40-year long-term study of microbial parameters near Helgoland (German Bight, North Sea): historical view and future perspectives
Since 1873, the waters at Helgoland Roads (sampling station “Kabeltonne”) have been sampled daily to determine temperature and salinity. In 1962, microbiological parameters were determined for the first time to establish microbiological long-term studies on marine bacteria, starting with the colony-forming units (CFU). In the following years, several other microbiological parameters were integrated for different periods of time (e.g. activity parameters like ATP and ectoenzymatic activity, marine yeasts, oil-degrading bacteria, flagellates and molecular methods like PCR followed by denaturing gradient gel electrophoresis). To date, the total count of bacteria, flagellates and viruses have been acquired using fluorescent DNA dyes and epifluorescence microscopy. Here we present both a historical overview of the methods used and examples of results obtained over the past 40 years. Furthermore, we try to evaluate challenging new methods for marine microbial ecology, appropriate for long-term studies of marine bacteria.
KeywordsMarine bacteria Long-term studies Bacterial diversity Seasonal succession
Overview of the parameters acquired from the Microbiology Department of the Biologische Anstalt Helgoland at the sampling station “Kabeltonne”, Helgoland Roads from 1962 until today. Samples were mostly taken at weekly intervals. AODC acridine orange direct counting; BOD biological oxygen demand; CFU colony-forming units; DGGE denaturing gradient gel electrophoresis; F-DC flagellates direct counting; FBC flowcytometric bacterial counts; ODB oil-degrading bacteria; TOC total organic carbon; VLP-DC virus-like particles direct counting
pouring plate technique
spread plate technique
0.2 μm–3 μm
This is an attempt to evaluate the results from a historical point of view, and also considering the knowledge acquired by the scientific community during the last 15 years.
Samples were collected weekly from 1-m depth or from the neuston at the location “Kabeltonne” between the island of Helgoland and the adjacent dune island by the research boats Ellenbogen or Aade. For incubation experiments and cultivation of bacteria, samples were taken aseptically using a sterile ZoBell sampler. Neuston samples were taken with a fly-screen. Table 1 shows the recorded parameters and the corresponding sampling periods.
Bacteria and flagellate counts (AODC, F-DC) were carried out according to Zimmermann and Meyer-Reil (1974) as modified by Hobbie et al. (1977) using epifluorescence microscopy (Zeiss IM35 with acridine orange filter set). The estimation of bacterial biovolume and conversion in terms of carbon was performed according to Bratbak (1985). Prior to fixation with formalin (1% final concentration), samples were filtered through 60- or 20-µm nylon mesh to remove large planktonic cells.
Direct counts of virus-like particles (VLP-DC) were performed as described by Hennes and Suttle (1995), modified after Xenopoulus and Bird (1997). After removal of the bacterial fraction (0.15-µm pressure filtration) viruses were collected onto a 0.02-µm Anodisc membrane filter and rinsed twice with pre-filtered Nanopure water. Filters were stained with Yo-Pro-1 in the microwave (600 W) for 3–4 min and washed three times with Nanopure water after cooling down to room temperature. Epifluorescence microscopy was used to count viruses stained with Yo-Pro-1. For each sample, >200 viruses in 20 randomly selected fields were counted at a magnification of 1,600 using an epifluorescence microscope (Zeiss IM35) equipped with an acridine orange filter set.
Flowcytometric bacterial counts
Formalin-fixed (final concentration 1%) samples were stained with SYTOX (Molecular probes) according to the manufacturer’s instructions. As an internal standard, an aliquot of fluorescent beads (MF-RhB-beads; 2 µm; Specht GmbH) with known concentration was added. Samples were analyzed using an FACSCalibur Flowcytometer (Becton Dickinson) at 488 nm excitation wavelength and FL1 detection at 530/30 nm. The data were processed using the Paint a Gate Software (Becton Dickinson).
Colony-forming units (CFU)
Water samples were plated on marine broth 2216E (Oppenheimer and ZoBell 1952) using a Spiral Plater (Spiral Systems). For the cultivation of marine yeasts, samples were filtered on 0.45-µm membrane filters and the filters laid on top of an agar plate (Fell et al. 1960). The agar plates were incubated for 7 (yeasts) to 21 days (CFU) at 18°C in the dark. Bacterial or yeast colonies were counted using a dissection microscope or a magnifying glass.
The adenosine triphosphate (ATP) content of water samples was acquired according to Vosjan and Nieuwland (1987) using a LUMAC Biocounter M2500 and internal spiking with ATP.
