Serge Parent

Phosphorus budget as a water quality management tool for closed aquatic mesocosms.

Since the start-up of the St. Lawrence Mesocosm (SLM) at the Montreal Biodome in 1992, phosphorus has accumulated slowly, reaching about 18 mg P l(-1) in 2000. It was decided that this concentration should be lowered to about 2mg P l(-1) to maintain a safe nitrogen:phosporus (N:P) ratio of about 10. Before deciding what type of treatment to use for the removal of phosphorus, a P budget was estimated for 1998 in order to evaluate the different pathways of phosphorus in the mesocosm. The resulting budget had only a 1% difference between the inputs (CV = 12.9%) and the sum of the outputs and changes in P pools (CV = 12.5%). P inputs amounted to 40.5 kg for 1998: food for fish and invertebrates contributed 76% of the inputs while seabird guano contributed 20%. Filtration and general cleaning removed 51% of the inputs while water losses removed 22%. The slight but constant difference between total phosphorus and dissolved reactive phosphorus (DRP) in 1998 and previous years led us to believe that only DRP (mostly orthophosphate) accumulated in the system. The accumulation of DRP was 10% of the inputs in 1998. The budget showed that the importance of water losses is relative and depends on the DRP concentration in the SLM. Furthermore, it was possible to compare this P budget with an N budget of the SLM prepared in 1995. The comparison helped us understand why nitrate in closed-circuit mesocosms are characterized by a high, never-ending accumulation while DRP is characterized by a net increase in the first few years after start-up followed by a very small increase in the following years. Considering its low CV, this P budget was considered a useful water quality management tool in designing a P removal unit for the SLM. This budget may also serve as a guideline for managers of closed-circuit systems such as marine aquariums and aquacultures as well as for designers of P removal units.

Bacterial Diversity in a Marine Methanol-Fed Denitrification Reactor at the Montreal Biodome, Canada

The bacterial biota of a methanol-fed denitrification reactor used to treat seawater at the Montreal Biodome were investigated using culture-dependent and molecular biology methods. The microbiota extracted from the reactor carriers were cultivated on three media. Three isolate types were recovered and their 16S ribosomal DNA (rDNA) genes were determined. The analysis showed that the isolate types were related to α-Proteobacteria. They are members of the Hyphomicrobium and Paracoccus genera and the Phyllobacteriaceae family. Uncultured bacteria were identified through a 16S rDNA library generated from total DNA extracted from the microbiota. Clones were screened for different restriction profiles and for different DGGE (denaturing gradient gel electrophoresis) migration profiles. More than 70% of clones have the same restriction profile, and the sequence of representative clones showed a relation with the Methylophaga members of the Piscirickettsia family (γ-Proteobacteria). Sequences from other profiles were related to bacterial species involved in denitrification. The number of species in the denitrification reactor was estimated at 15. Bacterial colonization on newly added carriers in the denitrification reactor was monitored by PCR-DGGE. The DGGE migration profiles evolved during the first 5 weeks and then remained essentially unchanged. PCR-DGGE was also used to monitor the microbial profiles in various aquarium locations. As expected, bacterial populations differed from one location to another, except for the sand and trickling filters which presented similar DGGE migration profiles.

Addition of trace metals increases denitrification rate in closed marine systems.

We investigated the effect of trace metals (Fe, Mn, Cu, Zn and Mo) on the denitrification unit at the Montreal Biodome. Two dosages of the five trace metals were tested on a denitrifying bacterial population which was extracted from the denitrification unit and cultured in 250 mL chemostats with artificial seawater. The low dosage showed a 20% increase in the denitrification rate whereas the high dosage had a more pronounced effect with a 250% increase. No increase in bacterial growth was observed, suggesting that the trace metals had an effect on the denitrification activity. When the trace metals were tested separately, only iron had a significant effect similar to the increase in the denitrification rate observed when the five trace metals were added. The combination of Fe and Mn caused a small but significant increase compared to the five trace metals. We then tested the effect of adding Fe, Mn and Cu to the denitrification unit at the Montreal Biodome. A high dosage of these trace metals showed a 250% increase in the denitrification rate, which went from 200 to 700 g NO(x)-N/d. Our results showed that the addition of trace metals is crucial for denitrification activities.

Dissimilatory reduction of nitrate in seawater by a Methylophaga strain containing two highly divergent narG sequences.

Methylophaga spp. are methylotrophs commonly associated with marine environments and have been defined as strict aerobic methylotrophs. They have been shown previously to represent 50–70% of the bacterial population in the biofilm of the methanol-fed denitrification reactor treating a large seawater aquarium at the Montreal Biodome. It was therefore surprising to find such a high concentration of Methylophaga spp. in anoxic conditions. In this study, we showed by cultivation-independent and -dependent approaches that one Methylophaga strain present in the anoxic biofilm is involved in the denitrification process. DNA stable-isotope probing (SIP) experiments in which the biofilm was cultured under denitrifying conditions with 13C-methanol have revealed the enrichment of one particular taxon. By screening a 16S ribosomal RNA gene library derived from a 13C-DNA fraction of the SIP gradients, 62% of the library was composed of one sequence affiliated with the genus Methylophaga. One strain, named JAM1, representing this Methylophaga species was isolated. It grows aerobically but also under denitrifying conditions by reducing nitrate into nitrite. The nitrate-reducing activity was correlated with the presence and the expression of two highly divergent narG genes (narG1 and narG2). narG1 showed a high percentage of identity with the corresponding part of narG found in Thiobacillus denitrificans, which suggests a recent acquisition of narG in strain JAM1 by horizontal gene transfer. This study provides the first direct evidence of the adaptation of a Methylophaga species to an oxygen-limited environment.

