Comparison of the denitrification potential of aerobic granules cultivated with acetate and propionate

Simultaneous nitrification-denitrification has been observed in aerobic granular sludge sequencing batch bubble column reactors fed with either acetate or propionate with elimination rates of up to 60-70%. Several in-situ tests under anoxic conditions were performed to evaluate the capacity of the two granular sludges to reduce nitrate (NO3-) and nitrite (NO2-). In absence of dissolved carbon (C), the intracellularly stored carbon acts as electron donor for denitrification. Therefore, the NO3- and NO2- removal could mainly be associated to organisms with internal C-storage such as glycogen accumulating organisms (GAO) and phosphate accumulating organisms (PAO). It was found that in both reactors the specific NO2- uptake rate was much higher than the specific NO3- uptake rate. These results agree with recent findings that some denitrifying PAO (DPAO) can only reduce NO2- but not NO3-. Moreover the propionate reactor was more efficient for NO2- removal than the acetate reactor. These results indicated that propionate as carbon source favored the growth of PAO with NO2- reducing capacity

DENITRIFYING PAO AND GAO IN AEROBIC GRANULAR BIOFILM CULTIVATED WITH ACETATE AND PROPIONATE

Graciela Gonzalez Gil, Christof Holliger, Samuel Lochmatter, David Gregory Weissbrodt

Phosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO) play an important role in aerobic granular biofilm systems used for wastewater treatment. Both are slow growing organisms which can take up and store carbon under anaerobic conditions. The growth of PAO over GAO is preferred because of their capacity to remove phosphate. As some PAOs and GAOs can utilize nitrite or nitrate as electron acceptor in absence of oxygen, they act also as denitrifiers (i.e. DPAO/DGAO). The presence of active DPAOs in a treatment system is desirable because the same carbon source is used for dephosphatation and denitrification. The aim of this study was to investigate and to compare the denitrification potential of aerobic granular biofilms having already a good phosphate removal activity, and that were cultivated with either propionate or acetate as model contaminats. The results show that both biofilms had considerably higher specific uptake rates for nitrite than for nitrate (3.26 times for acetate and 4.79 times for propionate). These results agree with a study (Martin et al., 2006) which showed, that some DPAO have no nitrate reductase genes and can therefore only reduce nitrite but not nitrate. Therefore, DGAO were likely responsible for nitrate and DPAO together with DGAO for nitrite removal. This assumption is supported by the observation that the nitrite uptake rate decreased in the absence of phosphate, whereas the nitrate uptake rate was not influenced by phosphate presence or absence. Furthermore we observed a 27% higher specific nitrite uptake rate in the propionate reactor than in the acetate reactor. This supports the hypothesis that granules fed with propionate may favor the development of PAO with nitrite reducing capacity. We conclude that (i) to enhance the denitrification and create a link to the P-removal, nitrite oxidation to nitrate has to be inhibited, and that (ii) propionate leads to more robust DPAO populations than acetate. In addition to the biochemical results presented in this abstract, microbial community analysis of granules will be done to complete the results.

2009

Bacterial Resource Management for Nutrient Removal in Aerobic Granular Sludge Wastewater Treatment Systems

