The Effects of a Bacillus Biosurfactant on Methanogenic Hexadecane Degradation

The effects of microbially produced biosurfactants on hydrocarbon degradation have been examined previously by other researchers. However, almost all of these studies were conducted using rhamnolipid biosurfactants produced by various Pseudomonas species under aerobic conditions. The purpose of this study was to elucidate the effects of various levels of the Bacillus sp. JF2 lipopeptide biosurfactant on the degradation of hexadecane under methanogenic conditions. Hexadecane degradation did increase significantly when levels below critical micelle concentration of the pre-purified biosurfactant were added. However, at levels above this amount of biosurfactant, degradation of hexadecane appeared to be inhibited. The terminal electron accepting process, methanogenesis, was stimulated by surfactant addition. A review of the published literature revealed a wide variety of results, some which are similar to, but many that differ from those reported here. However, the results from this study were reproducible. Although there is no clear explanation for these results, more research on the effect of biosurfactants produced by Gram positive bacteria on the biodegradation of hydrocarbons is needed, as well as further studies under anaerobic conditions.     

The Effects of a Bacillus Biosurfactant on Methanogenic Hexadecane Degradation

The effects of microbially produced biosurfactants on hydrocarbon degradation have been examined previously by other researchers. However, almost all of these studies were conducted using rhamnolipid biosurfactants produced by various Pseudomonas species under aerobic conditions. The purpose of this study was to elucidate the effects of various levels of the Bacillus sp. JF2 lipopeptide biosurfactant on the degradation of hexadecane under methanogenic conditions. Hexadecane degradation did increase significantly when levels below critical micelle concentration of the pre-purified biosurfactant were added. However, at levels above this amount of biosurfactant, degradation of hexadecane appeared to be inhibited. The terminal electron accepting process, methanogenesis, was stimulated by surfactant addition. A review of the published literature revealed a wide variety of results, some which are similar to, but many that differ from those reported here. However, the results from this study were reproducible. Although there is no clear explanation for these results, more research on the effect of biosurfactants produced by Gram positive bacteria on the biodegradation of hydrocarbons is needed, as well as further studies under anaerobic conditions.     

Rapid quantification of a microbial surfactant by a simple turbidometric method

Quantification of the biosurfactants produced by a variety of microorganisms is a time taking and difficult task due to the lack of rapid, efficient and accurate methods. This work presents a simple turbidometric method for quantification of crude biosurfactants based on their property to become insoluble at low pH values. Biosurfactants obtained from a Bacillus sp. using different carbon substrates showed a good linear correlation (R(2)>0.99) between biosurfactant concentrations and turbidity in the range of 1 to 10 g L(-1) of crude biosurfactants. The substrate specific equations (SSE) and generalized equations (GE) developed in this work effectively predicted the amount of crude biosurfactant produced in different sets of fermentation experiments validating the method. A similar linear correlation was also observed with biosurfactants obtained from two other strains, Bacillus circulans and Pseudomonas sp. This simple method may prove to be effective in fast, accurate and inexpensive quantification of crude biosurfactants produced by diverse bacteria.     

Nitrate-reducing bacteria on rat tongues.

Nitrite-producing bacteria (NPB) were isolated from tongues of laboratory rats. The most commonly found nitrite-producing organism was Staphylococcus sciuri, followed by Staphylococcus intermedius, Pasteurella spp., and finally Streptococcus spp. Both morphometric quantification of bacteria on tongue sections and enumeration of culturable bacteria (CFU) showed an increase in the density of bacteria towards the posterior tongue. Up to 65% of bacteria were located in the deep clefts on the posterior tongue. The proportion of culturable NPB in the total culturable microbial population increased from 6% (10(5) CFU cm-2) on the anterior tongue to 65% (10(7) CFU cm-2) on the posterior tongue. Different species compositions of NPB were found on different tongue sections with S. intermedius populations decreasing and S. sciuri and Pasteurella populations increasing towards the posterior tongue. Nitrite production was sensitive to oxygen, and significant nitrite production was only detected on the posterior tongue where the majority of bacteria are situated in deep clefts in the tongue surface. This study suggests the importance of bacteria in nitrite production, from nitrate, on the tongue. Nitrite produced on the tongue may subsequently form nitric oxide in the acidic environment of the stomach. Because of the antimicrobial properties of nitric oxide, a key role for nitrate-reducing tongue bacteria in host animal defense against food-borne pathogens in proposed.     

