Autotrophic Cultures Research Articles

Morphophysiological analyses of Neochloris oleoabundans (Chlorophyta) grown mixotrophically in a carbon-rich waste product

Neochloris oleoabundans is considered one of the most promising oil-rich microalgae because of its ability to store lipids under nitrogen starvation. However, high biomass densities, required for applications on medium to large scale, are not reached in this condition of growth. As previous studies on other microalgae have shown that mixotrophy allows to obtain higher biomass in comparison to autotrophic cultures, we performed morphophysiological analyses in order to test the mixotrophic growth capability of N. oleoabundans. A carbon-rich manure derived from the apple vinegar production (AWP) was added to the medium. Cells were also cultivated under nutrient starvation (tap water), to observe the expected lipids accumulation, and combining AWP to water, to test the potential of this waste in a low-cost culture system. The results highlighted that AWP in the medium allowed to obtain the highest final cell density. Moreover, starch granules were stored inside chloroplast at the beginning of the experiment. The presence of AWP did not induce variations on light harvesting complex II (LHCII)-photosystem II (PSII) assembly, even if an interesting promotion of pigment synthesis in cells was observed. On the other hand, in starved cells, chloroplast degeneration, pigment content decrease, altered LHCII-PSII assembly and accumulation of high amount of lipid globules were observed, irrespective of the presence of AWP. The results suggest that mixotrophy promotes growth in N. oleoabundans and open up the possibility of using waste products from agri-food industries for this purpose. After growth, cells could be transferred under nutrient starvation to induce lipid accumulation.

NaCS–PDMDAAC immobilized autotrophic cultivation of Chlorella sp. for wastewater nitrogen and phosphate removal

The unicellular green microalgae, Chlorella sp., entrapped in sodium cellulose sulphate/poly-dimethyl-diallyl-ammonium chloride (NaCS–PDMDAAC) to create microalgae capsules were used to remove total nitrogen (T-N) and phosphate (PO43−-P) from artificial wastewater. Batch and semi-continuous cultivations with capsules recycling were carried out in shake flasks within an illuminated incubator. Experimental results showed that the NaCS–PDMDAAC capsules have desirable biophysical properties such as good mechanical stability, biocompatibility with the microalgae cells resulting in biomass enrichment within the capsules, and high substrate concentration tolerance. NaCS–PDMDAAC Chlorella sp. capsules displayed a considerable high T-N and PO43−-P removal rate of 12.56 and 10.24mg/g biomass per day respectively. The developed immobilized microalgae system has the potential for continuous wastewater treatment operation with reduced downstream operation cost due to the large size of the capsules being 5–6mm.

Bacterial Community Associated with Autotrophic and Heterotrophic Cultures of Medicinal Plant Smallanthus sonchifolius (Yacon)

Several unfruitful attempts to grow axenic shoot cultures of Smallanthus sonchifolius, also known as yacon, were made before healthy shoots grew in association with bacteria on half strength Murashigue and Skoog media supplemented with 2.2 µM benzylaminopurine. Twenty-one bacterial isolates were obtained from in vitro S. sonchifolius plantlets, eight of these isolates were identified as Flavimonas oryzihabitans, Curtobacterium pusillum, Sphingomonas paucimobilis, and Microbacterium imperiale. These microorganisms produced indole acetic acid (IAA) at amounts varying between 8.89 to 47.45 μg/mL, reason for being classified as plant growth promoting bacteria (PGPB). The results show that buds associated with bacteria cultured on sucrose free media produced 3.77 new roots measuring 18.33 cm in length after a 30-day growing period. In contrast, buds growing on sucrose supplemented media, the number of roots induced was higher (6.67 to 14 roots/explant) but shorter in length, 4.67 to 5.83 cm. During plant acclimatization to soil, photosynthesis and water efficiency were measured showing that the plants were healthy and vigorous. A slightly higher rate of photosynthesis and water use efficiency was recorded in the plants produced on heterotrophic conditions as compared to plants grown in sucrose free media. Plants adapted well in the soil demonstrating that the PGPB community associated to S. sonchifolius in shoot cultures was not harmful to plant production. The purpose of this study was to demonstrate that the bacteria associated with in vitro S. sonchifolius shoot cultures was not the result of microbial contamination, but rather from symbiotic associations that extended from cultivation in the greenhouse, to culture and back to soil. This is the first report to show that autotrophic cultures may represent a viable alternative to grow healthy plants without eliminating beneficial bacteria associated with the host.

