5 m) and each tank contained 12 fish (initial BW: 46.88 ± 0.38 g). Dietary florfenicol improved growth (P = 0.089), G:F (P = 0.036), and serum complement component concentrations (P < 0.001) of hybrid tilapia. However, the compound decreased the estimated intestinal bacterial count estimated by rpoB quantitative PCR (P < 0.001) and bacterial diversity (visual band numbers, Shannon diversity index, and Shannon equitability index based on 16S rDNA V3 denaturing gradient gel electrophoresis fingerprints) compared with the control. Although sequential use of florfenicol and DVAQUA improved growth and G:F numerically to a similar extent as dietary florfenicol, and increased intestinal bacterial count to normal quantities, the sequential use of florenicol and DVAQUA decreased intestinal bacterial diversity (visual band numbers, Shannon diversity index, and Shannon equitability index) as well as serum complement component concentrations (P < 0.001) compared with their respective use and the control. These findings might be negatively related to disease control and host defense, and the sequential use of florenicol and DVAQUA should be practiced with caution. Feeding DAVQUA to the fish improved nonspecific immunity and increased intestinal bacterial count and bacterial diversity, but further research, including challenge studies, should be conducted before recommendation of DVAQUA supplementation to hybrid tilapia diets.

Histological changes in intestine of Atlantic salmon (Salmo salar L.) following in vitro exposure to pathogenic and probiotic bacterial strains.

Furunculosis and vibriosis are diseases that cause severe economic losses in the fish-farming industry. The foregut of the Atlantic salmon (Salmo salar L.) was exposed in vitro to two fish pathogens, Aeromonas salmonicida (causative agent of furunculosis) and Vibrio anguillarum (causative agent of vibriosis), and to one probiotic strain, Carnobacterium divergens, at 6 x 10(4) or 6 x 10(6) viable bacteria per milliliter. Histological changes following bacterial exposure were assessed by light and electron microscopy. Control samples (foregut exposed to Ringer's solution only) and samples exposed only to C. divergens had a similar appearance to intact intestinal mucosal epithelium, with no signs of damage. However, exposure of the foregut to the pathogenic bacteria resulted in damaged epithelial cells, cell debris in the lumen, and disorganization of the microvilli. Co-incubation of the foregut with a pathogen and C. divergens did not reverse the damaging effects caused by the pathogen, although these were alleviated when probiotic bacteria were used. Based on these results, we suggest that the probiotic bacterium, C. divergens, is able to prevent, to some extent, pathogen-induced damage in the Atlantic salmon foregut.

Translocation of viable Aeromonas salmonicida across the intestine of rainbow trout, Oncorhynchus mykiss (Walbaum).

The pathogenic bacterium Aeromonas salmonicida is the causative agent of the destructive disease furunculosis in salmonids. Horizontal transmission in salmonids has been suggested to occur via the skin, gills and/or intestine. Previous reports are contradictory regarding the role of the intestine as a route of infection. The present study therefore investigates the possibility of bacterial translocation across intestinal epithelia using Ussing chamber technology, in vitro. Intestinal segments were exposed for 90 min to fluorescein isothiocyanate-labelled pathogenic A. salmonicida. Sampling from the serosal side of the Ussing chambers showed that bacteria were able to translocate across the intestinal epithelium in both the proximal and distal regions. Plating and subsequent colony counting showed that the bacteria were viable after translocation. During the 90 min exposure to A. salmonicida, the intestinal segments maintained high viability as measured by electrical parameters. The distal region responded to bacterial exposure by increasing the electrical resistance, indicating an increased mucus secretion. This study thus demonstrates translocation of live A. salmonicida through the intestinal epithelium of rainbow trout, suggesting that the intestine is a possible route of infection in salmonids.

Effects of natural winter pasture and commercial pellet on the ultrastructure of small intestinal epithelium in reindeer.

Segments of small intestine (duodenum, jejunum and ileum) from slaughtered reindeer (Rangifer tarandus tarandus) grazing natural winter pastures (n=3) and reindeer fed commercially available pellets (RF-80) in winter (n=5) were collected and immediately fixed in McDowell's fixative. Transmission electron microscopy was employed to investigate the ultrastructural features of the epithelium and lamina propria along the small intestine and to relate these to the different diets. Major differences in ultrastructural features were observed between the small intestinal enterocytes of reindeer fed the two diets. Enterocytes in reindeer fed the natural diet displayed a normal appearance with a dense cytoplasm and distinct microvilli. In contrast, reindeer fed the commercial diet showed damaged enterocytes amongst the normal cells. Abnormal changes included disintegration and loss of microvilli, cytoplasmic swelling, loss of membrane integrity and increases in the width of intercellular spaces, especially in the jejunum.

