Young nis scientist fellowships programme - scientific part of application

Young NIS Scientist Fellowships Programme 2002
First Name:
Patronic Name:
Family Name:

Institute of Biology of the Southern Seas (IBSS), National Academy of Sciencies of Ukraine (NASU), 2, Nakhimov ave., Sevastopol 99011, Scientific Field & Expertise (half a page maximum)
In broad terms, I specialise in marine microbial ecology. A crucial realisation for this field in recent years is
the structural (in terms of biomass) and functional (in terms of matter/energy fluxes) dominancy of
bacterioplankton in the aquatic food webs. This power is encapsulated, in particular, by the concept of the
microbial loop’, the crux of which is in a unique ability of bacterioplankton to utilize non-living
particulate organic substances and up to 65% of the phytoplanktonic primary production (about 90 gigatons
C, yearly!) in the form of excreted dissolved compounds, and thereby to recycle the lost organic matter to
higher-order consumers back into the food web. My primary interest is in the possible contribution of the
much neglected but greatly abundant (up to 90% total bacterioplankton) ultramicrobacteria to the marine
food webs. They were discriminated from the ‘conventional’ bacterioplankton in Sieburth’s nomenclature
and classified as femtoplankton. Then, they were defined as metabolically inactive (dormant) cells. This
view is confirmed using indirect methods of marking metabolism, e.g. cyanoditolyl tetrazolium chloride for
‘respiring’ cells. In common with all plankton, the direct measurement of ultramicroplankton metabolism
and, as a result, of carbon/energy fluxes is heretofore an unsolved problem. A few years ago, I decided to
harness my expertise and understanding of the structure of the plankton, their allometrics and their
energtics within a thermodynamic basis to concentrate on solving the problem of the exact status of the
ultramicroplankton in the plankton community. Within the INTAS project 96-1961, we have proposed a
solution, directly measuring instantaneous energy fluxes by microcalorimetry to quantify total heat and
specific heat flux in natural bacterioplankton (paper in preparation for Appl. Environ. Microb.). Employing
the novel method in field studies, we have surprisingly found that planktonic femtofraction involving
ultramicrobacteria and non-metabolizing viruses reliably produces more heat per seawater volume than
picoplankton presented by heterotrophic bacteria, cyanobacteria and picoalgae! This phenomenon could be
explained by intense ultramicrobacterial metabolism or. by some extracellular processes. This can just
serve a subject for further inquiry (with unexpected implications for modern concepts!). I would like to
give the ultramicrobacteria a chance, in form of this application, to stand upon their rights and, possibly, to
augment the deserts of bacterioplankton as a whole.
Research Objectives (half a page maximum)
List the objectives in the form "To determine, to investigate etc."
The finding that the heat production of the femtoplankton is surprisingly high has determined the subject and overall aim of this study. To gain insight into the nature of this phenomenon and to estimate its possible implications for the present-day concepts of bacterioplankton energetics, the following particular objectives will be pursued: • to identify the source(s) of the heat energy dissipated by the planktonic femto-fraction (0.01 to 0.2 µm), differentiating between metabolism of the femtoplanktonic procaryotes and extracellular biochemical processes; • to quantify carbon/energy fluxes through the bacterial component of the femtoplanktonic community; • to update the conceptual model of the ‘microbial loop’ and its energy budget according to a novel status of the femtoplankton if the same is confirmed. Work Programme (maximum of 3 pages)
The first objective of the application (identification of the heat sources) implies some preliminary conceptual basis. Consequently, I have generated three alternative hypotheses (with the respective implications for the concept of aquatic microbial food web) which allow, firstly, to explain the phenomenon of high femtoplankton heat production and, secondly, to determine the research strategy: Hypothesis ‘INTRA’. The registered heat flow is associated with [intracellular] metabolism of living biomass contained in the femtofraction. The overall enthalpy change of extracellular biochemical reactions in the femtofraction is out of microcalorimeter detectability and can be neglected. Implications: The role of the femtoplankton in the pelagic microbial loop is not limited only by the impact of viruses and phages on bacterial and microalgal populations. Overall energy flux through the femtoplanktonic community (whose metabolism is represented by the ultramicrobacteria) exceeds those of picoplankton, including more abundant in terms of biomass heterotrophic bacteria, cyanobacteria, and picoalgae. If the hypothesis is confirmed, one has to refuse the paradigm of the picoplankton dominancy in carbon/energy cycling in the