Anoxic Iron Cycling Bacteria from an Iron Sulfide- and Nitrate-Rich Freshwater Environment

In this study, both culture-dependent and culture-independent methods were used to determine whether the iron sulfide mineral- and nitrate-rich freshwater nature reserve Het Zwart Water accommodates anoxic microbial iron cycling. Molecular analyses (16S rRNA gene clone library and fluorescence in situ hybridization, FISH) showed that sulfur-oxidizing denitrifiers dominated the microbial population. In addition, bacteria resembling the iron-oxidizing, nitrate-reducing Acidovorax strain BrG1 accounted for a major part of the microbial community in the groundwater of this ecosystem. Despite the apparent abundance of strain BrG1-like bacteria, iron-oxidizing nitrate reducers could not be isolated, likely due to the strictly autotrophic cultivation conditions adopted in our study. In contrast an iron-reducing Geobacter sp. was isolated from this environment while FISH and 16S rRNA gene clone library analyses did not reveal any Geobacter sp.-related sequences in the groundwater. Our findings indicate that iron-oxidizing nitrate reducers may be of importance to the redox cycling of iron in the groundwater of our study site and illustrate the necessity of employing both culture-dependent and independent methods in studies on microbial processes.

Autotrophic cultivation of Spirulina platensis for CO2 fixation and phycocyanin production

Microalgae biotechnology provides a new industrial paradigm that simultaneously yields various vital products and captures CO2 in a single process. Development of optimal strategies to harness microalgal resources is critical to full-scale production and application of microalgae-derived products. This work parametrically investigated the autotrophic cultivation of Spirulina platensis for enhanced simultaneous CO2 fixation and phycocyanin production using conical flasks and newly designed photobioreactors. In the absence of culture aeration in the conical flasks, a maximum biomass concentration of 3.20g/L was achieved under alkaline conditions with initial pH of ∼9. In the presence of culture aeration, a maximum biomass concentration of 5.96g/L was obtained under intermittent CO2 supply at 20mM/(L2d) (that is 20mM/L every other day). Continuous sparging of 0.1L/min compressed air in combination with 20mM/(Ld) intermittent CO2 supply into a customarily designed photobioreactor resulted in biomass and crude phycocyanin concentrations of 5.92g/L and 1.06g/L respectively. Further photobioreaction optimization resulted in biomass and phycocyanin concentrations of 7.27g/L and 1.22g/L respectively using 20mM/Ld CO2 intermittent aeration supplemented with continuously supplied compressed air under specific flow conditions. The highest volumetric titre of phycocyanin obtained in this work is amongst the highest reported in literature. These results indicate that high biomass, phycocyanin concentration and CO2 fixation rate could be obtained with minimal extra effort simply by optimizing bioprocess conditions. The growth results of S. platensis cultivation fitted well to the logistic rate equation.

Effects of Carbon and Nitrogen Sources on Fatty Acid Contents and Composition in the Green Microalga, Chlorella sp. 227

In order to investigate and generalize the effects of carbon and nitrogen sources on the growth of and lipid production in Chlorella sp. 227, several nutritional combinations consisting of different carbon and nitrogen sources and concentrations were given to the media for cultivation of Chlorella sp. 227, respectively. The growth rate and lipid content were affected largely by concentration rather than by sources. The maximum specific growth was negatively affected by low concentrations of carbon and nitrogen. There is a maximum allowable inorganic carbon concentration (less than 500~1,000 mM bicarbonate) in autotrophic culture, but the maximum lipid content per gram dry cell weight (g DCW) was little affected by the concentration of inorganic carbon within the concentration. The lipid content per g DCW was increased when the microalga was cultured with the addition of glucose and bicarbonate (mixotrophic) at a fixed nitrogen concentration and with the lowest nitrogen concentration (0.2 mM), relatively. Considering that lipid contents per g DCW increased in those conditions, it suggests that a high ratio of carbon to nitrogen in culture media promotes lipid accumulation in the cells. Interestingly, a significant increase of the oleic acid amount to total fatty acids was observed in those conditions. These results showed the possibility to induce lipid production of high quality and content per g DCW by modifying the cultivation conditions.

