Use of BODIPY FL substrates to detect PHOs in activated sludge
BODIPY dye-labeled proteins have previously mainly been used to study the protease activity of purified enzymes (e.g. Welder et al., 2002), and only recently have they been applied also to study protease activity directly in a bacterial culture (Yoshioka et al., 2003). Our study has shown that BODIPY FL proteins can also be used successfully to label and identify PHOs in combination with FISH in activated sludge and thus gain more knowledge regarding the organisms involved in hydrolysis and consumption of proteins in complex microbial communities.
Both casein and BSA are used widely in studies of protein hydrolysis in relation to wastewater systems and are assumed to represent the proteins present in wastewater fairly well (Morgenroth et al., 2002). According to the provider (Molecular Probe, VWR International, Roedovre, Denmark) BODIPY FL casein can be used to detect a wide range of proteases including serine, acid, metallo and sulfhydryl proteases, thus ensuring reliable labeling of most bacterial PHOs. Information regarding the enzymes that are detected by BODIPY FL BSA is not available, but they may be similar to BODYPI FL casein as we found the same organisms in the samples investigated to be active for both proteins.
Several studies have previously shown that many exoenzymes in microbial aggregates are mainly associated with the cell surfaces (Confer & Logan, 1998; Goel et al., 1998; Kloeke & Geesey, 1999; Nielsen et al., 2002) and this was also observed for the proteases. Once quenched BODIPY dye-labeled casein and BSA were hydrolysed, fluorescent precipitates attached onto the surfaces of bacteria excreting the proteases thus enabling labeling of PHOs. However, some hydrolysates were released to bulk water, as also observed by Yoshioka et al. (2003), so bacteria able to take up these oligopeptides were also labeled. Furthermore, this release caused an increase in background fluorescence. Although protease activity is mainly associated with cell surfaces, the possibility that some bacteria primarily excrete protease into bulk liquid (and thus provide some background activity) cannot be ruled out. These bacteria would be missed from our PHO screening.
Inhibitors were successfully used to block the metabolic activity of all bacteria in the activated sludge, so potential consumers of the hydrolysates were inactivated and production of new exoproteases inhibited, allowing only detection of active PHOs at the time the incubation was initiated. Iodoacetate, fluoroacetate and azide were employed to inhibit glycolysis, the citric acid cycle (TCA cycle) and the electron transport chain, respectively, and activated sludge samples were incubated with inhibitors for at least 20 min before a BODIPY FL substrate was added. Rhodocyclus-related PAOs and some GAOs belonging to the Gammaproteobacteria (GB bacteria) were not stained with BODIPY FL casein or BSA in the presence of these inhibitors, but were fluorescent in the absence of inhibitors, indicating that they were not PHOs, but amino acid or oligopeptide consumers. This result agrees with the findings from previous studies (Kong et al., 2004, 2006) where microautoradiography combined with FISH showed that these two groups are capable of taking up certain amino acids. However, it was also noticed that some amino acid-utilizing actinobacterial PAOs (Kong et al., 2005), which were also present in the sludge samples examined, were not positively stained (results not shown). It is not clear whether it was because the amino acids they took up were not labeled by BODIPY dye or that they could not take up amino acids conjugated with BODIPY dye. By contrast, the epiflora and different filaments were still stained in the presence of the inhibitors, indicating strongly that extracellular proteases of these bacteria were present before the incubations started as proteases could not be produced in the presence of the inhibitors. Thus, the protease activity detected in this study for activated sludge was the in situ activity and reflected the real activity in wastewater treatment plants.
Some of the inhibitors used in this study not only inhibited the uptake of oligopeptides or amino acids, but also to some extent the activity of exoproteases. It was observed that the fluorescence intensity of the stained PHOs decreased with increased inhibitor concentrations. It is known that iodoacetate is an effective inhibitor of cysteine proteases (Vincents et al., 2004) whereas the effect of fluoroacetate and azide on protease activity is less clear. A decrease in the fluorescence intensity of the stained PHOs was often seen after 45–60 min of incubation. This may be due to dissolution of precipitated hydrolysates and inhibition of proteases caused by accumulation of hydrolysates. Therefore, the results show that it is important to test the inhibitor concentrations before they are applied to a new microbial community.
The background fluorescence levels increased with higher substrate concentrations due to the release of more hydrolysates with BODIPY dye into bulk liquid, thereby masking the fluorescence signals from PHOs. Therefore, for a new type of microbial community it is necessary to optimize substrate concentration and staining time. Another problem related to the quality of the FISH signals of Gram-negative PHOs and consumers of amino acids and/or oligopeptides was noticed. The staining generally caused the FISH signal to weaken and sometimes no FISH signal could be seen. This was probably due to problems associated with the inability of the probes to penetrate into the paraformaldehyde-fixed cells, and ethanol fixation was required to improve the penetration of the gene probes.