Biosurfactant Production by a Soil Pseudomonas Strain Growing on Polycyclic Aromatic Hydrocarbons

The capacity of polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria to produce biosurfactants was investigated. Twenty-three bacteria isolated from a soil contaminated with petroleum wastes were able to form clearing zones on mineral salt agar plates sprayed with solutions of PAHs. Naphthalene and phenanthrene were utilized as sole substrates. Biosurfactant production was detected by surface tension lowering and emulsifying activities from 10 of these strains grown in an iron-limited salt medium supplemented with high concentrations of dextrose or mannitol, as well as with naphthalene or phenanthrene. Glycolipid determinations showed that in cultures of Pseudomonas aeruginosa 19SJ on naphthalene, the maximal productivity of biosurfactants was delayed compared with that in cultures grown on mannitol. However, when small amounts of biosurfactants and naphthalene degradation intermediates were present at the onset of the cultivation, the delay was markedly shortened. Production of biosurfactants was accompanied by an increase in the aqueous concentration of naphthalene, indicating that the microorganism was promoting the solubility of its substrate. Detectable amounts of glycolipids were also produced on phenanthrene. This is the first report of biosurfactant production resulting from PAH metabolism.     

Identification and ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry characterization of biosurfactants,including a new surfactin,isolated from oil‐contaminated environments

Biosurfactant‐producing bacteria were isolated from samples collected in areas contaminated with crude oil. The isolates were screened for biosurfactant production using qualitative drop‐collapse test, oil‐spreading and emulsification assays, and measurement of their tensoactive properties. Five isolates tested positive for in the screening experiments and displayed decrease in the surface tension below 30 mN m^?1. The biosurfactants produced by these isolates were further investigated and their molecular identification revealed that they are bacteria related to the Bacillus genus. Additionally, the biosurfactants produced were chemically characterized via UHPLC‐HRMS experiments, indicating the production of surfactin homologues, including a new class of these molecules.     

Bacterial Diversity within Feedlot Manure

In this study, we identified the predominant culturable anaerobic bacteria and enumerated the total culturable anaerobic bacterial population present in samples of feedlot manure from Southern Queensland. Sixteen bacterial isolates were cultured from feedlot pad material with species of Lactobacillus, Clostridium and Bacillus predominating. From a library of 123 clones, produced by the amplification, cloning and partial DNA sequencing of the 16S rRNA gene, only 3% were closely related to previously described species and 21% to known genera. Of the total clone library, 96% were apparently Gram-positive and fell within families whose members were generally anaerobes. The majority (71%) of the clone library was related to either members of the family Clostridiaceae or lactic acid-producing bacteria (Lactobacillus or Lactosphaera). It was concluded that Gram-positive clostridial and lactic acid-producing bacteria predominate in feedlot pad manure. The overwhelming majority of species are novel and have not been obtained in culture. It would appear that the most likely source of the sickly-sweet nuisance odours (particularly from butyric acid) that emanate from feedlots is the by-product of anaerobic fermentation by clostridia. Gut-inhabiting and Gram-negative bacteria do not appear to survive for lengthy periods of time under the environmental conditions present in feedlot manure.     

Microbiology of the oil fly, Helaeomyia petrolei

Helaeomyia petrolei larvae isolated from the asphalt seeps of Rancho La Brea in Los Angeles, Calif., were examined for microbial gut contents. Standard counts on Luria-Bertani, MacConkey, and blood agar plates indicated ca. 2 x 10(5) heterotrophic bacteria per larva. The culturable bacteria represented 15 to 20% of the total population as determined by acridine orange staining. The gut itself contained large amounts of the oil, had no observable ceca, and maintained a slightly acidic pH of 6.3 to 6.5. Despite the ingestion of large amounts of potentially toxic asphalt by the larvae, their guts sustained the growth of 100 to 1,000 times more bacteria than did free oil. All of the bacteria isolated were nonsporeformers and gram negative. Fourteen isolates were chosen based on representative colony morphologies and were identified by using the Enterotube II and API 20E systems and fatty acid analysis. Of the 14 isolates, 9 were identified as Providencia rettgeri and 3 were likely Acinetobacter isolates. No evidence was found that the isolates grew on or derived nutrients from the asphalt itself or that they played an essential role in insect development. Regardless, any bacteria found in the oil fly larval gut are likely to exhibit pronounced solvent tolerance and may be a future source of industrially useful, solvent-tolerant enzymes.     