Identification and characterization of Carnobacteria isolated from fish intestine.

Eleven bacterial strains were isolated from the gastrointestinal tract of four fish species, Atlantic salmon (Salmo salar L.), Arctic charr (Salvelinus alpinus L.), Atlantic cod (Gadus morhua L.) and wolffish (Anarhichas lupus L.). All the strains were Gram-positive rods, non-sporing, catalase and oxidase-negative, able to grow at pH 9.0 but not on acetate containing media (pH < or = 5.4), and were fermentative. They had a high content of oleic acid (18:1 n-9) in cellular lipid, and were found to belong to the genus Carnobacterium by phenotypic criteria. The eleven carnobacteria strains were further identified on the basis of 16S rDNA sequence analysis and AFLP(TM) fingerprinting.

Epithelium-associated bacteria in the gastrointestinal tract of Arctic charr (Salvelinus alpinus L.). An electron microscopical study.

AIMS: The primary aim was to use transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to define the location of epithelium-associated bacteria in the digestive tract of the salmonid fish, Arctic charr (Salvelinus alpinus). METHODS AND RESULTS: TEM and SEM examination of the gastrointestinal tract demonstrated substantial numbers of ovoid and rod-shaped bacterial cells associated with the microvillous brush borders of enterocytes. Bacteria were found at the tips of microvilli as well as between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in two regions (pyloric caeca and midgut). CONCLUSION: Electron microscope examination of the gut is an important tool for evaluating the microbial ecology of the fish digestive tract ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the current study clearly demonstrate that the intestine is involved in bacterial endocytosis.

Lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.).

The present study reports the effect of excessive handling stress and starvation on the lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.). A relatively low population level (approximately 2 x 103 bacteria per gram wet tissue) of viable adherent heterotrophic bacteria was associated with the digestive tract (foregut, midgut and hindgut). Of the 752 bacterial isolates isolated from diet, water and the digestive tract, 201 isolates belonged to the carnobacteria. Of these isolates, one from the diet, one from the rearing water and 80 from the gastrointestinal tract, were further identified on the basis of 16S rDNA sequence analysis. All these isolates were identified as being Carnobacterium piscicola-like. Daily repeated stress and starvation of the fish over 11 d had no influence on the total culturable bacterial numbers or population level of C. piscicola associated with the digestive tract. C. piscicola-like isolates colonizing the various intestinal regions (foregut, midgut and hindgut) were also screened for their ability to produce growth inhibitory compounds active against the fish pathogen Aeromonas salmonicida. Of the 199 C. piscicola isolates tested, 139 inhibited growth of the pathogen.

Identification and characterization of carnobacteria associated with the gills of Atlantic salmon (Salmo salar L.).

Using the surface plate technique, the population level of aerobic bacteria, occurring in the gills of Atlantic salmon (Salmo salar L.), was determined to be approximately 3 x 10(4) g(-1). Of 100 isolates investigated, 58 were gram-negative. Out of the 42 gram-positive isolates, 26 belonged to the carnobacteria, of which ten were further identified on the basis of 16S rDNA sequence analysis and AFLP fingerprinting. All were identified as Carnobacterium piscicola-like. These carnobacteria strains were also screened for their ability to produce growth inhibitory compounds active against the fish pathogens Aeromonas salmonicida subsp. salmonicida, Vibrio anguillarum and Vibrio salmonicida. Nine out of the ten C. piscicola isolates tested strongly inhibited growth of the three pathogens.

The effect of diet on aerobic bacterial flora associated with intestine of Arctic charr (Salvelinus alpinus L.).

