High-throughput cultivation of heterotrophic bacteria during a spring phytoplankton bloom in the North Sea

On-going studies of phytoplankton-bacterioplankton interactions at the long-term ecological research site Helgoland Roads have indicated that many of the heterotrophic bacterial taxa have not yet been cultivated. A high-throughput approach combining whole cell matrix-assisted laser desorption ionization – time of flight mass spectroscopy with 16S rRNA gene sequencing was applied to the spring bloom of 2016. Aiming at an assessment of cultivability during a spring bloom, cultivation on solid marine media had to be used since dilution to extinction would not have been feasible for a high-throughput approach, as performed in this study. A total of 5023 isolates were obtained from nine weekly samples on eight different solid media between the early-bloom and post-bloom periods. Most of the 4136 strains identified affiliated with Bacteroidetes (13.3%), Gammaproteobacteria (26.9%), Alphaproteobacteria (40.6%) and Actinobacteria (6.7%). Of the 271 operational phylogenetic units (OPUs) identified, 13 are likely to represent novel genera and 143 novel species. A comparison with 16S rRNA gene tag data indicated that most of the isolates were rather rare in surface waters, with the exception of five OPUs affiliating with Rhodobacteraceae, Polaribacter, Psychromonas and Pseudoalteromonas. The effort yielded many novel isolates, yet most of the abundant heterotrophic bacteria still remained elusive. The large strain collection obtained will not only provide insights into the succession of the cultivable fraction of the bacterioplankton, but also enable fine-tuned taxonomic and physiological follow-up studies for improving our knowledge on heterotrophic bacteria in North Sea waters.     

Second derivative UV absorbance analysis to monitor nitrate-reduction by bacteria in most probable number determinations

Heterotrophic and autotrophic nitrate-reducing bacteria (NRB) play important roles in many environments. These bacteria are often enumerated by most probable number (MPN) methods. Measuring NO(3)(-) depletion in the MPN cultures is the definitive way to determine the presence of NRB. Media used for MPN determinations of NRB in oil field waters usually contain high Cl(-) concentrations, matching those in the water samples. Many methods for measuring NO(3)(-) concentrations, such as ion chromatography (IC), cadmium reduction and ion electrode methods, are adversely affected by high concentrations of Cl(-) and organic compounds. A second derivative UV absorbance method proved to be a fast and reliable means for measuring NO(3)(-) depletion in MPN media used for enumerating autotrophic and heterotrophic NRB, without interferences from Cl(-) or the organic components in the latter medium. The MPN results for heterotrophic NRB determined by the second derivative UV absorbance agreed well with those determined by the production of nitrous oxide, and were often higher than those determined by measuring nitrate depletion by the diphenylamine spot test.     

Optimization of procedures for the recovery of heterotrophic bacteria from marine sediments

A study was undertaken of the various factors affecting the recovery of heterotrophic bacteria from marine sediments. The dilution medium and culture medium were found to be of great importance in the recovery of heterotrophic colony forming units (CFU). Statistical analysis of the total viable counts obtained under the test conditions showed that artificial seawater (ASW) without further supplementation was equal to or superior to ASW plus 0.1% peptone or ASW plus 0.1% peptone and 0.1% glycerol. The addition of a surfactive agent, on the other hand, resulted in 95% inhibition of the recoverable CFU. The elapsed time (up to 12 hr) between recovery of a sedimentary core and completion of plating procedures was found to have little effect provided the sedimentary sample was removed from the core, placed in ASW, and stored in a refrigerator until actual plating occurred. It was further noted that lower organic nutrient concentrations, approximately one-tenth of those generally in use, resulted in significantly higher total viable counts. Finally, replicate contiguous sampling at three depths in a core resulted in no significant changes in the number of CFU from the surface samples, indicating a greater surface homogeneity than that previously suspected. The same pattern was not true, however, for samples obtained a lower positions in the core, thus indicating pockets of microbial concentration. Person to whom correspondence and request for reprints should be sent.     