Carbon dioxide fixation by Chlorella sp. USTB-01 with a fermentor-helical combined photobioreactor

A promising microalgal strain isolated from fresh water, which can grow both autotrophically on inorganic carbon under lighting and heterotrophically on organic carbon without lighting, was identified as Chlorella sp. USTB-01 with the phylogenetic analysis based on 18S ribosomal ribonucleic acid (rRNA) gene sequences. In the heterotrophic batch culture, more than 20.0 g·L−1 of cell dry weight concentration (DWC) of Chlorella sp. USTB-01 was obtained at day 5, and which was used directly to seed the autotrophic culture. A novel fermentor-helical combined photobioreactor was established and used to cultivate Chlorella sp. USTB-01 for the fixation of carbon dioxide (CO2). It showed that the autotrophic growth of Chlorella sp. USTB-01 in the combined photobioreactor was more effective than that in the fermentor alone and the maximum DWC of 2.5 g·L−1 was obtained at day 6. The highest CO2 fixation of 95% appeared on day 1 in the exponential growth phases of Chlorella sp. USTB-01 and 49.8% protein was found in the harvested microalgal cells.

Biodiesel production from Stichococcus strains at laboratory scale

AbstractBACKGROUND: This paper reports the results of an experimental campaign of autotrophic cultures of Stichococcus strains aiming at selecting the most promising strain for biofuel production. The strain selected—S. bacillaris 158/11—was cultivated in 1 L lab‐scale bubble column photobioreactors under fed‐batch and semi‐continuous conditions. A Bold basal medium supplemented with NaNO3 as nitrogen source was adopted. Tests were carried out at 23 °C, 140 µE m−2 s−1, and air flow rate ranging between 0.4 and 4 vvm. Cultures were characterized in terms of pH, concentration of total nitrogen, total organic carbon, total inorganic carbon, biomass, lipid fraction and methyl‐ester distribution of transesterified lipids.RESULTS: S. bacillaris 158/11 proved to be the best strain to produce biodiesel. Methyl‐ester distribution was characterized by a large fraction of methyl palmitate, methyl linolenate, methyl linoleate, and methyl oleate along with phytol. The process photosynthetic efficiency—fraction of available light stored as chemical energy ‐ was about 1.5%. Specific biomass productivity was ∼60 mgDM L−1 day−1 under the semi‐continuous conditions tested. Total lipid productivity was 14 mg L−1 day−1 at a dilution rate of 0.050 L day−1.CONCLUSION: S. bacillaris 158/11 is a potential strain for massive microalgae cultures for biofuel production. Higher biomass/total‐lipid productivity could be obtained in sunlight. Copyright © 2011 Society of Chemical Industry

Synthesis of distinctly different sets of antimicrobial activities by elicited plant cell suspension cultures

The aim of this study was to evaluate the potential of plant suspension cultures for the production of antimicrobial activities. The extracellular, intracellular and cell wall bound fractions of 16 heterotrophic, photoautotrophic and photomixotrophic plant cell suspension cultures each treated with nine different elicitors were tested for the elicitor dependent production of antimicrobial activities. Distinctly different patterns of bioactivities directed against a panel of human isolates including Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria as well as fungi (Candida maltosa) were identified for all except the two autotrophic cell cultures. The intracellular fractions of elicited cell cultures were more active than extracellular fractions while cell wall bound fractions showed almost no activities. The intracellular fraction of heterotrophic Lavendula angustifolia cells elicited with a preparation of Pseudomonas syringae was the most active fraction against Candida maltosa. The intracellular fraction of photomixotrophic Arabidopsis thaliana cells elicited with salicylic acid was active against all test isolates. An antimicrobial protein could be identified and partially purified from this culture. Our findings suggest that elicited plant cell cultures may present a new promising alternative source of antimicrobial proteins.

The Comparison of Lutein Production by Scenesdesmus sp. in the Autotrophic and the Mixotrophic Cultivation

The results of this study indicate that an increase in CO(2) percentage to 30% can enhance Scenedesmus sp. growth in autotrophic cultivation to a maximum of 0.85 g/l as compared with 0.6 g/l obtained in the batch with air (after 6 days of cultivation). However, while the CO(2) was higher than 30%, it showed a negative impact on cell growth. A mixotrophic cultivation with 3 g/l of glycerol can achieve 0.38 g l(-1) day(-1) of the maximum biomass productivity compared with that of 0.21 g l(-1) day(-1) in autotrophic cultivation. Nevertheless, the lutein content of the mixotrophic cultivation was 0.08-0.1% lower than 0.2-0.25% obtained in autotrophic cultivation, which led to a lower lutein productivity of 0.36 mg l(-1) day(-1) in the mixotrophic batch compared with 0.44 mg l(-1) day(-1) obtained in the autotrophic batch. The limitation of cell growth in the mixotrophic cultivation would be the contributing factor regarding the lower lutein productivity. The mixotrophic cultivation of repeated batch to remove potential inhibitive metabolic products from glycerol catabolism does not show an obvious improvement on biomass. Conclusively, mixotrophic cultivation achieves higher biomass productivity with lower lutein content than that of autotrophic cultivation, which leads to lower lutein productivity. Therefore, the autotrophic cultivation is preferred in the lutein production.