marine ecosystems. Hypothesis ‘EXTRA’. The heat flow by femtoplankton is too low to be detected by a calorimeter and can be neglected during the calorimetric measurements. The registered heat flow is associated with extracellular biochemical reactions, namely the process of enzymatic hydrolysis of macromolecular organic substances concentrated in the femtofraction. Implications: The result would support a widespread suggestion that ultramicrobacteria form the pool of dormant bacteria with extremely low metabolic activity. At the same time, an opportunity occurs to adapt microcalorimetric method for measuring in situ extracellular enzyme activity in the sea. The studies in this field could improve the picoplankton heat flux estimates obtained by our original method (V. Mukhanov et al., 2000). Hypothesis ‘SUM’. Both processes, the in situ hydrolytic activity (controlled by bacterial extracellular enzymes) and the metabolic activity of the femtoplankton, contribute to the registered heat flow. Implications: It is the most uncertain situation in which the correctness and accuracy of the used methods (especially when the components of the heat flow are distinguished) will determine if the work is successful. The ultramicrobacterial cell-specific heat flux has to be precisely quantified before any generalizations become possible. The methods general for all the research tasks. Seawater samples will be collected at two stations in Sevastopol Bay (the Black sea, Ukraine), fractionated and concentrated using Sartorius filtration equipment (Mukhanov et al., 2000). Bacterioplankton (picobacteria, PicoB), ultramicrobacteria (femtobacteria, FemtoB), and virus-like particles will be counted using standard epifluorescence microscopy (Carl Zeiss microscope with HBO-202 Hg burner) and the fluorochromes DAPI and proflavin, as described in Weinbauer & Suttle (1997) and Turley (1993) for viral and bacterial enumeration, respectively. The living and dead bacterial cells will be distinguished using a fluorescent dye, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) ‘marking’ the cells that have an active electron transport system (Rodriguez et al., 1992), and some specific antimicrobial agents (see the particular tasks below). The picophototrophs (cyanobacteria and picoalgae) will be identified by the red autofluorescence of the photosynthetic pigments. The instantaneous rate of heat flow of the concentrated samples will be measured by the original method (Mukhanov et al., 2000), using a BioActivity Monitor 2277 (Thermometric AB, Sweden). The research programme will be as follows: Task 1. Identification of the source(s) of the heat energy dissipated by the planktonic femtofraction. Within this task, I will differentiate between the two heat flow components associated with extracellular and intracellular processes, respectively. The goal will be reached by inhibition (and the whole expulsion) of the cellular metabolism. The FemtoB will be killed using a number of antimicrobial agents (ciprofloxacin, ofloxacin, tobramycin, and carbenicillin), following Spoering & Lewis (2001). The more effective agents or their cocktails will be determined. All the control and treated subsamples will be examined by microcalorimetry (for the heat flow measurements) and epifluorescence microscopy after CTC and DAPI staining (for total and CTC+ cell counts). Schedule: 1 to 4 months Task 2.1. Determination of the FemtoB physiological status (for the hypotheses INTRA and SUM only). The heat flow rate by the femtofraction will be measured by the same method that was used earlier for picoplankton (Mukhanov et al., 2000, and in press). To identify the metabolically active bacterial cells, several independent methods are to be used in parallel: (i) respiring (CTC+) cells will be determined by fluorescent red-orange CTC-formazan crystals in them (Rodriguez et al., 1992); (ii) viable cells will be distinguished by the original direct viable counts (DVC) method (Kogure et al., 1979). The cells will be incubated with nutrients and nalidixic acid, a quinolone antibiotic, which prevents cell division – as a result, nutrient-responsive cells elongate and may be enumerated microscopically. (iii) viable cells will also be assessed by an improved DVC method using antibiotic cocktail instead of nalidixic acid alone (Joux & LeBaron, 1997). After the calorimetric measurements, volume-, surface- and cell-specific heat fluxes will be estimated for PicoB and FemtoB from the same samples. Along with epifluorescence microscopy, a particle counter (ELZONE 280PS System) will be employed to determine total particle count and their size. Schedule: 4 to 8 months. Task 2.2. Quantifying the fluxes of carbon and energy through the femtoplanktonic community (for the hypotheses INTRA and SUM only). From the data obtained in the Tasks 2.1 and 2.2 and using conventional conversion factors (wet weight – dry weight – carbon, the oxycaloric equivalent, bacterial gross growth efficiency, etc.), the carbon and energy fluxes through the FemtoB community will be calculated per seawater volume and