Optimization of procedures for the recovery of heterotrophic bacteria from marine sediments

A study was undertaken of the various factors affecting the recovery of heterotrophic bacteria from marine sediments. The dilution medium and culture medium were found to be of great importance in the recovery of heterotrophic colony forming units (CFU). Statistical analysis of the total viable counts obtained under the test conditions showed that artificial seawater (ASW) without further supplementation was equal to or superior to ASW plus 0.1% peptone or ASW plus 0.1% peptone and 0.1% glycerol. The addition of a surfactive agent, on the other hand, resulted in 95% inhibition of the recoverable CFU. The elapsed time (up to 12 hr) between recovery of a sedimentary core and completion of plating procedures was found to have little effect provided the sedimentary sample was removed from the core, placed in ASW, and stored in a refrigerator until actual plating occurred. It was further noted that lower organic nutrient concentrations, approximately one-tenth of those generally in use, resulted in significantly higher total viable counts. Finally, replicate contiguous sampling at three depths in a core resulted in no significant changes in the number of CFU from the surface samples, indicating a greater surface homogeneity than that previously suspected. The same pattern was not true, however, for samples obtained a lower positions in the core, thus indicating pockets of microbial concentration.     

Distribution of Vibrio vulnificus in the Chesapeake Bay.

Vibrio vulnificus is a potentially lethal human pathogen capable of producing septicemia in susceptible persons. Disease is almost always associated with consumption of seafood, particularly raw oysters, or with exposure of wounds to seawater. An oligonucleotide DNA probe (V. vulnificus alkaline phosphatase-labeled DNA probe [VVAP]), previously shown to be highly specific for V. vulnificus, was used to enumerate this species in environmental samples collected from the Chesapeake Bay between April 1991 and December 1992. Total aerobic, heterotrophic, culturable bacteria were enumerated by plate counts on nonselective medium. The number of V. vulnificus organisms was determined by colony lifts of spread plates for subsequent hybridization with VVAP. V. vulnificus was not detected in any samples collected during February and March (water temperature of < 8 degrees C) but was found in 80% of the water samples collected during May, July, September, and December (water temperature of > 8 degrees C), with concentrations ranging from 3.0 x 10(1) to 2.1 x 10(2)/ml (ca. 8% of the total culturable heterotrophic bacteria). In a multiple regression analysis, increased V. vulnificus concentrations were correlated with lower salinities and with isolation from samples collected closer to the bottom. Isolation from oysters was demonstrable when water temperatures were 7.6 degrees C, with concentrations ranging from 1.0 x 10(3) to 4.7 x 10(4)/g (ca. 12% of total culturable bacteria). In samples collected in May and July, V. vulnificus was identified in seven of seven plankton samples and four of nine sediment samples. Our data demonstrate that V. vulnificus is a widespread and important component of the bacterial population of the Chesapeake Bay, with counts that are comparable to those reported from the Gulf of Mexico.     

Correlation of Direct Viable Counts with Heterotrophic Activity for Marine Bacteria

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly ¹⁴C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.     

Quantitative analysis of bacterial communities from Mediterranean sapropels based on cultivation-dependent methods

Microbial communities of ancient Mediterranean sapropels, buried sediment layers of high organic matter, were analyzed by most probable number (MPN) approaches. Mineral media containing different carbon sources in sub-millimolar concentrations were used. MPN numbers were elevated in sapropels and at the sediment surface, which mirrored total cell count distributions. Highest MPN counts were obtained with a mixture of different monomeric and polymeric substrates, with amino acids or with long-chain fatty acids as sole carbon sources. These values reached up to 2 x 10(7) cm(-3), representing 3.3% of the total cell count. A total of 98 pure cultures were isolated from the highest positive dilutions of the MPN series, representing the most abundant microorganisms culturable by the methods used. The strains were identified by molecular biological methods and could be grouped into 19 different phylotypes. They belonged to the alpha-, beta-, gamma-, and delta-Proteobacteria, to the Actinobacteria and the Firmicutes. However, about half of the number of isolates was closely related to the genera Photobacterium and Agrobacterium. Regarding the high cultivation success, these organisms can be assumed to be typical sapropel bacteria, representing a substantial part of the culturable indigenous microbial community.     