David Gregory Weissbrodt

The approach of wastewater treatment has shifted towards a holistic view in order to achieve sustainability in addition to environmental protection. The aerobic granular sludge (AGS) technology, which relies on the use of fast-settling granular biofilms called granules, is progressively becoming a new standard for high-rate biological nutrient removal (BNR) and secondary clarification in single sequencing batch reactors (SBR). The AGS intensive process has been related with definite savings in land area, construction, and operation costs. In an economical analysis, theoretical savings of CHF 0.45 per m3 (1 Swiss franc CHF ≈ 0.83 €) were computed for a Swiss wastewater treatment plant (WWTP) of 200’000 capita removing all nutrients biologically. Besides scale-up, fundamental research was required to understand and tailor the structure of underlying bacterial communities that form granules and remove nutrients. Mechanisms of bacterial selection were investigated in a systems approach to propose strategic axes for optimal management of the bacterial resource for efficient process performances. This research led to the following advances. After having designed a flexible reactor infrastructure for AGS research, a mathematical modeling methodology was developed to understand the hydraulic and biological processes involved during plug-flow transport of wastewater across the settled AGS bed during the feeding phase. BNR relies on the preferential selection of polyphosphate-accumulating organisms (PAO) by proper uncoupled supply of electron donors and electron acceptors. A bed height to diameter ratio of 6 was deduced to be optimal for full bed loading and efficient anaerobic acetate uptake under reference simulation conditions (20°C, pH 7.0). A bioinformatics methodology called PyroTRF-ID was developed for the identification of bacterial relatives involved in BNR processes by combination of terminal-restriction fragment length polymorphism (T-RFLP) and pyrosequencing data. This procedure provided high resolution of bacterial community structures and dynamics of AGS systems. Electrical conductivity and polyphosphatase assays were proposed for rapid and low-cost assessment of the fractions of active PAO and of the dephosphatating potential of activated sludge and granular sludge. Positive linear correlations were obtained between the fraction of PAO, the biomass specific rate of conductivity evolution measured in anaerobic metabolic batch tests, and the polyphosphate-hydrolyzing enzymatic activity of cell extracts. Wash-out conditions used to stimulate granule formation were shown to exert a selection pressure not only on physical properties of early-stage granules, but also on the underlying bacterial community composition. Operation with constant volumetric organic loading rates (OLR) during wash-out resulted in the formation of slow-settling fluffy granules dominated by filamentous Burkholderiales affiliates formed under low aeration (< 2 cm s-1) and high mesophilic temperature (30°C), and of fast-settling dense granules dominated by Zoogloea spp. under high aeration (4 cm s-1) and with an inoculum originating from a BNR-WWTP. These exopolysaccharide producers were considered as model organisms for granulation. However, their proliferation over PAO and nitrifiers correlated with poor BNR. PAO estab-lished at mature stage as soon as sufficient AGS was present to fully remove volatile fatty acids (VFA) during the anaerobic feeding phase. Without purge of excess sludge, glycogen-accumulating organisms (GAO) competed with PAO at a later stage of reactor operation. Granulation was shown to occur in stirred-tank SBRs operated under steady-state condi¬tions to cultivate PAO and GAO enrichments. The Accumulibacter- and Competibacter-dominated communities displayed granulation potential without involvement of Zoogloea spp. Fast-settling nuclei formed despite initial operation with a settling time as high as 60 min, and evolved towards granules after decrease of settling time to 10 min. Dynamic control of the OLR, anaerobic contact time, and sludge retention time (SRT) selected for active PAO in early-stage granules. Confocal laser scanning microscopy (CLSM) revealed that granula¬tion mechanisms depend on the predominant organisms involved. Fast-growing Zoogloea spp. formed smooth biofilm continuum matrices. The slower-growing PAO and GAO formed het¬erogeneous granular structures by proliferation in compact colonies around flocs. Fluctuations in operation variables were shown by multivariate statistics to impact on process performance and bacterial community structures in anaerobic-aerobic AGS-SBRs. The size of granules impacted on nitrification and dephosphatation by affecting oxygen mass transfer. Efficient BNR was obtained with at least 500 mgCOD L-1of acetate, 30 gCOD gP-1, and 10 gCOD gN-1. Although clades of PAO can denitrify, nitrogen removal was thus not restricted only to PAO. A broad denitrifying community was identified. The bacterial community continuum comprised two major opponent clusters, composed of members of the core microbiome of full-scale BNR-WWTPs. The first mainly comprised Accumulibacter, Nitrospira, Xanthomonadaceae, and Aminobacter affiliates selected by conditions of efficient BNR. The second mainly comprised Competibacter, Sphingobacteriales, Cytophaga, and Tetrasphaera affiliates correlating with periods of low BNR. Bacterial selection in AGS systems was mainly impacted by pH, according to multifactorial experiments. Accumulibacter and BNR were favored with alkaline pH, low mesophilic temperatures, and in the presence of propionate. Competibacter proliferated with acidic pH, temperature close to 30°C, and only acetate. Tetrasphaera spp., potential major PAO of certain full-scale plants, withstood Competibacter-selective conditions. Enhanced BNR was obtained under non-limiting conditions with food-to-microorganism ratios above 25 mgCODs gCODx-1. With fixed 2-h starvation, dephosphatation was only complete with full aeration. Since alternating aerobic-anoxic starvation conditions led to partial conversions, the length of starvation phases should be controlled in function of the redox conditions applied. Overall, optimal bacterial resource management in AGS systems requires full control of system behavior by taking advantage of the flexibility of the SBR technology. A methodology is proposed to this end, comprising milestones for wastewater characterization, SBR design, “anaerobic selector” design, control of the starvation phase length, shaving of fluctuations in operation variables, purge of excess sludge, proper selection for a stable, active and cooperative bacterial community, as well as efficient BNR. As key research perspective, investigations with real wastewater should validate the knowledge gained on bacterial behaviors under low-complexity conditions. Significant differences in predominant bacterial relatives were detected between lab-scale and full-scale BNR sludge. Metagenomics will provide a broader view on key organisms and functions of BNR and AGS microbiomes.

