Jürgen Marxsen

Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes

ABSTRACT The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions.

Microbial communities in streambed sediments recovering from desiccation

Climate change affects running waters not only by increasing temperatures but also by increasing discharge variability as more frequent and severe floods and more frequent and longer droughts occur, especially in upper reaches. Mediterranean streams are known to experience droughts, but Central European headwaters are also beginning to be affected. The development of bacterial communities (abundance, composition) and the recovery of microbial functions (bacterial production, extracellular enzyme activity) were explored after rewetting desiccated streambed sediments via a sediment core perfusion technique. The bacterial community composition changed only slightly in the sediments from the Central European stream Breitenbach (Germany), but distinctly in the Mediterranean Mulargia River (Sardinia, Italy) during 4 days of experimental rewetting. Breitenbach sediments probably enabled survival of bacterial communities more similar to indigenous streambed communities, because they were less dry. High activity of enzymes involved in polymer degradation at the beginning of rewetting in both sediments indicated the persistence of extracellular enzymes during drought. After 4 days, nearly all microbial activities reached a level similar to unaffected sediments for the Breitenbach, but not for Mulargia. Here, much more intense drying resulted in a more distinct change and reduction of the microbial community, responsible for slower recovery of structure and functions.

Pioneering bacterial and algal communities and potential extracellular enzyme activities of stream biofilms

Microbial biofilms are important for the turnover of organic matter in small streams. A rapid colonization of the epilithic surface will become more important given the predicted increase of flood events. Here, we elucidated the pioneering community structure and activity of stream biofilms. Colonization of glass slides exposed in a small stream for 1, 4, 8, 12, and 24 h was compared with those exposed for 7 days or 5 months. Forty thousand microbial cells and 10 algae cm(-2) attached to the glass slides within 1 h of exposure. Catalyzed reporter deposition-FISH demonstrated that the pioneer community that settled within 12 h was dominated by Cytophago-Flavobacteria. Later stages were characterized by an enrichment of Gammaproteobacteria and Betaproteobacteria. However, a major fraction of the detected bacterial cells could not be identified beyond the domain level. Green algae dominated the pioneering algal groups, but were outnumbered by filamentous algae after the attachment period. Potential activity of alkaline phosphatase was already detected after 4 h, beta-glucosidase after 8 h, and beta-xylosidase only after 7 days of biofilm formation. Thus, biofilm formation occurred rapidly and the functionality of the assemblages was evident within a few hours. However, the ratios of beta-xylosidase : beta-glucosidase suggested that the initial biofilms relied more on autochthonous than on allochthonous carbon sources in contrast to mature biofilms.

Significance for Extracellular Enzymes for Organic Matter Degradation and Nutrient Regeneration in Small Streams

Extracellular enzymatic hydrolysis is the first step in the microbial degradation of macromolecular organic matter. Investigations into this process in lotic systems are scarce (e.g., Duddridge and Wainwright, 1982; Boon, 1989; Marxsen and Witzel, 1990), even though it may limit microbial substrate uptake and production. The first publications on extracellular enzymes in aquatic systems appeared more than 20 years ago (Overbeck, 1961; Reichardt et al., 1967; Chapter 1), but for a long time afterwards there were few investigations into this subject. One reason was probably the lack of an appropriate method. However, in recent years artificial substrates that release colored (Meyer-Reil, 1981; 1983; 1984; Hoppe et al., 1983) or fluorescent (Pettersson and Jansson, 1978; Hoppe, 1983; Somville and Billen, 1983; Rego et al., 1985; Chrost, 1990) compounds after enzymatic hydrolysis have been introduced into the field of aquatic microbial ecology. In particular, the fluorogenic model substrates have made sensitive, simple, and rapid measurements of enzyme activity possible.

Extracellular phosphatase activity in sediments of the Breitenbach, a Central European mountain stream

Activity of extracellular phosphatases (phosphomonoesterases) was measured in sandy streambed sediments of the Breitenbach, a small unpolluted upland stream in Central Germany. Fluorigenic 4-methylumbelliferyl phosphate served as a model substrate. Experiments were conducted using sediment cores in a laboratory simulation of diffuse groundwater discharge through the stream bed, a natural process occurring in the Breitenbach as well as many other streams.

Organic Matter Dynamics in the Breitenbach, Germany

1969, when the Breitenbach became the focus of investigations at the Limnological River Station of the Max Planck Institute of Limnology (Limnologische Flupstation des Max-Planck-Instituts fir Limnologie) in Schlitz. The stream fauna has been studied intensively since this time (e.g., I1lies 1971, Meijering 1971, Zwick 1984, Wagner 1986, Becker 1990), and only more recently have aspects such as chemistry, hydrology, bacteria, algae, and POM and DOM dynamics been considered (e.g., Brehm and Meijering 1982, Marxsen 1980, 1988, 1996, Cox 1990, Koch 1990, Marxsen and Witzel 1991, Fiebig and Marxsen 1992, Fiebig 1992). The stream catchment is almost completely forested, chiefly by Fagus sylvatica and Pinus sylvestris. The main channel of the Breitenbach is

Extracellular enzyme activities during slow sand filtration in a water recharge plant