Biosurfactants,an help in the biodegradation of hexadecane? The case of <Emphasis Type="Italic">Rhodococcus</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis> strains

The roles of the extracellular biosurfactants produced by two bacterial strains, Pseudomonas aeruginosa GL1 and Rhodococcus equi Ou2, in hexadecane uptake and biodegradation were compared. For this purpose, cell hydrophobicity and production of glycolipidic biosurfactants were evaluated during bacterial growth on hexadecane, as well the effects of these biosurfactants on culture supernatants properties i.e., surface and interfacial tensions, and emulsification and pseudosolubilization capacities. The results showed that the role of biosurfactants was different in these two strains and was directly related to the hydrophobicity of the bacterial cells concerned. Extracellular biosurfactants produced by strain R. equi Ou2 had only a minor role in hexadecane degradation. Direct interfacial accession appeared to be the main mechanism for hexadecane uptake by the hydrophobic cells of strain R. equi Ou2. On the contrary, the biosurfactants produced by P. aeruginosa GL1 were required for growth on hexadecane, and their pseudosolubilization capacity rather than their emulsification capacity was involved in substrate degradation, allowing uptake from hexadecane micelles by the hydrophilic cells of this bacterium. The roles of biosurfactants thus differ widely among bacteria degrading hydrophobic compounds. J.-P. Vandecasteele—in retirement     

茅尾海红树根际土壤可培养细菌多样性及抑菌活性研究

为挖掘茅尾海红树植物根际土壤可培养细菌资源,研究其抑菌活性,该文使用培养基对茅尾海5种红树植物(红海榄、黄槿、无瓣海桑、桐花树、阔苞菊)的7份根际土壤进行富集培养,选用6种不同分离培养基对富集样品进行可培养细菌的分离纯化,基于菌株16S rRNA基因序列信息进行物种多样性分析,并采用纸片法筛选具有抑制表皮葡萄球菌、耐甲氧西林金黄色葡萄球菌、铜绿假单胞菌活性的菌株。结果表明:(1)从7份红树植物根际土壤样品中分离到120种可培养细菌,隶属于35科47属,其中链霉菌属(Streptomyces sp.)占菌种总数的 14.2%,同时发现5种潜在新菌株。(2)通过抑菌活性初筛,发现9种细菌的发酵粗提物对至少一种致病菌具有抑菌活性。综上表明,茅尾海红树植物根际土壤可培养细菌多样性丰富,并且部分菌株具有抑制人类致病菌活性。该研究结果为新型抗生素的开发与利用提供了菌种资源。     

生物表面活性剂产生菌的筛选

从１０００份土壤和水等样品中,经富集培养、血平板分离、摇瓶培养和排油活性测定等方法筛选出１０株能产生各种生物表面活性剂的菌株（包括细菌,酵母和霉菌）。其中一株细菌产海藻糖脂,一株细菌产鼠李糖脂,两株细菌分别产长碳链不饱和脂肪酸和壬二酸,两株酵母产生的脂多糖具有良好的乳化性能     

Diversity and metabolic potential of culturable root-associated bacteria from Origanum vulgare in sub-Himalayan region