EPFL2013

Identification and characterization of proteins supporting dehalorespiration in Desulfitobacterium hafniense strain TCE1

Laure Prat

Respiration is a fundamental catalytic process in the aerobic and anaerobic energy metabolism of many prokaryotic and most eukaryotic organisms. The major difference between these organisms is that various organic and inorganic substrates can be used to donate or accept electrons in the bacterial and archaeal kingdoms, in various ranges of redox potentials in order to drive aerobic (with oxygen as electron acceptor) and anaerobic respiratory pathways. During the last few decades, several bacterial strains have been reported to use chlorinated compounds as terminal electron acceptor under anaerobic conditions, in a process called dehalorespiration. Among more than thirty dehalorespiring bacterial strains isolated to date, the Dehalococcoides group and the Gram-positive genus Desulfitobacterium comprise the largest number of reductively dehalogenating pure cultures. While most Dehalococcoides isolates appeared as highly specialized bacteria that depend strictly on dehalorespiration for growth, members of the genus Desulfitobacterium are very versatile microorganisms with regards to the electron donors and acceptors utilized. Globally, this remarkable prokaryotic respiratory flexibility is guaranteed by an elaborate reservoir of complex redox enzymes. Regarding the specific electron transport chain involved in dehalorespiration, rather little knowledge has been obtained so far with the exception of the key enzyme, the reductive dehalogenase, well described in several dehalorespiring bacteria, and of little information on b-type cytochromes and menaquinones. The overall objective of this thesis was to identify new possible components of the electron transport chain by comparative proteomic analysis, to investigate the presence/absence of a regulatory pathway leading to the biosynthesis of the reductive dehalogenase, and finally to characterize newly identified proteins that might support the dehalorespiration process. A comparative 2D proteomic analysis was performed on Desulfitobacterium hafniense strain TCE1, a bacterium capable of using tetrachloroethene (PCE) as electron acceptor. Soluble and membrane-associated proteins were analyzed from cells cultivated on different couples of electron donors and acceptors, lactate – fumarate (LF), lactate – PCE (LP), hydrogen – fumarate (HF), and hydrogen – PCE (HP). Cells growing on LP or HP revealed 72 and 93 protein spots in membrane fraction, and 49 and 12 spots in soluble fraction that correspond to increasingly expressed proteins compared to their fumarate-grown counterparts. More than 80 proteins were identified by mass spectrometry analysis, among which the key enzyme in the dehalorespiration process, the PCE reductive dehalogenase (PceA), as well as interesting candidates which could support dehalorespiration. The possible role of these proteins was analyzed in further details. Involved in energy and carbon metabolisms, electron transfer flavoproteins (ETF) and proteins related to the Wood-Ljungdahl pathway of CO2 fixation were identified in HP growth conditions. Proteins associated to stress response such as the phage shock protein PspA, and to regulatory pathways, the transcriptional repressor CodY, were also observed and discussed. Although only slightly induced by PCE, an additional protein displaying homology to sulfur-transferases was found in a large abundance in the fraction of membrane-associated proteins. This protein was named PhsE, as the corresponding gene belongs to a gene operon with homology to the molybdoenzyme thiosulfate/polysulfide reductase operons (phs/psr) found in Salmonella typhimurium or Wolinella