Functional Diversity in the Denitrifying Biofilm of the Methanol-Fed Marine Denitrification System at the Montreal Biodome

Nitrate is a serious problem in closed-circuit public aquariums because its accumulation rapidly becomes toxic to many lifeforms. A moving bed biofilm denitrification reactor was installed at the Montreal Biodome to treat its 3,250-m3 seawater system. Naturally occurring microorganisms from the seawater affluent colonized the reactor carriers to form a denitrifying biofilm. Here, we investigated the functional diversity of this biofilm by retrieving gene sequences related to narG, napA, nirK, nirS, cnorB, and nosZ. A total of 25 sequences related to these genes were retrieved from the biofilm. Among them, the corresponding napA1, nirK1, cnorB9, and nosZ3 sequences were identical to the corresponding genes found in Hyphomicrobium sp. NL23 while the narG1 and narG2 sequences were identical to the two corresponding narG genes found in Methylophaga sp. JAM1. These two bacterial strains were previously isolated from the denitrifying biofilm. To assess the abundance of denitrifiers and nitrate respirers in the biofilm, the gene copy number of all the narG, napA, nirS, and nirK sequences found in biofilm was determined by quantitative PCR. napA1, nirK1, narG1, and narG2, which were all associated with either Methylophaga sp. JAM1 or Hyphomicrobium sp. NL23, were the most abundant genes. The other genes were 10 to 10,000 times less abundant. nirK, cnorB, and nosZ but not napA transcripts from Hyphomicrobium sp. NL23 were detected in the biofilm, and only the narG1 transcripts from Methylophaga sp. JAM1 were detected in the biofilm. Among the 19 other genes, the transcripts of only two genes were detected in the biofilm. Our results show the predominance of Methylophaga sp. JAM1 and Hyphomicrobium sp. NL23 among the denitrifiers detected in the biofilm. The results suggest that Hyphomicrobium sp. NL23 could use the nitrite present in the biofilm generated by nitrate respirers such as Methylophaga sp. JAM1.

Inhibition of biological phosphorus removal in a sequencing moving bed biofilm reactor in seawater.

A new process was developed to achieve denitrifying biological phosphorus removal in wastewaters containing high levels of nitrate and phosphate with a low level of organic matter. This could particularly be useful in recirculating systems such as aquariums or fish farms to prevent accumulation of nitrate and phosphates and to avoid regular cost extensive and polluting water replacement. Phosphorus (P) was removed from the influent in a sequencing moving bed biofilm reactor, stored in the attached biomass and then cyclically removed from the biomass by filling the reactor with anaerobic water from a stock tank. Phosphate was accumulated in the stock tank which allowed for use as fertilizer. The feasibility of the experimental design was demonstrated by using the activated sludge model No. 3 (ASM3) complemented by the EAWAG Bio-P module implemented in the WEST simulation software. A pilot scale experiment was conducted in two identical reactors in two runs: one to treat water from a marine mesocosm, the other to treat a synthetic freshwater influent. No biological phosphorus removal was achieved during the seawater run. During the freshwater run, average P removal efficiency was 20%, of which 80% was attributed to biological removal and 20% to chemical precipitation. The absence of efficiency in seawater was attributed to the high concentration of calcium.

Are meiofauna transient or resident in sand filters of marine aquariums

A paradoxical situation was found in the sand filters of a cold marine mesocosm: meiofaunal masses which were large enough to inhibit the mineralization and nitrification processes coexisted with nitrogen cycling bacteria. To test whether the copepod-dominated meiofauna were resident and actively feeding or transient and carried passively through the sand filters, residence times (RTs) were measured for various meiofaunal groups in a newly started filter and in a long established one. Most meiofauna colonized the newly started filter in less than 6 h, but their RTs were less than 24 h. In contrast, RTs were 147d for halacarids, 291 d for harpacticoid copepods and 1228d for nematodes in the long established filter. Mesocosm periphyton. which occupied a large fraction of the mesocosm surface area and was characterized by high meiofaunal densities, was probably the main source of meiofauna in the sand filters. Pool sediments, consisting of gravel or sand, were second to periphyton and contributed hydrozoans and mesopsammic species to the filters. The small copepod Pseudonychocamptus proximus progressively replaced the large Tisbe furcata in sand filters during the fall of 1995 and was responsible for the large increase in meiofaunal biomass observed after spring 1996. This replacement was presumably facilitated by the copepod size selection process operated by the filters. Large copepods were retained by the surface layer of sand or brought up by the backwash water and then exit the mesocosm through the drain. High meiofaunal populations did not significantly affect nitrogen cycling bacteria in sand filters probably because meiofauna also fed on other abundant food sources which were carried in by the water flow.