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.

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.