Electro-stimulation of tofu wastewater for the production of single cell protein from various microorganisms

Tofu wastewater can be utilized as a substrate for microorganisms that produce single-cell proteins (SCPs). Because different microorganisms have different cellular components, the composition of SCPs varies. Electro-stimulation has the potential to speed up fermentation and increase product yield. The goal of this study was to find the best way to produce SCPs from Aspergillus awamori, Rhizopus oryzae, and Saccharomyces cerevisiae in the tofu wastewater substrate using electro-stimulation. The experimental method was used in the study, the data were analyzed using independent t-test statistical analysis, and the best treatment was identified using the effective index method. This treatment consisted of producing SCP with electro-stimulation of −1.5 V and without electro-stimulation for 72 h for the yeast and 96 h for the mold at 25 °C in tofu wastewater that had already been conditioned to a pH of 5. The parameters measured included measurement of population of microorganism, change in pH, dry biomass weight, carbohydrate content, and protein content. Electro-stimulation reduced the optimum fermentation time of A. awamori SCP from 56 to 32 h, resulting in 0.0406 g/50 mL of dry biomass, 30.09% carbohydrate content, and 6.86% protein content. Meanwhile, the optimal fermentation time on R. oryzae and S. cerevisiae were not accelerated by electro-stimulation. The best treatment was A. awamori without electro-stimulation, which produced 0.0931 g/50 mL of dry biomass, 20.29% carbohydrate, and 7.55% protein.     

Effect of Sex and Age on the Supercooling Point of the Winter-Active Diamesa mendotae Muttkowski (Diptera: Chironomidae)

Supercooling points (SCP) of adult Diamesa mendotae Muttkowski, 1915 (Diptera: Chironomidae) were determined at 3, 5, 9, 12 and 17 days post-collection. Supercooling points were recorded using surface contact thermometry and a cooling rate of ca. 1°C min^?1. Female SCPs averaged ?22.81, ?23.76, ?23.85, ?23.65, and ?20.87°C on each date post-collection and did not differ significantly. Male SCPs were statistically similar and averaged ?21.75, ?23.53, ?23.68, ?23.66, and ?22.92°C on each date post-collection. Paired comparisons of female/male SCPs on each date post-collection did not show significant differences over time. The overall mean SCP of D. mendotae (?23.05°C) is substantially lower than values of ?5.3°C and ?5.7°C published for adults and larvae of Belgica antarctica Jacobs 1900 collected at Palmer Station (Antarctica) and ?14.2°C for larvae of Paraclunio alaskensis Coquillett 1900 collected at Vancouver Island, British Columbia. In addition, the SCP of this species appears to be lower than that of other winter-active insect species reported in the literature. Although no adults survived after the SCP was recorded, further studies are necessary to determine if D. mendotae is a freeze-intolerant insect. Nevertheless, our results suggest that a low SCP could be used as a mechanism to facilitate emergence and adult activity of this species during winter conditions.     

Overwintering strategy of two weevils infesting three gorse species: When cold hardiness meets plant-insect interactions

The cold hardiness of two closely related weevil species, Exapion ulicis and E. lemovicinum was studied in relation to their life cycles. These two seed-eating weevils reproduce on Ulex plant species with different fruiting phenologies. E. ulicis lays eggs in spring and overwinters as an adult while E. lemovicinum lays eggs in autumn and overwinters as a larva. Adult weevils were collected in natural populations of Brittany (Western France) and characterized with morphological and molecular tools before experiments. We showed that both weevil species exhibited low supercooling points (SCPs) with mean seasonal values below −17 °C. Fresh mass, moisture content and sex were not correlated to supercooling ability. Weevils died upon freezing and the lower lethal temperatures (LLT) were within the range of SCP, indicating that both species are freezing intolerant. Comparison between species for SCP, LLT and survival to exposure at −8 °C in winter showed a higher cold resistance for E. ulicis than for E. lemovicinum. In addition, the seasonal evolution of cold hardiness differed depending on the species. These features suggest that response to cold of weevils is linked to their life cycles, and thus to the life history of their host plants.     