Biological oxygen demand
The measurement of biological oxygen demand (BOD) is best described in Deutsche Einheitsverfahren für Wasser, Abwasser und Schlammuntersuchungen (1983). Samples were incubated for 21 days at 18°C in the dark.
The ectoenzymatic activity (leucine aminopeptidase; LAPase) was measured according to Somville and Billen (1983) and Hoppe (1993). As an experimental extension, additional to the incubation at in situ temperatures, a temperature range from 0°C to 20°C (in eight steps) was applied by using a thermatron (Christian and Karl 1995). MCA-leucine was used as the artificial substrate.
The abundance of oil-degrading bacteria was determined by an MPN method according to Gunkel (1973) using crude oil as the carbon source. Samples were incubated for 12 weeks at 18°C in the dark.
Bacterial diversity and seasonal succession
Bacterial diversity and seasonal succession was examined by PCR of the 16S-rDNA gene, followed by denaturing gradient gel electrophoresis (DGGE). Natural bacterioplankton was harvested by pressure filtration (5 litres) onto a filter cascade (3-µm cellulose nitrate filter, Sartorius, followed by 0.22-µm Sterivex GS capsule filter, Millipore). After filtration, the filters were stored frozen (−20°C). Nucleic acid extraction was performed using the modified protocol of Anderson and McKay (1983), omitting the denaturing NaOH step. When filters displayed a brownish colour caused by humic acids, PVPP was added (Holben et al. 1988). All DNA extracts served as template DNAs in the PCR. They were finally kept in TE buffer and stored at –20°C until used. Before PCR amplification, the DNA extracts were analyzed using agarose gel electrophoresis. A part of the 16S rDNA (V3 region) was amplified using primers P2/P3 according to Muyzer et al. (1993). The amplification of PCR products of the proper size (233 bp) was confirmed by electrophoresis through a 1.4% agarose gel. DGGE was performed with a DCODE system (Bio-Rad) according to Muyzer et al. (1993). PCR products of P2/P3 were applied on 6% (weight/volume) polyacrylamide (Appligene) gels in 0.5× TAE buffer with denaturing gradients of 15%–55% urea/formamide. The electrophoresis was run at 60°C and 140 V for 3 h. After electrophoresis, the gels were removed from the glass plates, soaked for 15 min in Nanopure water containing ethidium bromide (0.5 mg l−1), rinsed in Nanopure water overnight (12 h), and photographed with UV-transillumination (302 nm) using Polaroid MP4 equipment. Photographs were electronically digitized using a Linotype Saphir Ultra 2 scanner and Adobe Photoshop software.
Results and discussion
Direct counts and flowcytometric measurements
Bacterial diversity and seasonal succession
Samples for the analyses of the composition of the bacterial community by DGGE on the basis of the 16S-rDNA were taken weekly from August 1996 until the end of 1999. Water samples were fractionated by membrane filtration in two subsets representing “attached bacteria” (>3 µm) and “free-living bacteria” (<3 µm to >0.2 µm). DNA was successfully extracted from all filter samples. PCR amplification with the primers P2 and P3 resulted in DNA fragments of the expected size (233 bp), verified by agarose gel electrophoresis. To resolve the genetic variability of fragments of identical size, the DNA samples were subjected to DGGE. Since only a limited number of samples can be analysed on a single DGGE gel (limited slots), we decided to include standards generated from five randomly chosen bacteria, resulting in five distinct DGGE bands. It should be mentioned that, due to the nonlinear running behaviour of DNA fragments during DGGE, these standards cannot be used in the same way as molecular weight markers applied in native electrophoresis methods. In literature, there is a growing number of publications containing image-analysed DGGE results and derived UPGMA plots. But the transformation DGGE to UPGMA is problematic and should receive much more attention. Bands which are out of range of the standard cannot be interpolated. An interpolation algorithm itself implies that fragments are resolved in a linear manner, because software packages were developed for the analyses of native gels. Furthermore, it should be evaluated whether the analyses should be based on band indices (absence/presence) or, more likely, based on correlation of densitometric values. Hence, we decided to align the individual gels by eye until the pattern of standards, and also the general band pattern, of the corresponding samples overlapped.