Given the requirements and optimizations discussed above, the new approach of combining FISH with BODIPY FL proteins is a very powerful method to identify the main functioning PHOs in activated sludge plants and other ecosystems. Furthermore, we also tried to quantify the protease activity per probe-defined population and compare this with the overall protease activity of the total community, as measured by the increase in fluorescence over time produced from BODIPY FL proteins or from methylumbelliferyl (MUF)-labeled substrates (Frolund et al., 1995). However, this proved to be very difficult owing to the increase in background fluorescence level and large variations in the exoenzymatic activity of the individual cells within a certain population, resulting in large variations in cellular fluorescence signal.
Identity and ecophysiology of the PHOs
A range of oligonucleotide probes were selected based on data obtained from our long-term study of these treatment plants, and these were then used in FISH probing to identify the PHOs. The dominant PHOs in three of the four plants investigated were rods attached as epiflora on different types of filamentous microorganisms. Most of them hybridized with probe SAP-309 targeting the family Saprospiraceae, phylum Bacteroidetes. They typically consisted of 8–12% of the bacterial biovolume. Epiphytic growth on filamentous microorganisms is commonly observed in activated sludge treatment plants, but their identity has never been revealed (Eikelboom, 2002; Thomsen et al., 2002). The SAP-309-targeted epiphytic bacteria have most likely not been detected in previous studies because they did not hybridize with the commonly applied probe (CF319a+b) targeting the Cytophaga–Flavobacterium group of the Bacteroidetes. However, coverage of the probe SAP-309 is relatively broad (Schauer & Hahn, 2005) and in addition to the epiflora, the probe also hybridized with some filamentous bacteria in the wastewater treatment plants investigated in this study. The detailed phylogeny of the epiphytic Saprospiraceae, therefore, awaits more detailed studies. However, they seem to form a unique ecological niche where by attaching themselves to filaments they form a three-dimensional ‘screen’ suited to trapping particulate organic matter, facilitating their role as PHOs (Fig. 1a–d).
The filaments attached with epiflora-PHOs were diverse, belonging to at least the phyla Chloroflexi and TM7 (candidate phylum) and class Proteobacteria. They could be identified as morphotypes commonly present in activated sludge from numerous studies worldwide (e.g. Eikelboom, 2002) and described as Eikelboom's Type 0041 (primarily from candidate phylum TM7), Type 1851 (primarily from Chloroflexi) and Type 1701 (mainly Aquaspirillum-related bacteria; Thomsen et al., 2006). Hence, the results strongly indicate that most filamentous bacteria in activated sludge with epiphytic growth are involved in protein degradation. However, the extent to which they were involved is not yet clear, and requires more data regarding the ecophysiology of the epiflora and their host, in particular the ecological relationship between them.
FISH probing also showed that several different filamentous bacteria were PHOs. Being less abundant but more diverse than the epiflora-PHOs in most of the sludge samples examined, the filamentous PHOs belonged again to the phylum Chloroflexi, candidate phylum TM7 and Aquaspirillum-related bacteria (Table 1). Most of these probes are very broad so the phylogeny and detailed ecophysiology of the filamentous PHOs require further investigation. For example, the filamentous PHOs hybridizing with CFX109 were only present in Aabybro, an N-removal plant consisting of nitrification and denitrification tanks, but not in the three other biological P removal plants containing not only nitrification and denitrification tanks but also anaerobic tanks (Biodenipho configuration). This suggests that a configuration-related selection may exist for the PHOs hybridizing with CFX109. The ecophysiology of Aquaspirillum-related bacteria has recently been investigated (Thomsen et al., 2004, 2006) and these filamentous organisms seem to be specialized in the consumption of amino acids, fitting well with the results obtained here showing them to be PHOs. Common to all these filamentous PHOs was that in most plants only a few of them had epiphytic bacteria. Whether this is due to the presence of different closely related filamentous species with different preferences for epiphytic growth (most probes applied were broad) or other factors, such as growth conditions, is not known.
PHOs with a morphotype of small cocci were also observed occasionally, but in much lower numbers than the epiflora and filamentous PHOs. They were not important PHOs in the sludge samples tested and no further effort was attempted to identify them. However, we realize that the amount of epiflora, filamentous and coccoid PHOs in each plant only represent the situation over a limited period (4–5 months), so the relative importance of these PHOs in a specific plant may change with time. However, the results obtained in this study indicate that only a few bacterial groups or populations were specialized PHOs and thus seem to be the key players in the turnover of protein in many activated sludge plants.