compared with the same indices obtained for the PicoB. Schedule: 8 to 9 months. Task 3.1. Measurement of bacterial extracellular enzyme activity (for the hypotheses EXTRA and SUM only). The hydrolytic capacity of the unaltered (after screening the sample through a membrane with pore size of 0.2 µm) and concentrated onto a membrane (nitrocellulose, 0.01 µm pore size) femtoplankton is to be measured by Hoppe’s method (1993), using fluorogenic model substrates. Schedule: 4 to 8 months Task 3.2 Calculation of the heat flux to enthalpy flux ratio by the enthalpy balance method (for the hypotheses EXTRA and SUM only). From the 1st Low of Thermodynamics and according to the enthalpy balance method (Kemp et al., 1997), the heat flux from a set of reactions (extracellular hydrolysis of organic substancies, in our case) must equal its enthalpy flux ratio calculated for the whole of the studied processes. The overall enthalpy of the process will be approximated from the data on its kinetics obtained in the Task 3.1. and the available data on composition and concentration of dissolved organic matter in different seawater fractions. Assuming the heat flux to enthalpy flux ratio equals unity (i.e. the enthalpy is balanced), the theoretical heat flux of the process will be estimated and compared with the experimental one obtained in the Task 1. Besides providing the necessary information on energy of the extracellular processes, this will be a validation of the Task 1 results. Schedule: 8 to 9 months. Task 4. The experimental session in Dr.Kemp’s laboratory (Wales University, UK). A month training in Aberystwyth (Wales, UK) is to involve the following activities: 1. Mastering the flow cytometry of natural planktonic samples using DAPI, CTC, SYBR Green I, 2. Conducting replicate experiments with pico- and femtoplanktonic samples collected in the in the coastal waters of the shoreline near Aberystwyth (Cardigan Bay) to reproduce the key results obtained in Sevastopol. The experiments will be combined with the flow cytometry (DAPI and CTC staining) of the pico- and femtoplankton as descibed in Sieracki et al. (1999). The results obtained will be discussed at an internal seminar. Task 5. Data generalization and presentation. The final theoretical generalizations will be mainly focused on energy flow and carbon cycling in planktonic ‘microbial loop’, with special emphasis on the role of femtoplankton. They will be based on mass-balanced flow diagrams (trophic webs) taking into account the magnitude of flows between the web compartments. According to the results obtained, the modified concept of the ‘microbial loop’ structure and its energy/carbon budget will be proposed. The task involves the performance of necessary statistical analysis and theoretical generalizations, preparing the data for the final report, presentation at conferences, and publication in a Western journal(s). Schedule: 10 to 12 months. References: Hoppe H.-G. (1993). Use of fluorogenic model substrates for extracellular enzyme activity (EEA) measurement of bacteria. In: Kemp P.F., Sherr B., Sherr E., Cole J.J. (eds.) Handbook of methods in aquatic microbial ecology. Lewis Publishers, Boca Raton, p. 423-432. Joux F., LeBaron Ph. (1997). Ecological implications of an improved direct viable count method for aquatic bacteria. Appl. Environ. Microb. 63(9): 3643-3647.
Kemp R.B., Evans P.M., Guan Y. (1997). An enthalpy balance approach to the study of metabolic activity in mammalian cells. J. of Thermal Anal. 49: 755-770.
Kogur K., Simidu U., Taga N. (1979). A tentative direct microscopic method for counting living marine bacteria. Can. J. Microbiol. 25: 415-420.
Mukhanov V.S., Rylkova O.A., Lopukhina O.A., Kemp R.B. The microcalorimetry of the natural bacterioplankton: a methodological investigation. 3rd INTAS Interdisciplinary Symposium on “General Biochemistry, Biotechnology and Environment”, Moscow University, Moscow, 14-17 December, 2000, pp. 12-13. Mukhanov V.S., Rylkova O.A., Lopukhina O.A., Kemp R. B. Productivity and thermodynamics of marine bacterioplankton: an inter-ecosystem comparison. Thermochimica Acta, in press. Rodriguez G.G., Phipps D., Ishiguro K., Ridgway H.F. (1992). Use of fluorescent redox probe for direct vizualization of actively respiring bacteria. Appl. Environ. Microbiol. 58: 1801-1808.
Sieracki M.E., Cucci T.L., Nicinski J. (1999). Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures. Appl. Environ. Microb. 65(6): 2409-2417.
Spoering A.L., Lewis K. (2001). Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J. of Bacteriol. 183(23): 6746-6751.
Turley C.M. (1993). Direct estimates of bacterial numbers in seawater samples without incurring cell losess due to sample storage. In: Kemp P.F., Sherr B., Sherr E., Cole J.J. (eds) Handbook of methods in aquatic microbial ecology. Lewis Publishers, Boca Raton, p. 143-147. Weinbauer M.G., Suttle C.A. (1997). Comparison of epifluorescence and transmission electron microscopy for counting viruses in natural marine waters. Aquatic Microb. Ecol. 13: 225-232.


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