For a long time in marine microbiology, CFU was the primary method in experimental work for the quantification and identification of marine bacteria. Beginning in 1962, Koch’s plate technique was applied to weekly sampling at the Biologische Anstalt Helgoland for the following 38 years (!). Until April 2000, when we stopped using plate techniques for bacterial quantification purposes, different media were applied and different habitats were sampled (see Table 1). All these efforts were based on the assumption that the bacteria which could be cultivated are somehow important to the ecosystem, while “the others” are dormant or inactive. Since the introduction of the microscopic direct count method by Hobbie et al. (1977) it has been obvious that there were indeed “others”. In 1985, Staley and Konopka (1985) coined the term “great plate count anomaly” for the discrepancy between bacterial counts derived from plating approaches on agar media and those from microscopic examination. By introducing molecular tools to marine microbiology, it can be assumed that the ability to form colonies on agar plates is probably restricted to certain fast-growing γ-proteobacteria. Besides these, some α-proteobacteria (Roseobacter), Bacteroidetes and gram-positive bacteria are commonly co-isolated, but the majority of bacteria which can be cultivated clearly belong to the genera Pseudoalteromonas, Alteromonas and Vibrio. In fact, compared to “the others”, these cultivable bacteria only occur in small numbers in marine waters and can hardly be detected by modern molecular tools (Eilers et al. 2000a, 2000b, 2001). On the other hand, it can be hypothesized that due to their ability to conserve high levels of ribosomes during periods of non-growth (Flärdh et al. 1992; Eilers et al. 2000b) these genera have the potential to outgrow other bacterial genera and are able to react preferentially to nutrient re-supply in the environment. For this opportunistic life strategy, Poindexter (1982) coined the term “feast-and-famine existence”.
Measurements of ATP and ectoenzymatic activity
organisms other than bacteria are co-extracted in spring;
“autumn” bacteria are less well extracted, or
“spring” bacteria actually contain more ATP.
Although the general mathematical conversion of ATP values to bacterial carbon cannot be recommended, it is obvious that either bacterial metabolic or population changes (or both) take place during early spring. This underpins the findings of the PCR-DGGE profiles, and also those of the ectoenzymatic activity measurements (see below).
The filtration of water samples generally leads to reduced activity in filtered fractions. On average, 29% (median 21%) of the initial activity could be measured for the <3 µm fraction, and 23% (median 17%) for the <0.2 µm fraction. Hence, at least two thirds of the proteolytic activity was particle-associated and was removed by filtration on 3-µm filters. Furthermore, since there was no measurable difference between the <3 µm and <0.2 µm fractions, one third of the total activity cannot be directly linked to bacterial cells and must be “free-enzyme”. This underpins the unique role of marine aggregates as “hotspots” in the water column, and also sources of enzymatic activity as already stated by different authors (Smith et al. 1992; Middelboe et al. 1995). It may even be possible that, during the colonisation of aggregates by bacteria, the expression of ectoenzymes can be “quorum-sensing” dependent (Gram et al. 2002), but this needs to be examined in future studies.
“Historical” parameters: oil-degrading bacteria, “marine” yeasts, biological oxygen demand, surface tension and TOC measurements
After the stimulating publication of Pomeroy (1974) on oceanic food webs, many methods were developed trying to elucidate this new idea. However, it seems that marine microbiologists are still at the stage of continuous method evolution to find tools for the description of the role of bacteria in the marine food web. In recent years, several promising molecular biology approaches have been adapted from medical science and have revolutionized marine microbial ecology. The most popular tools are based on 16S-rDNA signature sequences and are either probe- (FISH techniques) or primer-based (PCR techniques). For the first time, these tools permitted (and still permit) new insights, in terms of diversity and identity, into the in situ characteristics of marine bacterial populations. On the other hand, modern molecular methods can also be strongly biased. Hence multiphasic approaches, covering different molecular methods, are favourable.
One of these recently developed and promising methods combines FISH techniques with catalyzed reporter deposition (Pernthaler et al. 2002a) in order to enhance the hybridization signal (CARD-FISH; catalysed reporter deposition). By using an incubation technique with bromodeoxyuridine (BrdU) as a thymidine analogue, it is even possible to discriminate between DNA-synthesizing bacterial cells and dormant cells (Pernthaler et al. 2002b). The development of automated enumeration of FISH counts by the combination of epifluorescence microscopy and image analysis (Pernthaler et al. 2003), and the introduction of microfluidics-based flowcytometers to marine microbial ecology (Gerdts and Lüdke, in preparation) are also promising (and time-saving). A combination of FISH and micro-autoradiography could make it feasible to perform function analysis of specific bacterial communities or habitats in the environment (Lee at al. 1999).