succinogenes. PhsE protein architecture resembles to the class of tandem-domain rhodaneses, with the particularity to contain two active-site cysteines. It is also characterized by the presence of a typical lipoprotein signal peptide (LSP), which anchors PhsE on the outside of the cytoplasmic membrane. In the genome sequence of D. hafniense strain Y51, phsE (DSY3892) is part of an apparent operon encoding one out of about sixty different members of the complex iron-sulfur molybdoenzyme (CISM) family. PhsA, the catalytic molybdoenzyme, displays significant sequence homology with the thiosulfate/polysulfide reductase (PhsA/PsrA) subfamily. So far no other CISM operon has been described with a sulfur-transferase encoding gene. Despite the numerous functions proposed for rhodanese in cyanide detoxification, Fe/S cluster formation, oxidative stress protection, sulfur mobilization and involvement in general sulfur metabolism, the question of the physiological role of PhsE remains open. The expression of the Phs complex appeared to be influenced by the addition of sulfide in the culture medium, generally used as reducing agent in anaerobic cultures. Sulfide is known to affect the intracellular redox status. Therefore, it is hypothesized that PhsE might be involved in the control of the redox balance of the cell, notably by scavenging potentially toxic reduced sulfur species. To support data obtained at the protein level during proteomic analysis, genes and the corresponding messenger RNA from D. hafniense strain TCE1 were studied. While in silico DNA sequence analysis did not reveal any obvious features regulating the transcription of pceA, puzzling results were obtained in proteomic analysis showing an almost complete absence of PceA in cells grown on fumarate. To characterize this phenomenon, starting from a routinely PCE-grown inoculum, strain TCE1 was repeatedly transferred on medium containing fumarate as electron acceptor. The pool of pceA gene in the total culture DNA decreased significantly already after fifteen successive sub-cultures on fumarate, suggesting a shift in strain TCE1 population upon relief of the PCE selection pressure. Consequently the level of pceA mRNA and PceA protein also followed the same trend. Secondly, a PCE pulse experiment was performed in an anaerobic continuous culture of strain TCE1 on fumarate. It revealed that PCE itself has no power of induction on genetic level, except for the positive effect observed on pspA transcripts which encodes a protein involved in cell membrane integrity. Finally the expression study of the phsFABCDE gene cluster revealed that all phs genes are co-transcribed, and most likely build an operon. Thus phsE seemed to be strictly coregulated with the phs operon, and not under the influence of another promoter. In conclusion, proteomic analysis enabled to investigate the general physiological status of Desulfitobacterium hafniense strain TCE1 during dehalorespiration. A tentative model of the dehalorespiration pathway is proposed in summarizing recent data obtained on the topology of the PCE reductive dehalogenase and on sequence similarity with proteins involved in other anaerobic respiratory pathways. Regarding the possible support of PhsE towards the dehalorespiration, the complicated chemistry of sulfur does not allow to clearly attribute a function to the Phs complex, but there are indications that it might build a 'sulfur' oxidoreductase which could catalyze the conversion of sulfide to polysulfide with concomitant electron transfer to the quinone pool. The sulfide oxidation activity might be useful to D. hafniense to scavenge toxic sulfur species, avoiding that they disturb the redox balance of the cell, and redox-sensitive enzymatic complexes.

EPFL2009