Final Report Summary - HUMADE (Structural and functional biodiversity of humic matter degrading freshwater microbial communities) Humic substances (HS) are an important carbon and energy source for microorganisms in aquatic ecosystems. However, we still lack a detailed understanding of the enzymatic diversity used by those microorganisms to attack the complex structure of these polymers. Considering the environmental and economic importance of HS it is extremely important to understand the biological mechanisms involved in the conversion of complex polymers in nature. HS are a complex and heterogeneous mixture of polymers formed in soils, sediments and natural waters by physico- and biochemical transformation of plant biomass (humification). HS represent an important carbon source in freshwater ecosystems accounting for up to 80 % of the total dissolved organic carbon (DOC) pool. Based on current structural concepts, it is assumed that HS cannot be degraded by simple enzymatic reactions, but require a large number of different enzymes.In order to investigate microbial contribution to HS degradation in freshwater ecosystems, we investigated the genomic and metabolic diversity of natural microbial communities using next-generation sequencing technologies. Using cultivation-independent metagenomic and metatranscriptomic approaches, we aimed to resolve the structural and functional biodiversity of freshwater microbial communities on a very fine scale. To enable comparative analysis between different microbial communities, we sampled two lakes in northeastern Germany at various occasions: Lake Stechlin and Lake Grosse Fuchskuhle. Although both lakes are located in the same area (Stechlin-Ruppiner Land Nature Park in the Mecklenburg-Brandenburg Lake District, Germany). They are very different in their limnological features. Lake Stechlin is a large oligotrophic lake with DOC concentrations of 4.3 mg L-1 whereas Lake Grosse Fuchskuhle is a small shallow lake adjacent to a peat bog area with DOC concentrations up to 24.8 mg L-1. In total we took 62 samples covering oxic versus anoxic conditions, low versus high HS concentrations as well as different seasons. Nucleic acids were extracted from the samples and subjected to sequencing. In addition to the molecular samples, we also measured a variety of environmental parameters for multivariate statistical analysis to link microbial communities to certain biogeochemical functions in the lakes.By the time of termination of this project, DNA from almost all samples has been extracted and sequencing of the first metagenomes was completed using either the Roche-454 or Illumina sequencing technology. Unfortunately, we did not had any transcriptome data available by the termination of the project due to difficulties in obtaining high-quality RNA suitable for sequencing. However, in the last weeks of HUMADE a protocol was developed overcoming these issues and scientist from the hosting Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB) will process those samples in the coming months to complete this part of the project even beyond the official funding period. We furthermore sequenced the genomes of three bacterial isolates that are potentially involved in freshwater HS turnover: Polynucleobacter sp. (1 isolate), Sphingomonas sp. (2 isolates). The Polynucleobacter strain was isolated from Lake Grosse Fuchskuhle. This bacterium is highly abundant in freshwaters (up to 20 % of the total community) and has been shown to persisting UV radiation as well as reactive oxygen species (ROS) that are produced in freshwaters by photolytic reactions of organic matter including HS. The two Sphingomonas isolates were selected for sequencing as they are phylogenetically very closely related but isolated from two very different lakes (oligotrophic vs. dystrophic). Both isolates showed active HS degradation in lab experiments and indicated to be capable of performing aerobic-anoxygenic photosynthesis under oxic conditions. This energy source might be one solution for these organisms to gain additional energy for the break-down of complex polymers.Bioinformatic analysis of the first two metagenomes comparing oxic vs. anoxic conditions revealed HS degradation capabilities in both communities. Up to 10 % of the genes identified were related to carbohydrate metabolism including cellulases and hemicellulases. Thereby, endo-active enzymes (e.g. endoglucanases) were only found in anaerobic communities. We also identified several enzymes potentially involved in direct degradation of HS (e.g. phenol hydroxylases or catechol dioxygenases). Interestingly, we found very similar degradation pathways for complex polymers in both communities, although the environmental conditions were very different (e.g. ortho-protocatechuate pathway). The reason for this is still unknown and will be further investigated. Additional analysis are planned for the future to obtain a more detailed overview of the microorganisms involved in HS transformation in the studied lakes and what ecological role they play in these ecosystems. Our preliminary data suggest that microbial degradation greatly differs between oxic and anoxic environments and that stratification and mixing patterns have a severe effect on microbial HS degradation. Similar analysis will be performed on the other data sets generated so far (currently ca. 17 Gbp of sequence data) and the data that will be generated in future beyond this project.A fundamental understanding of microbial contribution to HS turnover in nature is becoming more and more important. As we can already observe today, global climate change is leading to an increase of organic matter input in freshwater ecosystems with unforeseeable consequences. This is also the reason why scientists from the IGB will continue working on HUMADE to really obtain new insights into the biology of HS degrading freshwater microorganism from a diversity point of view as well as from a physiological perspective.