Study of rhizospheric bacteria from important plants is very essential, as they are known to influence plant growth and productivity, and also produce industrially important metabolites. Origanum vulgare is a perennial medicinal aromatic plant rich in phenolic antioxidants. Present study investigates the diversity of culturable root-associated bacteria from this plant in Palampur, India, which constitutes a unique ecosystem due to high rain fall, wide temperature fluctuations and acidic soil. Both root endophytes and rhizospheric soil bacteria were isolated, which resulted in a total of 120 morphologically different isolates. They were found to group into 21 phylotypes based on restriction fragment length polymorphism analysis. Growth medium composition had significant effect on the diversity of the isolated bacterial populations. The isolates were characterized for various metabolic, plant growth promoting (PGP) and other biotechnologically useful activities, based on which they were clustered into groups by principal component analysis. Majority of the isolates belonged to ??-Proteobacteria and Firmicutes. Pseudomonas and Stenotrophomonas were the most dominant species and together constituted 27.5?% of the total isolates. Many isolates, especially ??-Proteobacteria, showed very high PGP activities. Few isolates exhibited very high antioxidant activity, which may find potential applications in food and health industries. Firmicutes were catabolically the most versatile group and produced several hydrolytic enzymes. To the best of our knowledge, it is the first study describing rhizospheric microbial community of O. vulgare.     

塑料饲养大蜡螟幼虫肠道可培养细菌多样性

【背景】昆虫肠道中存在大量的微生物,是昆虫正常生命活动所必需的。它们能够促进维生素的合成、脂肪和碳水化合物的吸收及利用,同时还可以保护宿主抵御天敌,忍受高温以及促进毒素或异生素的代谢,间接促进资源开发。【目的】研究PE塑料饲喂的大蜡螟幼虫肠道可培养细菌的多样性。【方法】利用16S rRNA基因序列分析技术,结合菌落形态和细胞形态及相关生理生化特征鉴定细菌种类。【结果】从大蜡螟幼虫肠道分离纯化的40株可培养细菌得到16种不同细菌遗传型,分别属于芽孢杆菌科(Bacillaceae)、肠球菌科(Enterococcaceae)、葡萄球菌科(Staphylococcaceae)、莫拉菌科(Moraxellaceae)4个科。其中芽孢杆菌科是肠道可培养细菌的优势细菌种类。结合菌落和细胞形态及生理生化特征,确定肠道可培养细菌为芽孢杆菌属9株、肠球菌属4株、葡萄球菌属2株以及不动杆菌属1株。【结论】通过研究大蜡螟幼虫肠道可培养细菌群落结构组成,可为开展大蜡螟肠道的微生态研究提供相关理论基础。     

Method for simple and rapid enumeration of total epiphytic bacteria in the washing solution of rice plants

The phyllosphere is one of the most common habitats for terrestrial bacteria. However, little is known about the populations of bacteria, including unculturable bacteria, that thrive on plant surfaces. Here, we developed a fluorescent nuclear staining technique to easily and rapidly observe and enumerate populations of total and living epiphytic bacteria, with particular emphasis on the concentration by centrifugation and fixation of the epiphytic bacteria. An investigation on the optimal conditions for centrifugation and fixation revealed that centrifugation at 20 400g for 2 min and fixation with 0.5% glutaraldehyde solution were the optimum conditions for observation of the bacteria. Using this technique, we assessed the populations of the total and living bacteria on the surface of rice plants. When epiphytic bacteria were recovered from rice seeds (Oryza sativa 'Koshihikari'), the number of total and living bacterial cells was 7.36 and 6.85 log??·g?1 (fresh mass) in the seed washing, respectively. In contrast, the numbers of total and living bacterial cells in the leaf sheath washings were 5.5-5.8 and 5.3-5.7 log??·g?1, respectively. Approximately 5%-30% of the total bacteria in the washing solution of rice plant were culturable. The usefulness of the enumeration method and the amount of bacteria on the plant surfaces are discussed.     

Prevalence of potentially pathogenic culturable bacteria on eggshells and in cloacae of female Pied Flycatchers in a temperate habitat in central Spain