Assessment of the spatial variability of intertidal benthic communities by axial tomodensitometry: importance of fine-scale heterogeneity

At the water-sediment interface of aquatic ecosystems, the presence of biogenic structures produced by benthic invertebrates strongly affects biogeochemical processes. The quantification of these structures and the assessment of the vertical distribution of fauna are essential for determining the impact of communities in sediments. In the present study, computer axial tomodensitometry (CAT-scan) was used to measure the space occupied by an intertidal community of the St. Lawrence estuary. Three cores were sampled at a site that was considered homogeneous according to surface sediments. The vertical distribution of biogenic structures and gravel were measured in the three cores using CAT-scan; the vertical distribution of fauna was also analysed for each core. The biogenic structures were highest at the water-sediment interface and decreased with depth in the three cores. The number of invertebrates also decreased with depth. We observed similar distributions of biogenic structures in cores 1 and 2. However, fewer biogenic structures were observed below 90 mm in core 3. This result was correlated with a high quantity of gravel from 90 to 140 mm in core 3 whereas the other cores had lower quantities of coarse material. We found relationships among the distributions of biogenic structures, fauna, and sediment characteristics (gravel quantity) that can affect species distribution. The vertical distributions of Macoma balthica, Mya arenaria, Nereis virens, and small-sized gallery-producing species (nematodes and oligochaetes) could also be recorded with the CAT-scan method. Thus, CAT-scan is an excellent tool to determine the fine-scale heterogeneity in the space occupied by benthic invertebrates in sediments.     

Diurnal variation in supercooling points of three species of Collembola from Cape Hallett, Antarctica

Daily changes in microclimate temperature and supercooling point (SCP) of Collembola were measured during summer at Cape Hallett, North Victoria Land, Antarctica. Isotoma klovstadi and Cryptopygus cisantarcticus (Isotomidae) showed bimodal SCP distributions, predominantly in the high group during the day and in the low group during the night. There were no concurrent diurnal changes in water content or haemolymph osmolality. By contrast, Friesea grisea (Neanuridae) had a unimodal distribution of SCPs that was invariant between daytime and nighttime. Isotoma klovstadi collected foraging on moss had uniformly high SCPs, which shifted towards the low group when the animals were starved for 2-8 h. When I. klovstadi was acclimated for five days with lichen or algae, SCPs were higher than if they were supplied with moss, while those that were starved (with free water or 100% relative humidity) displayed a trimodal SCP distribution. A variety of pre-treatments, including cold, heat, desiccation and slow cooling were ineffective at inducing SCP shifts in C. cisantarcticus or I. klovstadi. It is postulated that behavioural avoidance of low temperatures by vertical migration may be key in I. klovstadi's short-term survival of nighttime temperatures. These data suggest that the full range of thermal responses of Antarctic Collembola is yet to be elucidated.     

寄主对桔小实蝇耐寒性的影响

为了研究寄主营养对桔小实蝇耐寒性的作用,测定了以15种果蔬饲养的桔小实蝇1日龄蛹的过冷却点(supercooling points,SCP); 再选取南瓜、西红柿、柑桔、番石榴和杨桃等5种果蔬,测定了桔小实蝇3龄老熟幼虫、1日龄蛹、3日龄蛹、5日龄蛹、7日龄蛹和雌雄成虫的过冷却点,并观察了1日龄蛹的低温存活力。结果表明：（1）15种果蔬饲养所得的桔小实蝇1日龄蛹SCP均值在-11.03℃～-13.17℃,不同寄主发育的桔小实蝇SCP值存在显著性差异,其中以取食蒲桃的最高,为-11.03℃,取食苦瓜的最低,为-13.17℃。（2）5种果蔬饲养所得的桔小实蝇各虫态的SCP均值存在极显著差异(F_(4,863)＝35.6,P<0.01); 同一寄主上的桔小实蝇不同虫态之间SCP均值也达到极显著性差异(F_(6,863)＝392.9,P<0.01); 且寄主和发育龄期之间存在着极显著的交互作用(F_(24,863)＝9.4,P<0.01)。（3）桔小实蝇各发育阶段,SCP值表现一定变化： 老熟幼虫发育至1日龄蛹,SCP值变化不大; 蛹发育至3、5和7天过冷却能力明显增强,降至-20℃左右,但他们之间没有明显区别; 羽化后3～5天的成虫SCP值又升高至-10℃左右。老熟幼虫、1日龄蛹和2～3日龄成虫与3日龄、5日龄和7日龄蛹的SCP值之间有显著性差异。（4）将5种果蔬饲养所得的桔小实蝇1日龄蛹置于6℃和-3℃下进行较长时间(1～8天)和较短时间(1～8 h)的低温处理,发现番石榴、杨桃和南瓜发育的蛹经低温处理后的校正羽化率较西红柿和柑桔发育的蛹高; 同样在0℃、3℃、6℃和9℃处理(2天)的实验中,得出相似的结果。因此,本实验结果表明桔小实蝇幼虫由于生活寄主的不同使得其下一代蛹的耐寒性产生了差异,引起其差异的原因值得进一步研究。     