Sequential dark–photo fermentation and autotrophic microalgal growth for high-yield and CO 2-free biohydrogen production

Dark fermentation, photo fermentation, and autotrophic microalgae cultivation were integrated to establish a high-yield and CO 2-free biohydrogen production system by using different feedstock. Among the four carbon sources examined, sucrose was the most effective for the sequential dark (with Clostridium butyricum CGS5) and photo (with Rhodopseudomonas palutris WP3-5) fermentation process. The sequential dark–photo fermentation was stably operated for nearly 80 days, giving a maximum H 2 yield of 11.61 mol H 2/mol sucrose and a H 2 production rate of 673.93 ml/h/l. The biogas produced from the sequential dark–photo fermentation (containing ca. 40.0% CO 2) was directly fed into a microalga culture ( Chlorella vulgaris C–C) cultivated at 30 °C under 60 μmol/m 2/s illumination. The CO 2 produced from the fermentation processes was completely consumed during the autotrophic growth of C. vulgaris C–C, resulting in a microalgal biomass concentration of 1999 mg/l composed mainly of 48.0% protein, 23.0% carbohydrate and 12.3% lipid.

EFFICIENCY OF GROWTH AND NUTRIENT UPTAKE FROM WASTEWATER BY HETEROTROPHIC, AUTOTROPHIC, AND MIXOTROPHIC CULTIVATION OF CHLORELLA VULGARIS IMMOBILIZED WITH AZOSPIRILLUM BRASILENSE1

Heterotrophic growth of microalgae presents significant economic advantages over the more common autotrophic cultivation. The efficiency of growth and nitrogen, phosphorus, and glucose uptake from synthetic wastewater was compared under heterotrophic, autotrophic, and mixotrophic regimes of Chlorella vulgaris Beij. immobilized in alginate beads, either alone or with the bacterium Azospirillum brasilense. Heterotrophic cultivation of C. vulgaris growing alone was superior to autotrophic cultivation. The added bacteria enhanced growth only under autotrophic and mixotrophic cultivations. Uptake of ammonium by the culture, yield of cells per ammonium unit, and total volumetric productivity of the culture were the highest under heterotrophic conditions when the microalga grew without the bacterium. Uptake of phosphate was higher under autotrophic conditions and similar under the other two regimes. Positive influence of the addition of A. brasilense was found only when light was supplied (autotrophic and mixotrophic), where affinity to phosphate and yield per phosphate unit were the highest under heterotrophic conditions. The pH of the culture was significantly reduced in all regimes where glucose was consumed, similarly in heterotrophic and mixotrophic cultures. It was concluded that the heterotrophic regime, using glucose, is superior to autotrophic and mixotrophic regimes for the uptake of ammonium and phosphate. Addition of A. brasilense positively affects the nutrient uptake only in the two regimes supplied with light.

Oil Accumulation via Heterotrophic/Mixotrophic Chlorella protothecoides

Microalgal oil is a potential energy source because it can be easily converted to fatty acid methyl ester or hydrocarbon type of diesel, and it is produced with relatively higher productivity compared with oil from plants and animals. Heterotrophic growth of microalgae is superior due to its high final product concentration; however, the cost of the raw materials is unacceptable if sugar is utilized as the carbon source. The aim of this study is to optimize the lipid accumulation of Chlorella protothecoides by using carbon sources other than glucose in heterotrophic and mixotrophic cultures. Different factors such as different carbon sources, carbon to nitrogen ratio, initial pH level, salinity, and rotational speed are studied in affecting the cell growth and the oil accumulation. Our experiments revealed that the heterotrophic and mixotrophic cultures of C. protothecoides grew better than autotrophic cultures. C. protothecoides can grow on glycerol or acetate, as well as on glucose. Several stress factors were confirmed or discovered to significantly increase the lipid content of microalgae cells. The replacement of glycerol and acetate as carbon sources for microalgae cultivations provides potential for waste utilization: glycerol from biodiesel industry and acetate from biohydrogen production.

Mycoparasitism of arbuscular mycorrhizal fungi: a pathway for the entry of saprotrophic fungi into roots

Within the rhizosphere, arbuscular mycorrhizal (AM) fungi interact with a cohort of microorganisms, among which is the biological control agent, Trichoderma spp. This fungus parasitizes a wide range of phytopathogenic fungi, a phenomenon also reported in the extraradical mycelium (ERM) of AM fungi. Here, we question whether the mycoparasitism of the ERM could be extended to the intraradical mycelium (IRM), thus representing a pathway for the entry of Trichoderma harzianum within the root. Microcosm experiments allowing interactions between Glomus sp. MUCL 41833 placed in a clade that contains the recently described species Glomus irregulare and T. harzianum were set up under in vitro autotrophic culture conditions using potato as a host. A microscope camera-imaging system, coupled with succinate dehydrogenase staining, was used to assess the mycoparasitism in the ERM and IRM. Trichoderma harzianum colonized the ERM of the AM fungus and spread into the IRM, before exiting into the root cells. Intrahyphal growth of T. harzianum caused protoplasm degradation, decreasing the ERM and IRM viability. ERM of the AM fungus represented a pathway for the entry of T. harzianum into the roots of potato. It further sets off the debate on the susceptibility of the AM fungi of being infected by microorganisms from the rhizosphere.

Carbon isotope fractionation in phospholipid fatty acid biomarkers of bacteria and fungi native to an acid mine drainage lake

This study identifies isotope signatures associated with autotrophic and heterotrophic microbial communities that may provide a means to determine carbon cycling relationships in situ for acid mine drainage (AMD) sites. Stable carbon isotope ratios (δ13C) of carbon sources, bulk cells, and membrane phospholipids (PLFA) were measured for autotrophic and heterotrophic microbial enrichment cultures from a mine tailings impoundment in northern Ontario, Canada, and for pure strains of the sulfur oxidizing bacteria Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. The autotrophic enrichments had indistinguishable PLFA distributions from the pure cultures, and the PLFA cyc-C19:0 was determined to be a unique biomarker in this system for these sulfur oxidizing bacteria. The PLFA distributions produced by the heterotrophic enrichments were distinct from the autotrophic distributions and the C18:2 PLFA was identified as a biomarker for these heterotrophic enrichments. Genetic analysis (16S, 18S rRNA) of the heterotrophic cultures indicated that these communities were primarily composed of Acremonium fungi.Stable carbon isotope analysis revealed that bulk cellular material in all autotrophic cultures was depleted in δ13C by 5.6–10.9‰ relative to their atmospheric CO2 derived carbon source, suggesting that inorganic carbon fixation in these cultures is carbon limited. Individual PLFA from these autotrophs were further depleted by 8.2–14.6‰ compared to the bulk cell δ13C, which are among the largest biosynthetic isotope fractionation factors between bulk cell and PLFA reported in the literature. In contrast, the heterotrophic bulk cells were not significantly fractionated in δ13C relative to their carbon source and heterotrophic PLFA ranged from 3‰ enriched to 4‰ depleted relative to the isotopic composition of their total biomass. These distinct PLFA biomarkers and isotopic fractionations associated with autotrophic and heterotrophic activity in this laboratory study provide potential biomarkers for delineating autotrophic and heterotrophic carbon cycling in AMD environments.

FISH Analysis of Bacterial Attachment to Copper Sulfides in Bioleaching Processes

Bioleaching is the biological conversion of an insoluble metal compound into a water soluble form. In this process metal sulfides are oxidized to metal ions and sulfate by acidophilic microorganisms capable of oxidizing Fe2+ and/or sulfur-compounds. The metal solubilization from sulfide minerals is a chemical process which requires Fe3+ reduction. It is an environmentally friendly technique and an economical method for recovering metals that requires low investment and operation costs. In this work we studied the bioleaching of two kinds of acid-soluble copper sulfides, one easily leached by mesophilic bacteria (covellite), and the other one refractory to their activity (chalcopyrite), in acidic media with or without Fe2+ ions. We studied attached and planktonic populations of autotrophic bacteria, such as Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans in pure or mixed cultures. The influence of a heterotrophic microorganism, Acidiphilium cryptum, was also studied. Attachment was evaluated with fluorescence staining and FISH using four specific probes. L. ferrooxidans showed highest initial attachment in all cases. The presence of Ap. cryptum increased the cell attachment compared with the autotrophic pure cultures. It was possible to correlate experimental data with a mechanism of bacterial-metal sulfide oxidation, the polysulfide pathway for acid- soluble metal sulfides.

