The Chemical Transformation of the Cellular Toxin INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) as an Indicator of Prior Respiratory Activity in Aquatic Bacteria.

In the ocean, the prokaryote respiration rates dominate the oxidation of organics, but the measurements may be biased due to pre-incubation size filtration and long incubation times. To overcome these difficulties, proxies for microbial respiration rates have been proposed, such as the in vitro and in vivo estimation of electron transport system rates (ETS) based on the reduction of tetrazolium salts. INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) is the most commonly applied tetrazolium salt, although it is toxic on time scales of less than 1 h for prokaryotes. This toxicity invalidates the interpretation of the rate of in vivo INT reduction to formazan as a proxy for oxygen consumption rates. We found that with aquatic bacteria, the amount of reduced INT (F; µmol/L formazan) showed excellent relation with the respiration rates prior to INT addition (R; O2 µmol/L/hr), using samples of natural marine microbial communities and cultures of bacteria (V. harveyi) in batch and continuous cultures. We are here relating a physiological rate with the reductive potential of the poisoned cell with units of concentration. The respiration rate in cultures is well related to the cellular potential of microbial cells to reduce INT, despite the state of intoxication.

Substrate-Limited and -Unlimited Coastal Microbial Communities Show Different Metabolic Responses with Regard to Temperature.

Bacteria are the principal consumers of dissolved organic carbon (DOC) in the ocean and predation of bacteria makes organic carbon available to higher trophic levels. The efficiency with which bacteria convert the consumed carbon (C) into biomass (i.e., carbon growth efficiency, Y) determines their ecological as well as biogeochemical role in marine ecosystems. Yet, it is still unclear how changes in temperature will affect Y and, hence, the transfer of consumed C to higher trophic levels. Here, we experimentally investigated the effect of temperature on metabolic functions of coastal microbial communities inoculated in both nutrient-limited chemostats and nutrient-unlimited turbidostats. We inoculated chemostats and turbidostats with coastal microbial communities into seawater culture medium augmented with 20 and 100 µmol L-1 of glucose respectively and measured CO2 production, carbon biomass and cell abundance. Chemostats were cultured between 14 and 26°C and specific growth rates (µ) between 0.05 and 6.0 day-1, turbidostats were cultured between 10 and 26°C with specific growth rates ranging from 28 to 62 day-1. In chemostats under substrate limitation, which is common in the ocean, the specific respiration rate (r, day-1) showed no trend with temperature and was roughly proportional to µ, implying that carbon growth efficiency (Y) displayed no tendency with temperature. The response was very different in turbidostats under temperature-limited, nutrient-repleted growth, here µ increased with temperature but r decreased resulting in an increase of Y with temperature (Q10: 2.6). Comparison of our results with data from the literature on the respiration rate and cell weight of monospecific bacteria indicates that in general the literature data behaved similar to chemostat data, showing no trend in specific respiration with temperature. We conclude that respiration rates of nutrient-limited bacteria measured at a certain temperature cannot be adjusted to different temperatures with a temperature response function similar to Q10 or Arrhenius. However, the cellular respiration rate and carbon demand rate (both: mol C cell-1 day-1) show statistically significant relations with cellular carbon content (mol C cell-1) in chemostats, turbidostats, and the literature data.

The Vitamin B1 and B12 Required by the Marine Dinoflagellate Lingulodinium polyedrum Can be Provided by its Associated Bacterial Community in Culture.

In this study we established the B1 and B12 vitamin requirement of the dinoflagellate Lingulodinium polyedrum and the vitamin supply by its associated bacterial community. In previous field studies the B1 and B12 demand of this species was suggested but not experimentally verified. When the axenic vitamin un-supplemented culture (B-ns) of L. polyedrum was inoculated with a coastal bacterial community, the dinoflagellate's vitamin growth limitation was overcome, reaching the same growth rates as the culture growing in vitamin B1B7B12-supplemented (B-s) medium. Measured B12 concentrations in the B-s and B-ns cultures were both higher than typical coastal concentrations and B12 in the B-s culture was higher than in the B-ns culture. In both B-s and B-ns cultures, the probability of dinoflagellate cells having bacteria attached to the cell surface was similar and in both cultures an average of six bacteria were attached to each dinoflagellate cell. In the B-ns culture the free bacterial community showed significantly higher cell abundance suggesting that unattached bacteria supplied the vitamins. The fluorescence in situ hybridization (FISH) protocol allowed the quantification and identification of three bacterial groups in the same samples of the free and attached epibiotic bacteria for both treatments. The relative composition of these groups was not significantly different and was dominated by Alphaproteobacteria (>89%). To complement the FISH counts, 16S rDNA sequencing targeting the V3-V4 regions was performed using Illumina-MiSeq technology. For both vitamin amendments, the dominant group found was Alphaproteobacteria similar to FISH, but the percentage of Alphaproteobacteria varied between 50 and 95%. Alphaproteobacteria were mainly represented by Marivita sp., a member of the Roseobacter clade, followed by the Gammaproteobacterium Marinobacter flavimaris. Our results show that L. polyedrum is a B1 and B12 auxotroph, and acquire both vitamins from the associated bacterial community in sufficient quantity to sustain the maximum growth rate.

INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) Is Toxic to Prokaryote Cells Precluding Its Use with Whole Cells as a Proxy for In Vivo Respiration.

Prokaryote respiration is expected to be responsible for more than half of the community respiration in the ocean, but the lack of a practical method to measure the rate of prokaryote respiration in the open ocean resulted in very few published data leaving the role of organotrophic prokaryotes open to debate. Oxygen consumption rates of oceanic prokaryotes measured with current methods may be biased due to pre-incubation size filtration and long incubation times both of which can change the physiological and taxonomic profile of the sample during the incubation period. In vivo INT reduction has been used in terrestrial samples to estimate respiration rates, and recently, the method was introduced and applied in aquatic ecology. We measured oxygen consumption rates and in vivo INT reduction to formazan in cultures of marine bacterioplankton communities, Vibrio harveyi and the eukaryote Isochrysis galbana. For prokaryotes, we observed a decrease in oxygen consumption rates with increasing INT concentrations between 0.05 and 1 mM. Time series after 0.5 mM INT addition to prokaryote samples showed a burst of in vivo INT reduction to formazan and a rapid decline of oxygen consumption rates to zero within less than an hour. Our data for non-axenic eukaryote cultures suggest poisoning of the eukaryote. Prokaryotes are clearly poisoned by INT on time scales of less than 1 h, invalidating the interpretation of in vivo INT reduction to formazan as a proxy for oxygen consumption rates.

Maintenance of coastal surface blooms by surface temperature stratification and wind drift.

Algae blooms are an increasingly recurrent phenomenon of potentially socio-economic impact in coastal waters globally and in the coastal upwelling region off northern Baja California, Mexico. In coastal upwelling areas the diurnal wind pattern is directed towards the coast during the day. We regularly found positive Near Surface Temperature Stratification (NSTS), the resulting density stratification is expected to reduce the frictional coupling of the surface layer from deeper waters and allow for its more efficient wind transport. We propose that the net transport of the top layer of approximately 2.7 kilometers per day towards the coast helps maintain surface blooms of slow growing dinoflagellate such as Lingulodinium polyedrum. We measured: near surface stratification with a free-rising CTD profiler, trajectories of drifter buoys with attached thermographs, wind speed and direction, velocity profiles via an Acoustic Doppler Current Profiler, Chlorophyll and cell concentration from water samples and vertical migration using sediment traps. The ADCP and drifter data agree and show noticeable current shear within the first meters of the surface where temperature stratification and high cell densities of L. polyedrum were found during the day. Drifters with 1m depth drogue moved towards the shore, whereas drifters at 3 and 5 m depth showed trajectories parallel or away from shore. A small part of the surface population migrated down to the sea floor during night thus reducing horizontal dispersion. The persistent transport of the surface bloom population towards shore should help maintain the bloom in favorable environmental conditions with high nutrients, but also increasing the potential socioeconomic impact of the blooms. The coast wise transport is not limited to blooms but includes all dissolved and particulate constituents in surface waters.

Marine heterotrophic bacteria in continuous culture, the bacterial carbon growth efficiency, and mineralization at excess substrate and different temperatures.

To model the physiological potential of marine heterotrophic bacteria, their role in the food web, and in the biogeochemical carbon cycle, we need to know their growth efficiency response within a matrix of different temperatures and degrees of organic substrate limitation. In this work, we present one part of this matrix, the carbon growth efficiencies of marine bacteria under different temperatures and nonlimiting organic and inorganic substrate supply. We ran aerobic turbidostats with glucose enriched seawater, inoculated with natural populations of heterotrophic marine bacteria at 10, 14, 18, 22, and 26 degrees C. The average cell-specific growth rates increased with temperature from 1.17 to 2.6 h-1. At steady-state total CO2 production, biomass production [particulate organic carbon (POC) and nitrogen (PON)], and viruslike particle abundance was measured. CO2 production and specific growth rate increased with increasing temperature. Bacterial carbon growth efficiency (BCGE), the particulate carbon produced per dissolved carbon utilized, varied between 0.12 and 0.70. Maximum BCGE values and decreased specific respiration rates occurred at higher temperatures (22 and 26 degrees C) and growth rates. This trend was largely attributable to an increase in POC per cell abundance; when the BCGE was recalculated, parameterizing the biomass as the product of cell concentration and a constant cellular carbon content, the opposite trend was observed.