Radiometric dating of sediment records from mountain lakes in the Tatra Mountains

Sediment cores from nine different lakes in the Tatra Mountains, collected as part of the EU funded AL:PE, MOLAR and EMERGE projects investigating natural environmental records stored in remote mountain lake sediment sequences, were dated radiometrically by ²¹⁰Pb and ¹³⁷Cs. At five sites, D?ugi Staw G?sienicowy and Zielony Staw G?sienicowy on the Polish side of the Tatra Mountains and Starolesnianske pleso, Ni?né Terianske pleso, and L’adové pleso on the Slovak side of the Tatra Mountains, the cores were sectioned at close intervals and analysed in detail to produce a high resolution chronology. For the remaining four sites, Zmarzly Staw G?sienicowy (Poland), and Vel’ké Hincovo pleso, Vy?né Temnosmre?inské pleso, Vy?né Wahlenbergovo pleso (Slovakia), it was sufficient to establish a low resolution sketch chronology and only a few samples were analysed from each core. At L’adové pleso, multiple cores were collected in order to establish spatial distribution of sediments over the bed of the lake. Cores from all sites had good records of the fallout radionuclides from which it was possible to construct reliable chronologies of the recent sediments.     

Localization of the small CAB-like proteins in photosystem II

The cyanobacterial small CAB-like proteins (SCPs) consist of one-helix proteins that resemble transmembrane regions of the light-harvesting proteins of plants. To determine whether these proteins are associated with protein complexes in the thylakoid membrane, an abundant member of the SCP family, ScpD, was marked with a His tag, and proteins co-isolating with His-tagged ScpD were identified. These proteins included the major Photosystem (PS) II components as well as FtsH, which is involved in degradation of the PSII complex. To ascertain specific interaction between ScpD and the PSII complex, the His-tagged protein fraction was subjected to two-dimensional blue native/SDS-PAGE. Again, PSII components were co-isolated with ScpD-His, and ScpD-His was found to interact most strongly with CP47. ScpD association was most prominent with the monomeric form of PSII, suggesting ScpD association with PSII that is repaired. Using antibodies that recognize both ScpC and ScpD, we found the ScpC protein, which is very similar in primary structure to ScpD, to also co-isolate with the PSII complex. In contrast, ScpE did not co-isolate with a major protein complex in thylakoids. A fourth member of the SCP family, ScpB, could not be immunodetected, but was found by mass spectrometry in samples co-isolating with ScpD-His. Therefore, ScpB may be associated with ScpD as well. No association between SCPs and PSI could be demonstrated. On the basis of these and other data presented, we suggest that members of the SCP family can associate with damaged PSII and can serve as a temporary pigment reservoir while PSII components are being replaced.     

Säynäjälampi and the difficulties inherent in the dating of sediments in a hard-water lake