In order to extend the knowledge on the possible effect of diet on the gastrointestinal microbial community of fish, Arctic charr (Salvelinus alpinus L.) were fed diets containing high (23.7%) and low (6.4%) levels of carbohydrate. The number of viable aerobic and facultative aerobic bacteria associated with the digestive tract were not influenced by dietary regimen. A wide range of bacterial species was isolated, and the predominant bacterial species of both rearing groups were identified as Staphylococcus. There were, however, some differences in bacterial composition between the rearing groups, as well as inter-individual variations. For example, atypical Aeromonas salmonicida were isolated from the small and large intestine of two fish fed low dietary carbohydrate, while Aer. caviae-like isolates were found in the small intestine of four fish fed high carbohydrate. Non-motile Aeromonas spp. were found in the rearing group fed high dietary carbohydrate, but at low frequencies. Dietary manipulation seemed to influence the species composition of carnobacteria, Gram-positive rods, oxidase and catalase-negative and fermentative metabolism. Carnobacterium piscicola-like bacteria were only found in the small intestine, while C. mobile-like and Carnobacterium spp. were isolated from the large intestine of fish fed high carbohydrate. On the contrary, C. divergens-like isolates were found associated with the small and large intestine of fish fed low dietary carbohydrate.

The effect of dietary fatty acids on lactic acid bacteria associated with the epithelial mucosa and from faecalia of Arctic charr, Salvelinus alpinus (L.).

Arctic charr, Salvelinus alpinus (L.), held in fresh water, were fed four experimental diets containing different polyunsaturated fatty acids (PUFA). In addition, one group fed a diet containing only coconut oil as sole lipid source served as control. The population of aerobic heterotrophic bacteria associated with the epithelial mucosa and the faecalia was estimated using the dilution plate technique. Generally, the population level of adherent bacteria increased along the digestive tract (stomach, small intestine and large intestine). Adherent Gram-negative and Gram-positive bacteria seemed to be present at equal levels in all parts of the alimentary tract. Lactic acid bacteria dominated among the Gram-positive bacteria, and they were detected in all regions of fish fed the PUFA supplemented diets. The frequency of lactic acid bacteria was highest in the digestive tract of fish fed diets with added 7.0% linolenic acid (18:3 n-3) or 4% of a PUFA mix. A lower frequency of lactic acid bacteria was found in fish fed dietary linoleic acid (18:2 n-6), and they were absent or present in low numbers in fish fed the coconut oil diet. It is suggested that dietary fatty acids affect the attachment sites for the gastrointestinal microbiota, possibly by modifying the fatty acid composition of the intestine wall. Numerical taxonomy procedures showed that the lactic acid bacteria Carnobacterium spp. and a Carnobacterium piscicola-like strain were predominant, with smaller numbers of Lactobacillus plantarum, Streptococcus spp. and Leuconostoc mesenteroides present. Seven strains of Carnobacterium spp. were further identified on the basis of 16S rDNA sequence analysis, and all these strains were identified as Carnobacterium piscicola.

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae.

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot (Scophthalmus maximus) larvae exposed to V. pelagius and/or Aer. caviae. The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10(3) ml-1 and 10(3) larva-1. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 x 10(2) bacteria larva-1 on day 3 post-hatching to approximately 10(5) bacteria fish-1 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 x 10(4) cfu larva-1 exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10(3) of the Vibrio spp. belonged to V. pelagius. When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10(4) larva-1), of which 9 x 10(3) belonged to Aer. caviae. Later in the experiment, at the time when high mortality occurred, 9 x 10(5) Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria (V. pelagius) to the rearing water.

Production of Eicosapentaenoic Acid (20:5 n-3) by Vibrio pelagius Isolated from Turbot (Scophthalmus maximus (L.)) Larvae.

Fourteen bacterial strains isolated from turbot, Scophthalmus maximus (L.), larvae were screened for eicosapentaenoic acid (20:5 n-3) (EPA) production. Gas chromatography analysis revealed that one bacterial species, Vibrio pelagius, contained a high proportion of EPA in cellular lipid. This finding was confirmed by gas chromatography-mass spectrometry analysis. A higher concentration of EPA was detected when the bacterium was cultured at 4 degrees C.

Lipids of arctic charr,Salvelinus alpinus (L.) II. Influence of dietary fatty acids on the elongation and desaturation of linoleic and linolenic acid.