High incidence of halotolerant bacteria in Pacific hydrothermal-vent and pelagic environments

The abundance of halotolerant microorganisms in hydrothermal-vent and pelagic waters in the North and South Pacific was estimated by the most probable number (MPN) technique using a heterotrophic 16% NaCl medium incubated at 20-24 degrees C. Based on these MPNs and direct counts with epifluorescence microscopy to enumerate the total microbial population, salt-tolerant microbes comprised from <0.01 to >28% of the total microbial community. Fourteen isolates from these MPN enrichments were identified by sequencing a portion of the 16S rRNA gene, and all were found to belong to the genera Halomonas and Marinobacter. The response to salt of mesophilic hydrothermal-vent microbial isolates obtained without selecting for salt tolerance was also examined. Forty-one of 65 strains cultured from hydrothermal plume waters, low-temperature hydrothermal fluids, sulfide rock and an animal specimen at approximately 2000-2200 m depth from the Endeavour Segment of the Juan de Fuca Ridge were subjected to increasing concentrations of NaCl, and over half grew at a NaCl concentration that is lethal to many commonly isolated marine bacteria. At least 36 of the 65 isolates (>/=55%) grew in the enrichment medium supplemented with 10% NaCl; at least 30 of 65 (>/=46%) grew with 16% NaCl; at least 20 of 65 (>/=31%) tolerated 22% NaCl. Based on phylogenetic analysis of the 16S rRNA gene in nine of these 65 isolates, four belonged to the genus Halomonas. These Halomonas strains tolerated 22-27% NaCl. It is possible that a majority of the other 16 isolates which grew with 22% NaCl are also Halomonas based on their degree of halotolerance, morphology, and apparent abundance as revealed by MPN enrichments. The four Halomonas strains obtained without selecting for halotolerance were further characterized physiologically and metabolically. Overall, they grew between -1 degrees C and 40 degrees C, were facultative aerobes, oxidized between 49 and 70 organic compounds according to Biolog plate substrate utilization matrices, grew with oligotrophic quantities of carbon (0.002% yeast extract) in liquid media, reduced nitrate to nitrite, and tolerated up to 0.05-3 mM Cd(2+). Halomonas is one of the most abundant culturable organisms in the ocean, and its success may be attributed to its metabolic and physiological versatility.     

Basal medium for the selective enumeration of rumen bacteria utilizing specific energy sources.

A 40% rumen fluid basal medium has been developed that without added substrate will support growth of about 10% or less of the total colony count obtained with 40% rumen fluid-glucose-cellobiose-starch-agar medium (RGCSA). The basal medium is prepared by anaerobic incubation of all ingredients in RGCSA medium except the carbohydrates, Na2CO3, and cysteine for 7 days at 38 degrees C. After incubation, substrate(s), Na2CO3 and cysteine are added and the medium is tubed and sterilized as in normal medium preparation. When xylose was included with glucose, cellobiose, and starch as added carbohydrates in the incubated medium, colony counts were comparable to those obtained with RGCSA medium. The addition of specific carbohydrates or other substrates as energy sources to the basal medium suggested that the percentage of the bacterial population capable of utilizing these energy sources was influenced by the ration of the animal; however, considerable animal variation and day-to-day variation in a given animal was observed. Comparison of the population in animals fed either orchardgrass hay or 60% corn-40% orchardgrass (60-40) indicated little or no difference for the percentage of bacteria utilizing glucose, pectin, xylan, or mannitol. Increases in the percentages of xylose-, cellobiose-, Glycerol-, and lactate-utilizing bacteria occurred with the orchardgrass hay ration, whereas the percentage of starch-digesting bacteria was increased significantly (P less than 0.01) in the animals fed the 60-40 ration. A limited number of bacterial strains were isolated from the basal medium without added substrate, most of which were atypical with respect to the predominant rumen bacteria. Growth of these strains, even in complex media, was very slow and limited. Based on these data with isolated strains and colony counts obtained in roll tube medium containing only minerals, resazurin, agar, Na2CO3, and cysteine, the selective medium overestimated the percentage of bacteria able to use a specific energy source. This overestimate was 6 to 7% of the total culturable count.     

Enhanced activity of oligotrophic endogenous bacteria in clay‐rich sediments by nutrient injection

A controlled field experiment was performed in which the microbiology and geochemistry of clay‐rich fluvial‐deltaic sediments were characterized both before and after nutrient injection into a shallow well. Acetate addition (with nitrogen and phosphate) initially increased the heterotrophic bacteria population in the groundwater within 21 days after nutrient addition. Consumption of oxygen and injected nutrient resulted in an expected stimulation of copiotrophic bacterial growth (31–48 days), then a noticeable “trough phase”; reflecting minimal or no bacterial growth followed by a secondary peak reflecting another bacterial population growth surge (62–85 days). This secondary surge was apparently supported by the nutrients generated by the decomposing biomass (initial population peak), and by oxygen replenishment supplied by continual ground water flow. During the intervening trough phase, bacterial counts by most‐probable‐number analysis of soil samples indicated that the denitrifying population increased to a greater degree than the remainder of the heterotrophic population. An increase in methane gas was detected in the head space of water samples collected during the trough phase, suggesting an increase in bacterial methanogenesis. There was no evidence of a concomitant increase in bacterial sulfate‐reducing activity. This rapid progression from aerobic to microaerophilic and anaerobic growth probably resulted from the microbial biodiversity present in clay‐rich sediments, which in turn is related to the development of microhabitats. These microhabitats developed because of the hydrogeologic character of the clay‐rich sediments in which flow is controlled by macropores while fluid in the clay matrix (micropores) is relatively isolated from the groundwater in the macropores.     