Previous measurements of the ¹⁴C activity of lake sediments and Potamogeton plants from Säynäjälampi, collected in 1970, indicated a severe hard-water effect (Donner et al. 1971). Samples from new cores, collected in 1977, were sliced into thin sections down to 18 cm. The ¹⁴C concentration in samples down to 7 cm was similar to that obtained by the Helsinki Laboratory. The corresponding apparent ¹⁴C age is almost 2 000 years. Light-coloured layers, apparently due to catchment drainage, were used together with density diagrams to correlate the cores. The drainage history gives a time-scale showing that the lowest samples dated to correlate the cores. The drainage history gives a time-scale showing that the lowest samples dated were just old enough to be pre-bomb sediments. Three samples of submerged plants were also examined, indicating some exchange with the air and an appreciable reservoir age. The activity of the plants was significantly lower in 1978 than in 1970. One sample, well below the lowest drainage stratum, indicates a reservoir age of ca. 1 100 years. This sample yielded an apparent age of the INS fraction which was significantly different from that of the SOL fraction. The difference was determined to ca. 600 years. Total ²¹⁰Pb was determined via ²¹⁰Po using isotope dilution while ²²⁶Ra was measured with the help of radon emanation. The ²²⁶Ra was used to estimate the supported ²¹⁰Pb. The unsupported ²¹⁰Pb showed a higher sediment accumulation rate for the top nine cm of one core (the length investigated was 16.5 cm). The varying accumulation rate was explained by human activities, as could be seen from the heterogenous sediment composition of the cores. Recent vegetational change is described from direct observations since 1957. Pollen analysis does not give any reliable indication of agriculture.     

The last occurrence of Proboscia curvirostris in the North Atlantic marine isotope stages 9-8

Well-preserved diatoms are present in high sedimentation rate Pleistocene cores retrieved on Ocean Drilling Program (ODP) Legs 151, 152, 162 and IMAGES cruises of R/V Marion Dufresne from the North Atlantic. Investigation of the stratigraphic occurrence of diatom species shows that the youngest diatom event observed in the area is the last occurrence (LO) of Proboscia curvirostris (Jousé) Jordan and Priddle. P. curvirostris is a robust species that can easily be identified in the sediments, and therefore can be a practical biostratigraphic tool. We have mapped its areal distribution, and found that it stretches from 40°N to 80°N in the North Atlantic. Further, we have correlated the LO P. curvirostris to the oxygen isotope records of six cores to refine the age of this biostratigraphic event. The extinction of P. curvirostris is latitudinally diachronous through Marine Isotope Stages (MIS) 9 to 8 within the North Atlantic. This is closely related to the paleoceanography of the area. P. curvirostris first disappeared within interglacial MIS 9 (324 ka) from the northern areas that are most sensitive to climatic forcing, like the East Greenland current and the sea–ice margin. It survived in mid-North Atlantic until the conditions of the MIS 8 (glaciation) became too severe (260 ka). In the North Pacific at ODP Site 883 the LO P. curvirostris falls within MIS 8. The observed overlap in age between the North Atlantic and the North Pacific strongly suggests that the extinction of P. curvirostris is synchronous between these oceans.     

Cold hardening processes in the Antarctic springtail, Cryptopygus antarcticus: clues from a microarray

The physiology of the Antarctic microarthropod, Cryptopygus antarcticus, has been well studied, particularly with regard to its ability to withstand low winter temperatures. However, the molecular mechanisms underlying this phenomenon are still poorly understood. 1180 sequences (Expressed Sequence Tags or ESTs) were generated and analysed, from populations of C. antarcticus. This represents the first publicly available sequence data for this species. A sub-set (672 clones) were used to generate a small microarray to examine the differences in gene expression between summer acclimated cold tolerant and non-cold tolerant springtails. Although 60% of the clones showed no sequence similarity to annotated genes in the datasets, of those where putative function could be inferred via database homology, there was a clear pattern of up-regulation of structural proteins being associated with the cold tolerant group. These structural proteins mainly comprised cuticle proteins and provide support for the recent theory that summer SCP variation within Collembola species could be a consequence of moulting, with moulting population having lowered SCPs.     

桔小实蝇不同发育阶段过冷却点的测定

对桔小实蝇Bactrocera dorsalis (Hendel)不同发育阶段的过冷却点进行了测定。结果表明：同一虫期个体间的过冷却点出现不同程度的变异,但均服从正态分布。不同虫期的过冷却点差异显著,其中蛹的过冷却点最低（-12.2℃～-15.0℃）。老熟幼虫的过冷却点为-8.1℃。成虫的过冷却点最低值为7日龄雄虫（-10.5℃）和雌虫（-10.1℃）,最高值为60日龄雄虫（-5.9℃）和雌虫（-6.4℃）,但同一发育时期的雌、雄成虫之间的过冷却点没有差异。测定结果提示蛹期最有可能是该虫在温带地区越冬的虫态。     

Diatom assemblages in sediments of Lake Juusa, Southern Estonia with an assessment of their habitat