Arctic charr,Salvelinus alpinus L. were fed five test diets containing 0% or 1% of different polyunsaturated fatty acids (PUFA) for 93 days. The fish were injected intraperitoneally with (1-(14)C)-18:2(n-6) or (1-(14)C)-18:3(n-3), and the bioconversion to longer chain PUFA studied. The conversion rate in neutral lipids was slow, with most label found as the fatty acid injected, while extensive modification took place prior to or during incorporation into polar lipids. Linolenic acid was preferred over linoleic acid as substrate for elongation and desaturation regardless of diet. In polar lipids, the predominant products of (1-(14)C)-18:2(n-6) metabolism were generally 20:3(n-6) and 20:4(n-6), while 18:4(n-3), 20:5(n-3) and 22:6(n-3) were the major products of (1-(14)C)-18:3(n-3) metabolism. The lack of radioactivity in 22:5(n-6) suggests that Δ 4 desaturation is specific for (n-3) PUFA. Feeding the PUFA deficient diet reduced the Δ 5 desaturation compared to fish maintained on PUFA supplemented diets. The Δ 6 desaturation was only reduced in fish fed C18 PUFA and injected with (1-(14)C)-18:3(n-3). Longer chain C20 and C22 PUFA, particularly those of the (n-3) family, exerted some inhibition on the elongation and desaturation of injected fatty acids compared to those fed C18 PUFA. The incorporation of radiolabelled fatty acids into polar lipids of fish fed a commercial diet was very low, and the desaturation neglectible in both polar and neutral lipids, showing that Arctic charr under culture conditions do not convert short chain PUFA to longer chain metabolites.

Lipids of arctic charr,Salvelinus alpinus (L.) I. Dietary induced changes in lipid class and fatty acid composition.

Arctic charr (Salvelinus alpinus L.) were fed either a commercial diet or six experimental test diets containing coconut oil and different polyunsaturated fatty acids (PUFA) at a level of 1% by dry weight. Best growth rates were observed with the commercial diet, worst with diet containing coconut oil with no PUFA. An increase in hepatic lipid, hepatic sterol esters and muscular moisture content, and a decrease in muscular lipid was generally found in fish fed the test diets compared to those maintained on the commercial diet.Phosphatidylcholine was the dominant polar lipid (PL) class in all tissues examined. Extensive modification of dietary saturated fatty acids into 18:1 (n-9) was observed in tissue triacylglycerols (TAG) of fish fed test diets. No changes occurred with the commercial diet.Dietary PUFA were essentially incorporated unchanged into tissue TAG of all fish in the present study. PUFA composition of hepatic phospholipids was significantly influenced by that contained in the diets. However both 18:2 (n-6) and 18:3 (n-3) in the test diets were extensively elongated and desaturated prior to incorporation into PL. The (n-9) PUFA content was always higher in liver of fish fed the test diets. When 18:2 (n-6) and 18:3 (n-3) were supplied together, the level of (n-3) PUFA exceeded those of (n-6) PUFA. Muscle PL were less influenced by diet than liver. In muscle (n-3) PUFA were always the predominant PUFA irrespective of diet. Only low amounts of (n-9) PUFA were found. It is suggested that (n-3) PUFA are the prime essential fatty acids for Arctic charr, and that they are used in preference to (n-6) PUFA for elongation, desaturation and incorporation into PL. The results suggest that the quantitative requirement of Arctic charr for EFA is may be higher than that of other salmonids.

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735.

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.

Trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735: Na+-stimulated anaerobic transport in cells and membrane vesicles.

Alteromonas putrefaciens NCMB 1735 required the presence of NaCl for anaerobic growth with serine, cysteine, and formate as substrate and trimethylamine oxide ( TMAO ) as external electron acceptor. When lactate was substrate, the organism grew equally well in the absence of NaCl. Anaerobic uptake of glutamate, aspartate, serine, cysteine, and lactate in resting cells was strongly stimulated with NaCl, and cytoplasmic membrane vesicles energized by electron transfer from formate to TMAO displayed active Na+-dependent uptake of serine. The data suggested that participation in transport processes was the only vital function of Na+ in A. putrefaciens. Formate- and TMAO -dependent anaerobic serine uptake in vesicles was sensitive to the protonophore carbonyl cyanide m-chlorophenyl-hydrazone and the ionophores valinomycin and gramicidin. Transport-active vesicles contained cytochromes of b and c type, and both serine uptake and TMAO reduction with formate were inhibited with the electron transfer inhibitor 2-heptyl-4-hydroxyquinoline N-oxide. Thus, reduction of TMAO to trimethylamine in A. putrefaciens appeared to be coupled with a chemiosmotic mechanism of energy conversion.