ABSTRACT Bacteria grow on avian eggshells and thus can potentially cause diseases in developing embryos. Little is known about culturable bacteria colonizing avian eggshells in free‐living birds, with most studies restricted to poultry. Our objective was to examine the culturable bacterial array growing on eggshells during incubation that could negatively affect hatching success of Pied Flycatchers (Ficedula hypoleuca) in a temperate montane habitat in central Spain. Cloacal culturable bacteria of females were also analyzed because bacteria can be vertically transmitted from females to eggs. We used fecal samples as surrogates of cloacal samples due to the small size of sampled birds. We found that eggshells and female cloacae of Pied Flycatchers harbored 24 and 40 bacterial families and species, respectively, but only a few in each clutch and each cloaca. Rod‐shaped gram‐negative bacteria and bacteria in the family Pseudomonadaceae were the most common bacteria on eggshells during early and late incubation and in female cloacae. Although based on small sample sizes, we found that females with rod‐shaped gram‐negative bacteria in their cloacae laid eggs that also had these bacteria, providing possible evidence for vertical transmission. We found no evidence for vertical transmission of Pseudomonadaceae, suggesting a possible environmental source for these bacteria. The prevalence of bacterial morphological types and major taxonomical categories on eggshells did not vary from early to late stages of incubation, providing support for the hypothesis that incubation may have bacteriostatic effects on bacterial proliferation on eggshells. Despite being primary egg invaders in poultry, we detected no effects of culturable Pseudomonadaceae or Pseudomonas luteola on hatching success. Our study represents the first to examine the culturable bacteria growing on the eggshells of a wild bird in a temperate habitat and additional studies based on culture‐independent techniques are required to confirm our results.     

西南地区高山湖泊中可培养细菌多样性及其所产胞外活性物质的特性

【目的】研究西南不同地区的高山湖泊中可培养细菌的多样性及其产胞外蛋白酶、纤维素酶和胞外多糖的能力。【方法】以西南4个不同地区的高山湖泊:雷波的马湖(LB)、中缅边境的凯邦亚湖(ZM)、沙德的莲花湖(SD)、腾冲的青海湖(TC)的水样为研究对象,利用稀释涂布平板方法对可培养细菌进行分离筛选,然后通过对可培养细菌的生理生化指标和16S r RNA基因序列进行分析,初步确定细菌属别;对分离得到的菌株进行产胞外蛋白酶和纤维素酶活性测定和产胞外多糖能力检测。【结果】从西南地区4个湖泊中共分离筛选得到41株细菌,其中LB 15株、ZM 13株、SD 7株、TC 6株。根据16S r RNA基因序列的系统进化分析,4个地区可培养细菌的组成和丰度存在明显差异,其中LB和ZM的优势菌属是芽孢杆菌属(Bacillus),其次是气单胞菌属(Aeromonas)和假单胞菌属(Pseudomonas),分离的TC菌株全部属于芽孢杆菌属(Bacillus),分离的SD菌株特异性较强。进一步酶活性和胞外多糖检测表明,分离得到的41株细菌中有28株菌的发酵产物具有蛋白酶活性,6株具有纤维素酶活性,17株可产胞外多糖(Exopolysaccharides,EPS)。其中有2株细菌同时产蛋白酶、纤维素酶和胞外多糖,10株细菌同时产蛋白酶和胞外多糖,2株细菌同时产蛋白酶和纤维素酶,1株细菌同时产纤维素酶和胞外多糖。【结论】西南4个高山湖泊中存在丰富的微生物菌种资源,且4个湖泊中筛选的可培养细菌受所处环境的影响大。其中莲花湖由于高海拔和较偏僻等特点,人为干扰小,分离得到的细菌类群与其他湖泊相比明显不同;而马湖、凯邦亚湖和青海湖3个湖泊的海拔相对较低,受人类活动影响较大,分离得到的细菌均较常见。此外高山湖泊中的可培养细菌具有分泌多种胞外活性物质特性,为工业化应用奠定了资源基础,极具更深入的开发和研究价值。     

Basis for formulating biosurfactant mixtures to achieve ultra low interfacial tension values against hydrocarbons

Biosurfactants could potentially replace or be used in conjunction with synthetic surfactants to provide for more cost-effective subsurface remediation. The design of surfactant formulations that are effective in lowering interfacial tension (IFT), which is necessary to mobilize entrapped hydrocarbons, requires information about the surface-active agent (surfactant) and the targeted non-aqueous phase liquids (NAPL). We hypothesized that biosurfactant and synthetic surfactant mixtures can be formulated to provide the appropriate hydrophobic/hydrophilic conditions necessary to produce low IFT against NAPLs, and that such mixtures will produce synergism that make them more effective than individual biosurfactants or synthetic surfactants. Our work tested the interfacial activity of biosurfactants from individual strains and mixtures of biosurfactants from different strains with and without a synthetic surfactant. Multiple regression analysis showed that, for lipopeptide biosurfactants produced by various Bacillus species, the interfacial activity against toluene depended on the relative proportions of 3-OH-C₁₄, C₁₅, C₁₆, and C₁₈ in the fatty acid tail. As the fatty acid composition became more heterogeneous the system produced lower IFT against toluene. In mixtures of lipopeptide biosurfactants with the more hydrophilic, rhamnolipid biosurfactant, the IFT against toluene decreased as the percentage of the 3-OH C₁₄ fatty acid increased in the lipopeptide. Mixtures of lipopeptide biosurfactants with the more hydrophobic synthetic surfactant, C12, C13-8PO SO₄Na, were able to produce low IFT against hexane and decane. In general, we found that lipopeptide biosurfactants with a heterogeneous fatty acid composition or mixtures of lipopeptide and rhamnolipid biosurfactants lowered the IFT against hydrophilic NAPLs. Conversely, mixtures of lipopeptide biosurfactants with a more hydrophobic synthetic surfactant lowered the IFT against hydrophobic NAPLs.     

Assessment of bacterial community structure in a long-term copper-polluted ex-vineyard soil

The influence of long-term copper contamination on the diversity of bacterial communities was investigated in an ex-vineyard soil. Two sites of the same area but exhibiting different 3-fold exchangeable copper (Ex-Cu) concentrations were analysed. Culturable bacterial community structure was assessed using a variety of approaches: determination of culturable bacteria number, analyses of 132 isolates, and denaturing gradient gel lectrophoresis (DGGE) patterns of bacterial biomass grown on agar plates and of soil DNA. There was no significant difference in the number of total heterotrophs at the two sites, whereas the percentage of fast-growing bacteria growing in 1 day, was lower at the site with the higher Ex-Cu content. A high percentage of Cu-tolerant bacteria was found in both sites (63-70%) and it was relatively independent of the Cu content. Shifts in species composition of the culturable bacterial community were detected by analysing isolates from the two soils, Gram-positive bacteria prevailed in the less-polluted soil while Gram-negative bacteria in the more-polluted soil. Each sample site had a community with a different metal resistance pattern. Our study seems to indicate that in this soil ecosystem, copper influenced the culturable bacterial communities, affecting the structural diversity and altering some of the metal resistance of the microorganisms. The Sorensen similarity index calculated on DGGE profiles of 16S rDNA of total and culturable bacterial communities indicated a different species composition at the two sites, although both sites had the same biodiversity degree and different dominance.     

Microbiology of the Oil Fly, Helaeomyia petrolei

Helaeomyia petrolei larvae isolated from the asphalt seeps of Rancho La Brea in Los Angeles, Calif., were examined for microbial gut contents. Standard counts on Luria-Bertani, MacConkey, and blood agar plates indicated ca. 2 × 10⁵ heterotrophic bacteria per larva. The culturable bacteria represented 15 to 20% of the total population as determined by acridine orange staining. The gut itself contained large amounts of the oil, had no observable ceca, and maintained a slightly acidic pH of 6.3 to 6.5. Despite the ingestion of large amounts of potentially toxic asphalt by the larvae, their guts sustained the growth of 100 to 1,000 times more bacteria than did free oil. All of the bacteria isolated were nonsporeformers and gram negative. Fourteen isolates were chosen based on representative colony morphologies and were identified by using the Enterotube II and API 20E systems and fatty acid analysis. Of the 14 isolates, 9 were identified as Providencia rettgeri and 3 were likely Acinetobacter isolates. No evidence was found that the isolates grew on or derived nutrients from the asphalt itself or that they played an essential role in insect development. Regardless, any bacteria found in the oil fly larval gut are likely to exhibit pronounced solvent tolerance and may be a future source of industrially useful, solvent-tolerant enzymes.     

Bacterial community associated with Pfiesteria-like dinoflagellate cultures

Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four ' Pfiesteria -like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the α-3 or SAR83 cluster.