Another approach is based on PCR techniques. DGGE, which has already been applied to our long-term studies of bacterial populations in the Helgoland Roads, was presented here. For future analysis of marine bacterial populations, we suggest a step-by-step approach beginning with PCR fingerprinting techniques based on native electrophoresis, such as ribosomal intergenic spacer analysis (RISA; Ranjard et al. 2001) or the analysis of repetitive extragenic palindromic sequences (repPCR; de Bruijn et al. 1996; Baker et al. 2003), which can be analyzed by automated image analysis software. With a PCR-DGGE approach, resolving larger 16S-rDNA fragments (Seibold et al. 2001; Schäfer et al. 2002; Wichels et al. 2004), it should be possible to identify OTUs by DGGE band sequencing and thus obtain a closer look at the bacterial community structure.
Recently it has been demonstrated that bacteriophages may be responsible for 20–50% of bacterial mortality in marine ecosystems, and are important in shaping microbial communities (Fuhrmann 1999; Wommack and Colwell 2000). To study the diversity of marine phages or viruses, pulsed field gel electrophoresis (PFGE) can be used to generate fingerprints of these viral communities (Wommack et al. 1999). At the Biologische Anstalt Helgoland, a culture collection of marine phage-host systems, containing more than 200 bacterial host strains and their specific bacteriophages has been built up during the last 15 years. Some host bacteria belong to the γ-subdivision of proteobacteria, and a few to the bacteroidetes phylum, but most remain unidentified. On the basis of the information provided by the complete sequence of the Roseophage genome SOI1 (Rohwer et al. 2000) the existence of similarities of functional genes for DNA polymerase, primase and endodesoxyribonuclease I was discovered. It would be interesting to screen the Helgoland culture collection of marine phage-host systems for these functional genes as a starting point for future specific primer and probe design. This approach has already been successfully used for phytoplankton viruses (e.g. Micromonas pusilla), pathogen viruses and coliphages (Gantzer et al. 1998; Griffin et al. 1999; Ikeda and Gray 1999). Furthermore, the data can provide exciting information on the potential transfer of functional genes between phages and bacteria. The expected insights into marine virioplankton diversity will grant the necessary knowledge to elucidate the influence of phages on the bacterial community structure.
The scientific work which has been performed at the Biologische Anstalt Helgoland reflects the development in general marine microbiology. Beginning in 1962, W. Gunkel initialized the microbial examinations at Helgoland Roads and the first water samples where analysed bacteriologically from the famous location “Kabeltonne”. Over the years, and of course according to the development of new methods and approaches (and fashions) by the scientific community, some methods were affiliated to the microbiological repertoire and data were recorded for a period of time. One might argue that the deeper reason for recording these data was, for example, to parameterize marine pollution or (as a view from today) global warming. Regarding oil-degrading bacteria or biological oxygen demand, the motivation of data collection is clear, but as pure basic science, some data were also recorded because it was possible to record these data and to evaluate methods suitable for marine microbial ecology. Some years ago, however, the molecular age changed our microbiological repertoire, and parameters like CFU were finally rejected. The aim of this review was to present an overview on the multitude of microbiological approaches performed at the Biologische Anstalt Helgoland during 40 years of studying bacteria in the German Bight.
As can be seen from the results presented in this study, each method used in the long-term studies in Helgoland Roads had its advantages and disadvantages, and only a well-tuned combination of new and already established methods will broaden our understanding of the role of bacteria in the marine food web and will help the scientific community to improve the knowledge of microbial oceanography. Based mainly on results of the PCR-DGGE profiling of bacterial diversity and seasonal succession over a longer period, we always observed two phases per year when dynamic shifts in the population structure occurred.
The oceanic food web, and especially the interactions between bacteria, phytoplankton and zooplankton, are still one of the most challenging systems. From the microbiological point of view, we are still at the beginning of understanding the marine microbial world, and hopefully we are becoming more aware of the tools and methods urgently needed. The integration of studies on marine bacteria into oceanographic long-term studies using new methods to identify organisms, structure and function of specific organisms, communities and habitats will be one of our greatest challenges in the future.
We are deeply indebted to Prof. W. Gunkel, who initiated the microbiological survey at the sampling station “Kabeltonne” near Helgoland in 1962. This long-term study was only possible due to his devoted work and a great number of laboratory technicians (E. Wollny, T. Hennemann, A. Sawall, E.H.J. Trekel, U. Henseleit) and crew members of the research boats “Ellenbogen” and Aade from the Biologische Anstalt Helgoland.
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