Inclusion of xylan in a medium for the enumeration of total culturable rumen bacteria.

The influence of the inclusion of xylan in a medium for enumeration of total culturable rumen bacteria was investigated. Maximum colony numbers were obtained on a medium, GCSX-2, which contained 0.033% each glucose and cellobiose and 0.067% each soluble starch and xylan. This medium gave higher colony counts than either medium 98-5 of Bryant and Robinson (J. Dairy Sci. 44:1446-1456, 1961), medium 98-5 of Chung and Hungate (Appl. Environ. Microbiol. 32:649-652, 1976), containing an added lucerne (hemicellulose + cellulose) fiber substrate, or medium GCSX-2 with the added lucerne (hemicellulose + cellulose) fraction. The time of collection of rumen fluid influenced the colony counts on the media containing the lucerne fiber substrate but was without effect on medium GCSX-2.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria

We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.     

Oxidative consumption of nitric oxide by heterotrophic bacteria in soil

Abstract: Uptake rate constants for nitric oxide were measured in a neutral calcic cambisol (KBE) and an acidic luvisol (PBE). The NO uptake was higher under oxic than under anoxic incubation conditions by a factor of about three. Gassing the soils with air containing 10 ppmv NO resulted in the accumulation of nitrate which accounted for 57–94% of the NO consumed. Aerobic heterotrophic bacteria were isolated on glucose-yeast extract medium from soil dilutions corresponding to a most probable number of 10⁸–10⁹ bacteria per gram dry weight soil. One of the isolates (strain PS88, a Pseudomonas sp.) exhibited NO consumption activity that was much higher under oxic than anoxic incubation conditions. When sterile KBE amended with strain PS88 was gassed with air containing 10 ppmv NO, 88% of the consumed NO was recovered as nitrate and nitrite. A screening of various bacteria obtained from culture collections showed a widespread ability for consumption of low NO concentrations. Our results indicate that NO consumption in soil is not only possible by reductive denitrification, but also by oxidation due to aerobic heterotrophic bacteria such as strain PS88.     

Total Counts of Marine Bacteria Include a Large Fraction of Non-Nucleoid-Containing Bacteria (Ghosts)

Counts of heterotrophic bacteria in marine waters are usually in the order of 5 x 10(sup5) to 3 x 10(sup6) bacteria ml(sup-1). These numbers are derived from unspecific fluorescent staining techniques (J. E. Hobbie, R. J. Daley, and S. Jasper, Appl. Environ. Microbiol. 33:1225-1228, 1977; K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980) and are subsequently defined as total counts of bacteria. In samples from the Baltic Sea, the North Sea (Skagerrak), and the northeastern Mediterranean Sea, we found that only a minor fraction (2 to 32%) of total counts can be scored as bacteria with nucleoids. Lack of DNA no doubt means inactive cells; therefore, a much lower number of bacteria that grow at rates higher than those previously estimated must be responsible for the measured bacterial production in these seas. The remaining bacterium-sized and/or -shaped particles included in total counts may be cell residues of virus-lysed bacteria (ghosts) or remains of protozoan grazing.     

Lithotrophic and Heterotrophic Bacteria in Deep Subsurface Sediments and Their Relation to Sediment Properties

Subsurface sediments obtained from three cores drilled to depths of 260 m below the surface in South Carolina were analyzed for heterotrophic bacteria; N₂‐fixing microaerophiles; and nitrifying, sulfur‐oxidizing, and H₂‐oxidizing lithotrophic bacteria. In addition, pore waters were extracted for chemical analysis of inorganic nitrogen species, sulfate, dissolved organic carbon, pH, and Eh. Autotroph populations were generally less than 10³ most probable number (MPN) g^‐1 dry sediment with sulfur‐oxidizing bacteria, detected in 60% of the sediment samples, being the most frequently encountered group. Nitrifying bacteria were detected mainly in sediments from one borehole (P28), and their populations in those sediments were correlated with pore‐water ammonium concentrations. Populations of heterotrophic bacteria in 60% of the sediments were greater than 10⁶ colony forming units (CFU) g^‐1 dry sediment and were typically lower in sediments of high clay content and low pH. Microaerophilic N₂‐fixing bacteria were cultured from >50% and bacteria capable of growth on H₂ were cultured from 35% of the subsurface sediments examined. Sediment texture, which controls porosity, water potential, and hydraulic conductivity, appears to be a major factor influencing microbial populations in coastal plain subsurface sediments.     