Diatom analysis of surface sediments and two sediment cores from different sedimentation areas of a small closed lake was undertaken with the aim of acquiring knowledge on the dependence of the distribution of diatom assemblages on lake bathymetry. Lake Juusa was selected for the study because we have for this lake a large data set about the lithological composition of sediments and macrofossil and cladoceran records for the Holocene. A high carbonate content (20–60%) in the sediment sequence indicates high carbonacity and relatively stable pH values during the Holocene. On the basis of comprehensive analysis, abrupt water-level fluctuations and changes in the trophic status were established. Results of this study showed that the fluctuations of the water-level were the leading factor determining the habitats of diatom assemblages in the lake. In the surface sediment samples planktonic species such as Cyclotella spp., Stephanodiscus spp. and Aulacoseira spp. had a depth optimum at 3–4 m and the most abundant periphytic taxa were distributed mostly at depths shallower than 3.5 m. The same regularity was established in sediment cores where a good correlation between planktonic species and lake water depth was found in sediments accumulated at water depths >4 m. Lake Juusa appears to be a proper site for detailed environmental reconstructions over the Holocene, and the results will give us a good opportunity to analyse the history of water-level fluctuations in other small Estonian lakes. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: K. Martens     

宽翅曲背蝗卵的过冷却能力与抗寒性

采用热电偶法,在室内测定了宽翅曲背蝗卵的过冷却能力及抗寒性.结果表明： 土壤含水量对滞育前卵的含水量有显著影响, 而对卵过冷却点(SCP)的影响不显著,卵含水量随着土壤含水量的升高而上升.不同发育时期卵的SCP、含水量和脂肪含量存在显著差异.随着卵的发育,其含水量从产卵当天的51.5%下降至120 d的46.8%,脂肪含量从10.5%(鲜质量)/19.0%(干质量)上升到14.5%(鲜质量)/28.9%(干质量),而SCP从-23.5 ℃下降至-30.0 ℃;卵SCP与其含水量及脂肪含量存在显著相关关系;深度滞育卵的SCP显著低于滞育前和滞育初期卵的SCP.不同低温强度和处理时间对滞育卵的存活率有显著影响.滞育卵暴露12 h的致死温度为-27.3 ℃,在-25 ℃低温处理的致死时间为22.73 d.滞育卵的SCP与致死温度相近,说明宽翅曲背蝗卵为不耐结冰类型,且SCP是衡量其抗寒性的可靠指标.     

Radiometrically determined dates and sedimentation rates for recent sediments in nine North African wetland lakes (the CASSARINA Project)

Sediment cores were collected from nine wetland lakes in Morocco, Tunisia and Egypt for the CASSARINA project investigating environmental change in Northern African wetlands. The cores were dated radiometrically by using natural (²¹⁰Pb) and artificial (¹³⁷Cs and ²⁴¹Am) radionuclides. At sites in Morocco and Tunisia with mean annual rainfall totals ranging from 500–1000 mm yr^–1, fallout records were generally satisfactory and it was possible to develop independent sediment chronologies based on the radiometric data alone. At the Egyptian sites, rainfall was less than 200 mm yr^–1 and fallout records were much less distinct. At these sites the radiometric data could only be used to give an indication of mean sedimentation rates during the past 30–40 years. By using a combination of fallout radionuclide, pollen, and macrofossil stratigraphic records it was however possible to determine a credible sediment chronology spanning the major part of the 20th century. Applying this chronology to records of spheroidal carbonaceous particles (SCP) from the same sediment cores, the onset of significant levels of atmospheric pollution in the Nile Delta is dated in all three cores to the mid 1950s. Results from a number of lakes (Sidi Bou Rhaba, Ichkeul and Korba) revealed high and accelerating siltation rates, threatening their continued existence beyond the next few decades. In contrast, sedimentation rates at all three Nile Delta sites appear to have declined in recent decades, most probably due to the impact of the Nile barrages.     