Effect of nitrogen and extraction method on algae lipid yield

Effects of nitrogen source and concentration as well as lipid extraction method on the lipid yield of autotrophic Scenedesmus dimorphus and heterotrophic Chlorella protothecoides were studied. Three concentration levels of nitrate, urea and glycine/yeast extract as the nitrogen source were investigated. The highest lipid yield of S. dimorphus in the 17-d autotrophic culture was 0.40 g/L from the 1.8 g/L urea medium, and the maximum lipid yield of C. protothecoides in the nine-day heterotrophic culture was 5.89 g/L from the 2.4 g/L nitrate medium. Four different cell disruption methods— bead-beater, French press, sonication and wet milling— were studied for their effectiveness in solvent extraction of algal lipids from S. dimorphus and C. protothecoides . Wet milling followed by hexane extraction was most effective for S. dimorphus lipid extraction, whereas bead-beater disruption followed by hexane extraction was best for C. protothecoides . Key words: algae, nitrogen source, lipid content, lipid extraction, Scenedesmus dimorphus , Chlorella protothecoides DOI: 10.3965/j.issn.1934-6344.2009.01.051-057 Citation: Ying Shen, Zhijian Pei, Wenqiao Yuan, Enrong Mao. Effect of nitrogen and extraction method on algae lipid yield. Int J Agric & Biol Eng, 2009; 2(1): 51

Comparison of autotrophic and heterotrophic cultivations of microalgae as a raw material for biodiesel production

Comparison of autotrophic and heterotrophic cultivations of microalgae as a raw material for biodiesel production

High efficiency production of astaxanthin by autotrophic cultivation of Haematococcus pluvialis in a bubble column photobioreactor

Haematococcus pluvialis was cultivated under photoautotrophic conditions in a bubble column with fed-batch addition of nutrients, especially nitrate, and a cell number above 5 × 10 6 cells mL −1 was attained after 300 h.The reduction of nutrient concentrations accompanied by dilution of the fermentation broth and an increase in the light intensity enhanced accumulation of astaxanthin. The final astaxanthin concentration of 390 mg L −1 was several times higher than ever reported. This combination of fed-batch addition of nutrients and dilution of broth for nutrient deficiency is a promising method for attainment of high cell and astaxanthin concentrations in a bubble column photobioreactor.

Functional Characterization of the Microbial Community in Geothermally Heated Marine Sediments

The microbial population of geothermally heated sediments in a shallow bay of Vulcano Island (Italy) was characterized with respect to metabolic activities and the putatively catalyzing hyperthermophiles. Site-specific anoxic culturing media, most of which were amended with combinations of electron donors (glucose or carboxylic acids) and acceptors (sulfate), were used for selective enrichment of metabolically defined subpopulations. The mostly archaeal chemoautotrophs produced formate at rates of 3.25 and 0.46 fmol cell(-1) day(-1) with and without sulfate, respectively. The glucose fermenting heterotrophs produced acetate (18 fmol cell(-1) day(-1)) and lactate (2.6 fmol cell(-1) day(-1)) and were identified as predominantly Thermus sp. and coccoid archaea. These archaeal cells also metabolized lactate (5.6 fmol cell(-1) day(-1)), but neither formate nor acetate. The heterotrophic culture enriched on formate/ acetate/propionate/sulfate utilized mainly formate (27 fmol cell(-1) day(-1)) and lactate (89-195 fmol cell(-1) day(-1)), and consumed sulfate (38-68 fmol cell(-1) day(-1)). These formate or lactate consuming sulfate reducers were dominated by Archaeoglobales (7% in situ) and unidentified Archaea. The in situ benthic community comprised 15% Crenarchaeota, a significant group only in the autotrophic cultures, and 3% Thermus sp., the putatively predominant group involved in fermentative metabolism. The role of Thermoccales (4% in situ) remained undisclosed in our experiments. This first comprehensive data set established plausible links between several groups of hyperthermophiles in shallow marine hydrothermal systems, their metabolic function within the benthic microbial community, and biogeochemical turnover rates.