Growth of infant fecal bacteria on commercial prebiotics

Fecal bacteria from 33 infants (aged 1 to 6 months) were tested for growth on commercial prebiotics. The children were born vaginally (20) or by caesarean section (13). Bifidobacteria, lactobacilli, gram-negative bacteria, Escherichia coli, and total anaerobes in fecal samples were enumerated by selective agars and fluorescence in situ hybridization. The total fecal bacteria were inoculated into cultivation media containing 2 % Vivinal® (galactooligosaccharides—GOS) or Raftilose® P95 (fructooligosaccharides—FOS) as a single carbon source and bacteria were enumerated again after 24 h of anaerobic cultivation. Bifidobacteria dominated, reaching counts of 9–10 log colony-forming units (CFU)/g in 17 children born vaginally and in seven children delivered by caesarean section. In these infants, lactobacilli were more frequently detected and a lower number of E. coli and gram-negative bacteria were determined compared to bifidobacteria-negative infants. Clostridia dominated in children without bifidobacteria, reaching counts from 7 to 9 log CFU/g. Both prebiotics supported all groups of bacteria tested. In children with naturally high counts of bifidobacteria, bifidobacteria dominated also after cultivation on prebiotics, reaching counts from 8.23 to 8.77 log CFU/mL. In bifidobacteria-negative samples, clostridia were supported by prebiotics, reaching counts from 7.17 to 7.69 log CFU/mL. There were no significant differences between bacterial growth on Vivinal® and Raftilose® P95 and counts determined by cultivation and FISH. Prebiotics should selectively stimulate the growth of desirable bacteria such as bifidobacteria and lactobacilli. However, our results showed that commercially available FOS and GOS may stimulate also other fecal bacteria.     

A method of the selective isolation and enumeration of marine Vibrionaceae

A noninhibitory medium and GasPack anaerobic culture system were employed for the selective enumeration and isolation of Vibrionaceae in seawater and marine sediments.Vibrio counts obtained by the new method for seawater and sediment samples were compared with vibrio numbers in the heterotrophic bacterial population appearing on a medium routinely employed in the laboratory for such counts. The ratio of the former to the latter counts ranged from 0.5 to 1.3, the average being 0.96. The seawater and sediment bacteria that grew and produced visible colonies on the medium under anaerobic conditions for 3 days at 20°C were almost exclusively vibrios.From the results reported here it is concluded that most of the vibrios present in seawater and sediment samples can be recovered by the new method developed in this study.     

Abundance and Biomass of Heterotrophic Flagellates, and Factors Controlling Their Abundance and Distribution in Sediments of Botany Bay

The abundance and biomass of heterotrophic flagellates were estimated monthly in sediments of Botany Bay during March 1999-February 2000. The annual abundance and biomass were in the ranges of 0.46-4.70 x 10(5) cells/cm(3) and of 0.30-8.61 micro g C/cm(3), respectively. The majority of heterotrophic flagellates (93-100%) were less than 10 mm in length and few flagellates were larger than 10 mm. Of the total microbial carbon biomass, heterotrophic flagellates made up about 5% (but at times up to 35%). The contribution of heterotrophic flagellates varied from month to month, and among the sites. The abundance of heterotrophic flagellates was negatively correlated with sediment grain size and positively correlated with the abundance of bacteria, algae (autotrophic flagellates and diatoms), and their probable grazers. A best subsets regression analysis showed that bacterial and algal abundance are the most important factors controlling the abundance of heterotrophic flagellates. When the previously reported grazing rates on bacteria were applied, heterotrophic flagellates would consume a maximum of 64% of bacterial standing stock daily in Botany Bay, suggesting that heterotrophic flagellates are important as bacterivores. However, the importance of heterotrophic flagellate grazing probably varies significantly among the sites and from month to month.