Activities of the extracellular enzymes beta-glucosidase and phosphatase and bacterial densities were investigated during the filtration process at several sites in a groundwater recharge plant at the Ruhr river (Hengsen recharge plant in Schwerte. Germany). Low numbers of microorganisms and low levels of activity in this type of habitat, compared to most surface waters, caused methodological problems when determining microbial activity. In this study, fluorigenic model substrates, which enable hydrolytic rates as low as 1 nmol (L x h)(-1) to be measured, were used to determine extracellular enzyme activities. Highest activities were determined in surface water (107 nmol (L x h)(-1) for beta-glucosidase and 252 nmol (L x h)(-1) for phosphatase). which decreased during the filtration process in the gravel prefilter and the main sand filter until the end of subsurface flow (1.6 nmol (L x h)(-1) and 6.8 nmol (L x h)(-1), respectively). Similarly, bacterial numbers decreased from 3.4 x 10(6) to 0.29 x 10(6) cells mL(-1). These data showed that microbial activity within the prefilter and the shallow layers of the sand filter had the greatest impact on water quality. In addition to its involvement in the continuous purification of surface water, the microbial community in the sand filter probably acts as a biological buffer against ephemeral increases in the loads of organic matter and nutrients in the recharge plant.

Extracellular enzyme activity associated with degradation of beech wood in a central European stream.

The degradation of beech wood (Fagus sylvatica L.) was followed over 16 months in a central European upland stream, the Breitenbach. 1 cm 3 cubes of beech wood were placed on the stream bed and sampled at monthly intervals. Besides mass loss, fungal biomass (ergosterol content) and lignin content, the activity of two extracellular enzymes was measured: β-D-glucosidase, an enzyme involved in the degradation of cellulose, and phenoloxidase, a ligninolytic enzyme. The suitability of the fluorigenic model substrate methylumbelliferyl-β-D-glucoside for measuring β-D-glucosidase activity in wood from aquatic environments was tested. This technique is much more sensitive than the conventional photometric method. The beech wood was degraded at a constant rate of k = 0.00272 d -1 across the entire 16-month incubation period. There was a rapid onset of microbial colonisation, as witnessed by the initial detection of enzyme activity, after only 7 days of exposure. Lignin and ergosterol content as well as β-glucosidase activity reached their highest values at the end of the 16-month incubation period. Phenoloxidase activity increased rapidly to a maximum after 6 weeks, and then decreased to almost zero by the end of the experiment. The combination of biochemical techniques for measuring extracellular enzyme activities with measurements of mass loss, chemical composition and microbial colonisation provided valuable insights into the decomposition of wood in aquatic environments.

Bacterial Community Composition and Extracellular Enzyme Activity in Temperate Streambed Sediment during Drying and Rewetting

Droughts are among the most important disturbance events for stream ecosystems; they not only affect stream hydrology but also the stream biota. Although desiccation of streams is common in Mediterranean regions, phases of dryness in headwaters have been observed more often and for longer periods in extended temperate regions, including Central Europe, reflecting global climate change and enhanced water withdrawal. The effects of desiccation and rewetting on the bacterial community composition and extracellular enzyme activity, a key process in the carbon flow of streams and rivers, were investigated in a typical Central European stream, the Breitenbach (Hesse, Germany). Wet streambed sediment is an important habitat in streams. It was sampled and exposed in the laboratory to different drying scenarios (fast, intermediate, slow) for 13 weeks, followed by rewetting of the sediment from the fast drying scenario via a sediment core perfusion technique for 2 weeks. Bacterial community structure was analyzed using CARD-FISH and TGGE, and extracellular enzyme activity was assessed using fluorogenic model substrates. During desiccation the bacterial community composition shifted toward composition in soil, exhibiting increasing proportions of Actinobacteria and Alphaproteobacteria and decreasing proportions of Bacteroidetes and Betaproteobacteria. Simultaneously the activities of extracellular enzymes decreased, most pronounced with aminopeptidases and less pronounced with enzymes involved in the degradation of polymeric carbohydrates. After rewetting, the general ecosystem functioning, with respect to extracellular enzyme activity, recovered after 10 to 14 days. However, the bacterial community composition had not yet achieved its original composition as in unaffected sediments within this time. Thus, whether the bacterial community eventually recovers completely after these events remains unknown. Perhaps this community undergoes permanent changes, especially after harsh desiccation, followed by loss of the specialized functions of specific groups of bacteria.

Importance of Extracellular Enzymes for Biogeochemical Processes in Temporary River Sediments during Fluctuating Dry–Wet Conditions

Climate change represents an emerging problem for temporary waters by increasing the frequency and duration of drought periods, with potentially important effects on fluvial biogeochemical processes. Although benthic microbial processes have been recognized to have a most important role in carbon, nutrient and energy flux, few studies have documented the response to dry–wet cycles in temporary waters. Data so far obtained demonstrate that microbial communities are significantly affected by water stress conditions and that their structure changes with a drastic reduction of cell abundance, vitality and metabolic activity. But hydrolytic enzymes constitute an exception to this trend. They are preserved even if the cells in which they originated become non-viable. We postulate that the preservation of these enzymes represents an important mechanism for the fast recovery of surviving microbial cells after flooding and the re-establishment of the metabolic functions of microbial communities.