Changes in the oceanic 13C/12C ratio during the last 140 000 years: High-latitude surface water records

Detailed records of the carbon and oxygen isotopic ratios of Neogloboquadrina pachyderma are compared between nine high-latitude sediment cores, from the Northern and Southern Hemispheres, covering the last 140 000 yrs. The strong analogies between the δ¹³C records permit to define a δ¹³C stratigraphic scale, with three clear cut transitions simultaneous with the oxygen isotopic transitions 6/5 (125 kyrs.), 5/4 (65 kyrs.), and 2/1 (13 kyrs.). The δ¹³C records of N. pachyderma in the high-latitude cores, which follow the changes in δ¹³C of the surface water TCO₂ near areas of deep water formation present trends similar to the benthic foraminifera δ¹³C records in cores V19–30 and M12-392, although amplitudes of the isotopic shifts are different. This implies that a large part of the observed variations represents global changes in the carbon distribution between biosphere and ocean.The ¹³C/¹²C ratios of N. pachyderma in the North Atlantic cores display larger regional variations at 18 kyrs. B.P. than at present. To explain these differences, we have plotted the 18 kyrs. B.P. δ¹³C values of N. pachyderma from 17 cores distributed N of 40°N. Comparison with published surface water temperature distribution at 18 kyrs. B.P. indicates that a strong divergent cyclonic cell, centered approximatively 55°N and 15°W, was active during most of the last ice-age maximum This hydrology, analogous to the present Weddell Sea, explains the published evidences of bottom water formation, if located on the northern flank of the gyre, and the strong polar front on the southern flank, probable location of intermediate water formation.     

A sexually dimorphic response in supercooling temperature, enhanced by starvation, in the lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae)

The effect of starvation on supercooling temperature (SCP) distribution was investigated in adults and larvae of Alphitobius diaperinus (Coleoptera: Tenebrionidae).

The mean values for SCPs of adults fed at 20°C were −14.5±2.4°C (31 males) and −10.3±1.3°C (29 females). The distribution of the SCPs of these control adults was unimodal. No significant differences were observed in either mean wet weight or mean dry weight between males and females.

The mean values for SCPs of adults starved for 1 month at 20°C were found to be bimodal due to sexual dimorphism. The mean SCPs for males was lower (−17±2.6°C; 28) than that for females (−11.2±1.8°C; 26). No significant differences were observed in either mean dry weight or wet weight between males and females.

The SCPs of both fed and starved larvae, kept for 1 month at 20°C were −12.3±2.4°C (fed) and −18.0±2.6°C (starved).     

Linkages between surface and deep circulations in the southern Japan Sea during the last 27,000 years: Evidence from planktic foraminiferal assemblages and stable isotope records

We analyzed planktic foraminiferal assemblages, oxygen and carbon isotope records, and the presence or absence of laminations to reconstruct the paleoenvironments of the southern Japan Sea since the last glacial period. Data were collected from two well-dated cores. One core (water depth 999 m) included thinly laminated mud layers, the other (water depth 283 m) contained nonlaminated sediments. Tephrochronology and accelerator mass spectrometry ¹⁴C dating of 14 horizons revealed that the two cores contained continuous records of the last 27 cal kyr. A total of 13 planktic foraminiferal species belonging to six genera were identified in down-core samples. The typical indicators of the Tsushima Current water, Globigerinoides ruber, Neogloboquadrina dutertrei, Pulleniatina obliquiloculata, Globigerinoides tenellus, and Globigerinita glutinata occurred since 9.3 cal kyr BP. Neogloboquadrina incompta, which was the dominant species in the Tsushima Current region of the modern Japan Sea, first occurred at 8.2 cal kyr BP and dominated the assemblage since 7.3 cal kyr BP. These results clearly indicate that the warm Tsushima Current started to inflow into the Japan Sea at 9.3 cal kyr BP, and the modern surface conditions in the southern Japan Sea were essentially established at 7.3 cal kyr BP. Our data and comparison of the presence or absence of laminated sediments in three locations from the southern Japan Sea suggest that deep circulation during the deglacial period was weaker than that at present. In addition, deep circulation in the modern Japan Sea, which supplies oxygen-rich water to the entire basin, started probably in association with the first inflow of the Tsushima Current beginning at 9.3 cal kyr BP.     

Microbial Communities from Methane Hydrate-Bearing Deep Marine Sediments in a Forearc Basin

Microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35°C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within the Euryarchaeota and Crenarchaeota domains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to the Bacteroidetes, Planctomycetes, Actinobacteria, Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments.