156
Denitrification of nitrous oxide is unaffected by electron competition in Accumulibacter Roy, S.*, Pradhan, N.*, Ng, H. Y.**, Wuertz, S*,***
*Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore 637551
**NUS Environmental Research Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411
*** School of Civil and Environmental Engineering, Nanyang Technological University, Singapore 639798
Keywords: Accumulibacter, electron competition, free nitrous acid Summary of key findings
Denitrifying phosphorus removal (DPR) is a promising technology. In this work we have focussed on identifying whether environmental factors or an incomplete denitrification pathway in dominant denitrifying organisms lead to nitrous oxide (N2O) accumulation in DPR systems. Under each test condition, Accumulibacter reduced two to three times more nitrous oxide than Competibacter. In batch tests, no significant decline in total NOx reduction rates was observed for Accumulibacter in the presence of multiple electron acceptors. Adequate PHA levels and free nitrous acid (FNA) concentrations lower than inhibitory levels (0.1 – 0.2 µg HNO2-N L-1) resulted in N2O accumulation in Accumulibacter and Competibacter at least 60% and 80% lower, respectively, than observed in previous studies. Validation with a metabolic model suggests that denitrification by Accumulibacter and Competibacter is not limited by electron competition. In fact, a metabolic model employed to identify accumulation of denitrification products and energy generated in each dentification step by both Accumulibacter enrichments revealed an increase in anoxic growth of 1.5 to 2 times on N2O (p-values of 0.03 and 0.0048 for Accumulibacternitrite and Accumulibacternitrate, respectively) in the presence of other nitrogen oxides, as compared to when N2O was the sole added electron acceptor. Overall, the reduction of nitrogen oxides by Accumulibacter is largely governed by their denitrification enzyme-specific affinity to electron carriers, the availability of sufficient internal storage polymers to provide energy for electron transfer, and sufficiently low levels of FNA to prevent inhibition.ll
Background and relevance
Denitrifying phosphorus removal (DPR) has been identified as an optimal treatment solution due to its reduced energy consumption, sludge wastage and carbon requirement for nutrient removal1. However, both denitrifying polyphosphate accumulating organisms (DPAOs) and denitrifying glycogen accumulating organisms (DGAOs) have been shown to release N2O, a potent greenhouse gas, under anoxic conditions. In terrestrial and aquatic ecosystems, the conversion of N2O to nitrogen gas by nitrous oxide reductase (Nos) is the only known biological N2O attenuation process in the biosphere2. Genomic evidence has revealed that many organisms possess only a subset of the denitrification pathway, sometimes lacking the nitrous oxide reductase gene (nos), resulting in the accumulation of N2O3. To this end, a genomic comparison of the denitrification pathway in Accumulibacter clades IA, IIA, IIB, and IIC showed significant differences and suggested that some clades were incapable of reducing N2O4. However, most metabolic models ignore these differences in denitrification capabilities of PAOs and GAOs and split the total nitrogen reduced between anoxic and aerobic phases5. Such an approach overlooks differences in phosphorus removal rates by different Accumulibacter clades that arise due to affinity towards certain nitrogen oxides. So far, models predicting N2O accumulation have been developed solely based on external nitrate dosage as terminal electron acceptor. However, various nitrogen oxides (NOx) occur simultaneously in biological wastewater treatment, and can serve as terminal electron acceptors. Finally, N2O accumulation is also affected by environmental conditions such as free nitrous acid (FNA) concentration, slow polyhydroxyalkanoates (PHA) degradation, and electron competition, which results in a higher flow of electrons to one step of the denitrification pathway, rather than electrons being distributed to all denitrification enzymes. Thus it is important to identify whether environmental factors or genomic potential for denitrification affects N2O reduction in DPR processes. Consequently, it is necessary to consider the effect of specific long-term enrichment conditions (nitrate or nitrite as electron acceptor) in modelling approaches.
157
In this study, we compared the denitrification kinetics of two Accumulibacter enrichments acclimated to utilizing either nitrite or nitrate in anoxic conditions. We hypothesized that the preference for certain NOx compounds would be driven either by electron competition or the extent of denitrification performed by the dominant organism in each enrichment culture. The results for Accumulibacter were contrasted with denitrification characteristics of Competibacter. Objectives for the study were to (i) identify any accumulation of intermediates in the denitrification process that would signify a preference for certain terminal electron acceptors, (ii) compare the role of electron competition in N2O accumulation, and (iii) validate an existing metabolic model6 to predict the mechanism of phosphorus uptake and nitrogen oxide accumulation in the presence of multiple electron acceptors.
Results
Accumulibacter enrichments had a high relative abundance of target organisms and a low presence of Competibacter (< 4% relative abundance) while the Competibacter enrichment was dominated by target organisms and a low abundance of Accumulibacter (Fig. 1A). We observed that enrichment-specific affinity for different electron acceptors affected the overall NOx reduction. For Accumulibacternitrate the reduction rates for nitrate, nitrite and N2O were 10.9 ± 0.3, 10.7 ± 1.2 and 10.6 ± 0.007 mg N gVSS-1 h
-1, whereas Accumulibacternitrite had similar reduction rates for nitrite and N2O but significantly lower rates for nitrate reduction (Fig. 1B). In addition to overall NOx reduction characteristics, electron distribution in the two Accumulibacter enrichments also suggests an enrichment-specific affinity for terminal electron acceptors. The ratio of phosphorus uptake to electron consumption was not affected by increasing concentrations of electron acceptors added in batch tests, but rather by the type of electron acceptor present (Fig. 1D). Competibacter enrichments showed preference for nitrate, and subsequent nitrite accumulation was observed during batch tests (Fig. 1B). It was interesting to observe that in comparison to previous studies measuring denitrification kinetics in DPAOs and DGAOs, N2O accumulation was not observed in this study when multiple electron acceptors were added simultaneously. Further, a metabolic model was employed in this study to predict anoxic growth rate at each denitrification step and associated phosphorus uptake for DPAOs using the measured nitrate, nitrite, and N2O reduction rates (Fig. 1C). Among the kinetic parameters analysed for sensitivity analysis and model calibration, four parameters (i.e., qPP, µDPAO1, µDPAO2, and µDPAO4) were found to significantly affect the predictability power of the model. N2O accumulation was not detected from the model and the anoxic growth rate modeled on N2O was calculated to be two to three times higher than the anoxic growth rate on nitrate or nitrite.
Discussion
The Accumulibacter enrichments in this study were dominated by target organisms while those utilized in previous studies included a significant fraction of GAOs, which could have led to confounding factors in understanding the denitrification kinetics of the target organism. Higher N2O utilization rates were observed for Accumulibacter than Competibacter, despite similar PHA storage, anoxic PHA utilization rates, and experimental conditions. This rules out the possibility of incomplete denitrification and N2O accumulation solely due to PHA serving as carbon source. It is likely that a combination of factors such as adequate PHA storage, availability of N2O, and FNA concentrations below inhibitory levels allowed for adequate nos transcription in Accumulibacter. The lack of nitrite and N2O reduction in Competibacter indicates a truncated denitrification pathway as observed in species such as Ca. Contendobacter odensis.
The metabolic model employed in this study was the first to consider denitrification as a four-step process and identify accumulation of intermediates during the process, allowing us to deduce whether electron competition limited N2O utilization. As per the model, increased availability of N2O and energy derived from the reduction of multiple electron acceptors was directed towards metabolic and growth-related activities. Compared to previous studies, a lower tendency for N2O accumulation and higher anoxic growth rate on N2O was observed when FNA concentrations were lower than suggested inhibitory levels. This identifies FNA as an important parameter that must be included in models developed to predict N2O accumulation based on nitrite concentration and pH.
158 References
1. Roy S, Pradhan N, Ng HY, Wuertz S. (2020) Denitrification kinetics indicates nitrous oxide uptake is unaffected by electron competition in Accumulibacter. bioRxiv: 2020.2005.2014.092429.
2. Graf, D.R.H., Jones, C.M. and Hallin, S. (2014) Intergenomic comparisons highlight modularity of the denitrification pathway and underpin the importance of community structure for N2O emissions. PLoS One 9(12), e114118.
3. Jones, C.M., Graf, D.R.H., Bru, D., Philippot, L. and Hallin, S. (2013) The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink. The ISME Journal 7(2), 417-426.
4. Camejo, P.Y., Oyserman, B.O., McMahon, K.D. and Noguera, D.R. (2019) Integrated omic analyses provide evidence that a “Candidatus Accumulibacter phosphatis” strain performs denitrification under microaerobic conditions. mSystems 4(1), e00193-00118.
5. Santos, J.M., Rieger, L., Lanham, A.B., Carvalheira, M., Reis, M.A. and Oehmen, A. (2020) A novel metabolic-ASM model for full-scale biological nutrient removal systems. Water Research 171, 115373.
6. Liu, Y., Peng, L., Chen, X. and Ni, B-J. (2015) Mathematical modeling of nitrous oxide production during denitrifying phosphorus removal process. Environmental Science & Technology 49(14), 8595-860
159 Presenting Author
Dr. Samarpita Roy
Is the presenting author an IWA Young Water Professional? Y /N (i.e. an IWA member under 35 years of age)
Bio: Samarpita obtained her PhD under the supervision of Prof. Stefan Wuertz at the Singapore Centre for Environmental Life Sciences Engineering (SCELSE). Thereafter, she continued her postdoctoral research at SCELSE to improve understanding of biological phosphorus removal and identify novel PAOs. Her research interests revolve around understanding microbiomes and engineering them for biotechnological applications to improve environmental and public health.
160
3 2
2 2
PAOs Got Talent?! Effect of the substrate composition on N2O formation in aerobic granular sludge
Dockx, L.*, Caluwé, M.*, Dobbeleers, T*. and Dries, J.*
*BioWAVE, Biochemical Wastewater Valorization and Engineering, Faculty of Applied Engineering, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
Keywords: AGS, d-PAO, N2O Summary of key findings
- AGS was formed in all SBRs but stable EPBR activity was only obtained with VFAs - N2O formation was strongly related to the presence of VFAs and Accumulibacter - NO --N accumulation lead to an increase in N O formation
Background and relevance
Aerobic granular sludge (AGS) is a recent innovation for biological wastewater treatment.1 Enhanced biological phosphorus removal (EBPR) occurs via the storage metabolism (cfr. polyhydroxyalkanoates (PHAs)) of polyphosphate accumulating organisms (PAOs), such as Candidatus Accumulibacter phosphatis, using alternating anaerobic and aerobic steps.2 EBPR can also be accomplished under anoxic conditions by denitrifying PAOs, which can remove nitrogen and phosphorus simultaneously using NO -or NO -.3 Furthermore, AGS is characterised by the formation of zones with different dissolved oxygen (DO) concentrations, which allows simultaneous nitrification and denitrification inside the granule.4 Of particular concern in biological N removal processes is the related emission of the greenhouse gas N2O.5 Recent whole-plant measurements indicate an underestimation of WWTP contribution to global N2O emissions (up to 75% of the total carbon footprint).6,7
Figure 1. Biologically driven N2O production during wastewater treatment for both aerobic and anoxic conditions in AGS
Previous lab-scale studies reported the accumulation of N2O in those systems where denitrification was conducted using internal storage polymers (e.g. PHAs). This would affect the activity of N2O reductase, causing electron competition between different denitrification enzymes.8 In this study, substrates other than the widely applied VFAs (e.g. actetate/propionate), such as amino acids and glucose, were applied wich allows to investigate the effect of these individual substrates on the N2O formation process in AGS.
Reactor Set-Up and Operating conditions
AGS-EBPR lab-scale sequencing batch reactors (12L) were inoculated with (flocculent) activated sludge and were operated with an anerobic feast/aerobic famine strategy. Two stages were performed, using synthetic wastewater (Table 1). Briefly, in both stages a reference SBR, fed with a mixture of acetate and propionate, was included (SBR A and D). During stage I, single substrates were fed. Stage II involved the feeding of a mixture of substrates (SBR E). A volume exchange rate of 12.5% was applied. A sludge retention time of 25 days was maintained by periodically removing excess biomass.
NH2OH oxidation
autotrophic nitrification pathway (AOB, NOB)
amoA nor
nosZ
nirK
NO- narG
N2O
ANOXIC
nirK norB
NO AEROBIC
161
4 4
4
4 3 2
Table 1. Summary of wastewater characteristics: carbon source (COD), NH +-N and PO 3--P in different reactors
Stage SBR VFAs (mg COD.L-1) Amino Acids (mg COD.L-1) Sugar NH4-N PO4-P COD/ COD/
(mg COD.L-1) (mg.L-1) (mg.L-1) N P
Na- acetate
Na-
propionate Leucine Aspartic Acid
Glutamic
Acid Glucose
I A 750 750 - - - - 134 30 11 50
I B - - 500 500 500 - - 30 11 50
I C - - - - - 1500 134 30 11 50
II D 750 750 - - - - 75 30 20 50
II E 250 250 - 250 250 500 13 30 20 50
Ex-situ batch experiments
To examine the influence of the individual substrates on the (d)EBPR activity and N2O formation, ex- situ batch tests were performed at the end of each stage, for each SBR. Batch tests were conducted dosing individual substrates to sludge from the corresponding SBR.
300mL of sludge was taken out the reactor at the end of the SBR cycle. After settling, the supernatant was discarded and the sludge was washed (3 times) with a wash buffer (0.1M NaHCO3 + 0.05M KCl). The individual substrates were dosed using the same COD loading as applied in the SBRs. The ML(V)SS concentration, DOC uptake (%) and P-release (mgP.gVSS
-1) during the anaerobic period, with a duration of 90min (equal to the duration applied in the SBRs) were determined for each batch test. Afterwards, the sludge was washed to remove excess carbon. K2HPO4 was dosed to obtain the desired PO 3--P concentration at the end of the anaerobic period. Subsequently, a concentration of 15 mg NOx-N.L-1 was initially added as a pulse.
Analyses
All samples were filtered over glass microfiber filters (particle retention of 1.2µm). Testkits (Hanna Instruments) were used to measure PO 3--P, NO --N and NO --N. DOC was measured using a Sievers InnovOx Laboratory Total Organic Carbon Analyzer. A Unisense® micro sensor (Aarhus, Denmark) with data logger was used to measure the N2O concentration in the liquid phase. 16S rRNA gene amplicon sequencing, targeting the V1-V3 region was carried out.9 Taxonomy prediction of the OTU sequences was carried out with MiDAS 3.7 as reference database.10
Results
Stable EBPR activity, due to the enrichment of Accumulibacter, was obtained when VFAs were present in the influent as sole substrate or as a fraction (SBR A, D and E). Amino acids resulted in the enrichment of specific fermenting organisms, especially Burkholderiaceae. Application of glucose promoted the enrichment of Saccharimonadaceae and GAOs rather than PAOs.
Batch tests (Figure 2) indicated that glucose lead to the lowest EBPR activity, in comparison to the other carbon sources applied.
162
Figure 2. DOC uptake (%) (bars) and P-release (mg PO43--P/gVSS) (•) in ex-situ batch experiments with sludge taken at the end of stage I and II. Individual substrates were dosed to sludge from SBR A (a), SBR B (b), SBR C (c), SBR D (d) and SBR E (e).
The highest N2O formation, compared to the total available nitrogen, was found in systems were VFAs were present. Moreover, the presence of NO2--N presumably causes an unbalanced denitrification pathway, in comparsion with the presence of NO3--N, leading to more N2O formation troughout the anoxic period (Figure 3). Almost no N2O formation was detected in SBR B and SBR C.
Figure 3. Overview of the total N2O-N formation (% N2O-N) compared to the total available nitrogen (NO3--N or NO2--N) at the start of the anoxic phase of the ex-situ batch test with sludge taken from the different reactors (SBR A (a), SBR B (b), SBR C (c), SBR D (d) and SBR E (e)) at the end of stage I and stage II.
Discussion
AGS was successfully formed in all reactor systems. Anaerobic batch tests (Figure 2) confirm the importance of both VFAs and (subsequently) the presence of Accumulibacter for the achievement of stable EPBR.11 Those systems were correlated to N2O production. A lower fraction of VFAs in the influent (SBR E) had a positive impact on N2O formation. Results regarding NO2--N dosage (Figure 3) confirm studies about the negative role of NO2--N accumulation on N2O formation.12 Additional research should focus on gene expression studies to investigate the presence and activity of key genes (nirS, nirK and nosZ in the genome of Accumulibacter) during the denitrification pathway.
163 References
1. Pronk, M., et al., Full scale performance of the aerobic granular sludge process for sewage treatment. Water Res, 2015. 84: p. 207- 17.
2. Nielsen, P.H., et al., Microbial communities involved in enhanced biological phosphorus removal from wastewater--a model system in environmental biotechnology. Curr Opin Biotechnol, 2012. 23(3): p. 452-9.
3. Ribera-Guardia, A., et al., Distinctive denitrifying capabilities lead to differences in N2O production by denitrifying polyphosphate accumulating organisms and denitrifying glycogen accumulating organisms. Bioresource Technology, 2016. 219: p. 106-113.
4. Nancharaiah, Y.V. and G. Kiran Kumar Reddy, Aerobic granular sludge technology: Mechanisms of granulation and biotechnological applications. Bioresour Technol, 2018. 247: p. 1128-1143.
5. EPA. Understanding Global Warming Potential. 2017; Available from: https://www.epa.gov/ghgemissions/understanding-global- warming-potentials.
6. Delre, A., J. Mønster, and C. Scheutz, Greenhouse gas emission quantification from wastewater treatment plants, using a tracer gas dispersion method. Science of The Total Environment, 2017. 605-606: p. 258-268.
7. Marques, R., et al., Denitrifying capabilities of Tetrasphaera and their contribution towards nitrous oxide production in enhanced biological phosphorus removal processes. Water Research, 2018. 137: p. 262-272.
8. Kampschreur, M.J., et al., Nitrous oxide emission during wastewater treatment. Water Res, 2009. 43(17): p. 4093-103.
9. Dobbeleers, T., et al., Biological nutrient removal from slaughterhouse wastewater via nitritation/denitritation using granular sludge: an onsite pilot demonstration. Journal of Chemical Technology & Biotechnology, 2020. 95(1): p.
111-122.
10. Nierychlo, M., et al., MiDAS 3: An ecosystem-specific reference database, taxonomy and knowledge platform for activated sludge and anaerobic digesters reveals species-level microbiome composition of activated sludge. Water Research, 2020. 182: p. 115955.
11. Weissbrodt, D.G., N. Shani, and C. Holliger, Linking bacterial population dynamics and nutrient removal in the granular sludge biofilm ecosystem engineered for wastewater treatment. FEMS Microbiol Ecol, 2014. 88(3): p. 579-12. 95. Rodriguez-Caballero, A., et al., Nitritation versus full nitrification of ammonium-rich wastewater: comparison in
terms of nitrous and nitric oxides emissions. Bioresour Technol, 2013. 139: p. 195-202.
Presenting Author
MSc. Lennert Dockx - PhD Candidate
BioWAVE, Faculty of Applied Engineering, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium Is the presenting author an IWA Young Water Professional? Y/N
Bio: Hi! In 2017, I graduated as Master of Science in Industrial Engineering (Biochemistry) at the University of Antwerp. Right afterwards, I became PhD student. My project is focussing on the microbial enrichments and nitrous oxide formation in aerobic granular sludge due to the application of different carbon sources.
#BioWAVE... Because there is no planet B!
164
Protective role of sialic acids in the extracellular polymeric substances of “Ca.
Accumulibacter phosphatis”
Tomás-Martínez, S.*, Chen, L.M.*, Kleikamp, H.B.C.*, Neu, T.R.**, Weissbrodt, D.G.*, van Loosdrecht, M.C.M.* and Lin, Y.*
*Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629 HZ, Delft, The Netherlands, Tel: +34 679602011; E-mail: S.TomasMartinez@tudelft.nl
**Microbiology of Interfaces, Department River Ecology, Helmholtz Centre of Environmental Research – UFZ, Brueckstrasse 3A, Magdeburg, Germany
Keywords: “Ca. Accumulibacter phosphatis”; Extracellular Polymeric Substances (EPS); Sialic acids.
Summary of key findings
Nonulosonic acids (NulOs) are a family of sugars that have mainly been studied in animal cells and pathogenic bacteria, due to their role in microbial-host interactions. Recently, they have been discovered in non-pathogenic bacteria, such as the ones present in aerobic granular sludge. In order to elucidate thir function in such bacteria, enrichments of “Ca. Accumulibacter” were obtained under saline and fresh water conditions. The difference in NulOs production from both enrichments suggested a protective role under saline stress due to their negative charge. Moreover, a protein degradation test was performed, which resulted in higher proteolytic activity when NulOs were removed.
Background and relevance
Nonulosonic acids (NulOs) are a family of acidic sugars with a nine-carbon backbone, which include many different related structures, such as sialic acids and other bacterial forms. They have mainly been studied for their relevance in animal cells and pathogenic bacteria, where they play an important role in stabilization of glycoproteins and microbial-host interactions.
Recently, NulOs have been discovered in glycoproteins within the extracellular polymeric substances (EPS) of salt-adapted aerobic granular sludge [1]. These monosaccharides are present in the terminus of glycoproteins and are important in the protection of the carbohydrate residues. “Ca. Accumulibacter phosphatis” was the dominant population in these granules and was suggested to be responsible of NulOs production. A further study revealed the presence of a variety of NulOs in a highly enriced culture of this bacterium. The analysis also revealed the potencial to produce different chemical structures of NulOs by “Ca. Accumulibacter” [2].
The aim of the current research was to explore the diversity and function of NulOs in the EPS of “Ca.
Accumulibacter”. For that, two enriched cultures were obtained under saline and non-saline conditions.
The effect of the salinity difference in the NulOs diversity was analyzed. Additionally, a protein degradation test was performed to evaluate the effect of NulOs in protein stability in the bioaggregates.
Results
Sialic acid-specific lectins (Fig. 1) showed a wider distribution of NulOs in the bioaggregates from the saline enrichment. Moreover, the higher intensity of the signal indicated a higher abundance in this type of biomass, which was further confimed by mass spectrometry analysis. With this technique, the different forms of NulOs in each sample was determined. Despite the difference in amount, both samples presented common NulOs, such as Kdn and the bacterial pseudaminic and legionaminic acids. The main difference was the presence of high amounts of neuraminic acid (NeuAc) in the saline enrichment.
165
In order to study the role of NulOs in protein stability, bioaggregates from both enrichments were treated with a neuramidase, an enzyme that specifically removes NeuAc. After this, treated and control granules were suspended to measure endogenous proteolytic activity. Peptide release was monitored by measuring absorbance at 280nm (Fig. 2). This release was higher (~2.6-fold) in the treated saline sample when compared to the control. In the case of the fresh water sample, treated sample and control presented a similar value. This difference between both types of samples can be associated to the specificity of the neuramidase. As NeuAc was only present in the saline enrichment, the enzyme could not remove any NulOs from the fresh water bioaggregates. Therefore, only when NulOs were removed (in the form of NeuAc), the protection against proteolytic activity was lost.
Discussion
These results showed that differences in salinity have an effect in NulOs production in “Ca.
Accumulibacter” enrichments. The higher amount of NulOs in the enrichment grown under saline conditions could act as a protective mechanism against this environment. The charge of the carboxyl- groups allows binding of cations under these conditions. Moreover, they can play an important role in the hydration of EPS. However, this does not exclude the presence of other anionic compounds that might present a similar function.
From this work, another protective role of NulOs can be suggested. The results showed that when NulOs (e.g. NeuAc) are removed by the action of specific enzymes, (glyco)proteins become sensitive to proteolytic activity. Moreover, the diversity of NulOs in the granules confers higher protection, as different specific enzymes would be needed to remove them.
In conclusion, the present work shows two potential protective role of NulOs in “Ca. Accumulibacter phosphatis”. However, further research should be conducted to elucidate the importance of these carbohydrates in non-pathogenic bacteria.
Figure 1. Confocal laser scanning microscopy with fluorescently labelled sialic acid specific lectins (HMA) of “Ca. Accumulibacter” enriched bioaggregates grown under saline (left) and fresh water (right) conditions.
166
Figure 2. Peptide concentrations over time of neuramidase treated (orange triangles) and non-treated (blue squares) bioaggregates. Left: saline conditions. Right: Fresh water conditions.
References
[1] Tomás-Martínez, S., Kleikamp, H.B.C., Neu, T.R., Pabst, M., Weissbrodt, D.G., van Loosdrecht, M.C.M and Lin, Y.
(submitted) Production of nonulosonic acids in the extracellular polymeric substances of “Candidatus Accumulibacter phosphatis”
[2] de Graaff, D.R., Felz, S., Neu, T.R., Pronk, M., van Loosdrecht, M.C.M. and Lin, Y. (2019) Sialic acids in the extracellular polymeric substances of seawater-adapted aerobic granular sludge, Water Research, 155, 343-351
Presenting Author
Sergio Tomás-Martínez PhD candidate
Delft University of Technology, Department of Biotechnology
Is the presenting author an IWA Young Water Professional? No (i.e. an IWA member under 35 years of age)
Bio: After completing my MSc in Life Science and Technology at TU Delft, I was hired as a PhD student at TU Delft on the Environmental Biotechnology group. During my PhD, I am involved in a NWO project: Nature inspired biopolymer nanocomposites towards a cyclic economy (Nanocycle). In my research, I study the extracellular polymeric substances (EPS) of polyphosphate accumulating organisms (PAOs). In this project, I analyse EPS from a composition and metabolic point of view.
167
Sulfated glycosaminoglycan-like polymers present in acidophilic biofilm
de Bruin, S.*, Vasquez-Cardenas, D.*, Machado de Sousa, DZ.**, Van Loosdrecht, M.C.M.* and Lin, Y.*
*Delft University of Technology
**Wageningen University and Research
Keywords: Mycobacterium; Anionic polymers; Extracellular polymeric substances Summary of key findings
Sulfated glycosaminoglycan-like polymers were discovered in the EPS of an acidophilic biofilm.
Quantification of the sulfated glycosaminoglycan-like polymers yieled a maximum concentration of 55.16 ± 2.06 µg/mg sample. FT-IR analysis of the sulfated polymers indicated a polymer consisting of predominantly polysaccharides. Further enzymatic treatment showed a 26% decrease in measured sulfated glycosaminoglycan-like content after chondroitinase ABC treatment.
Background and relevance
Inside the biofilm, organisms are immobilized in a self-produced extracellular matrix (ECM) consisting of extracellular polymeric substances(EPS) (Flemming and Wingender, 2010). Due to the ECM, microorganisms can grow in environments that are not be suitable for microbial life. One of these environments is found in a sulfur cave in Romania. Here, a biofilm grows by oxidation of S0 and H2S. The pH near the interface was measured to be <1, caused by the formation of sulphuric acid. In addition, water is only available through condensation on the cave wall. The biofilm is dominated by Mycobacterium spp., which are probably capable of oxidizing sulfur (Kusumi et al., 2011; Sarbu et al., 2018). Similar environments where acidophilic biofilms grow have been documented elsewhere (Jones et al, 2016; Li et al.,2017; Jiang et al., 2016). Negatively charged polymers concentrations in EPS have been shown to adapt to different conditions and thus contribute to e.g biofilm stability, osmotic
protection and water retention (Gagliano et al.,2018). Work on granular sludge has shown the presence of glycosylated proteins with glycan moieties similar to sulfated glycosaminoglycans
(sGAG)(Bourven et al., 2015; Varki, A., 2017; Lin et al., 2018; Boleij et al., 2020; Seviour et al., 2019; Felz et al., 2020;). Polyanionic molecules can contribute to the water balance in the tissues.
Therefore this work will investigate the presence of polyanionic molecules in the EPS matrix of the acidophilic biofilm of the sulfur cave. Insights in the presence of anionic molecules in extreme conditions can grant a better understanding into the function in biofilms.
Results
In order to analyse the presence of polyanionic components in the acidophilic biofilm, EPS was extracted and further characterized. The sulfated polymers present in the EPS were quantified using the BlyscanTM kit (Biocolor, UK) with chondroitin sulfate (CS) as standard. The method was developed to quantify the sGAG content of different types of vertebrate tissues with minimal interference from protein and polysaccharides. Additionally, the presence of O- and N-sulfation was determined. The untreated sample was used to determine the maximum amount of sGAG present in the sample. The measured concentration was 5.45 ± 0.02 µg/mg sample with 58% of O-sulfated compounds being measured. To verify that the sGAG-like molecules were polymers, EPS samples were also analysed. Concentrations of 21.52 ± 0.57 µg/mg and 55.16 ± 2.06 µg/mg were measured for S1 and S2, respectively. The ratio of O-sulfated compounds was measured to be 71.1% for S1. The sGAG-like molecules are concentrated in the extracted EPS samples indicating the sGAG-like molecules are present in the polymer fraction.
168
Table 1.1 Quantification of sulfated glycosaminoglycan-like polymers in biofilm and extracted EPS (n.m = not measured).
Sample sGAG µg/mg
sample Ratio O-sulfation % Ratio initial biofilm
Biofilm 5.45 ± 0.02 58 ± 2.1 0.545 %
S1 21.52 ± 0.57 71.1 ± 0.1 0.34
S2 55.16 ± 2.06 n.m. 0.19
FT-IR analysis was performed to analyse the sulfated polymer complexed with DMMB (sGAG complex) in the BlyscanTM kit (Biocolor, UK). The most prominent band in both spectra is found around 1023 and 1047 cm-1 which is indicative of C–O stretch in carbohydrates. Additionally protein presence was measured by the bands visible in the region around 1650 cm-1, amide I, and 1550 cm-1, amide II. The sGAG complex shows a decrease in the amide I and II bands, which is caused by the protease pretreatment used in the kit. Interestingly bands at around 1212 and 1167 cm-1 become apparent in this spectrum, which can indicates the presence of sulfate half-esters and either phosphate assymetric stretch and/or C–O–C stretch, respectively. This data suggests that the polyanionic
molecules are polysaccharides with negatively charged groups like e.g. sulfate and phosphate. (Talari et al., 2016; Baker et al., 2014; Devlin et al., 2019)
Figure 1.1 Normalized FT-IR spectra of extracted EPS and complexed sulfated polymers. Spectra were recorded from 4000 to 650 cm-1 and min-max normalized, absorbance is in arbitrary units (a.u).
DNA is a phosphorylated polysaccharide and could be part of the sGAG complex (Zheng and levenston, 2015). In order to test the similarity of the sGAG complex with CS, and DNA, samples were pretreated with chondroitinase ABC (ChABCase) and DNAse. The S2 with buffer solution showed a similar concentration as previously measured with 50.96 ± 0.70 µg/mg sample. The CS standard concentration was as expected 90.80 ± 0.51 µg/mg sample. The ChABCase on CS standard caused a 100% decrease in measured sGAG concentration, indicating that the ChABCase was active on CS standard. DNAse showed a 3.34% decrease in sGAG content implying a minimal DNA interference in the quantification. ChABCase addition decreased the sGAG concentration by 26.31%.
Finally, the combination of DNAse and ChABCase resulted in a 28.45% decrease. ChABCase is active on the the (1→3) glycosidic linkage between β-D-glucuronic acid and N-Acetyl-β-D- galactosamine-4-sulfate and β-D-glucuronic acid and N-Acetyl-β-D-galactosamine-6-sulfate.
Additionally ChABCase cleaves the (1→3) and (1→4) glycosidic linkages between β-D-glucuronic acid and N-Acetyl-β-D-glucosamine (Sigma-Aldrich, 2007; Felz et al., 2020). The sensitivity of ChABCase on the sulfated polymer shows the presence of a similar molecule. CS has a structured make-up and consists out of disaccharide blocks it is well possible that the sulfated polymers extracted here are less structured (Varki, A., 2017).
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Table 1.2 Sulfated glycosaminoglycan concentration after DNAse and ChABCase treatment. Samples were treated with
Sample sGAG µg/mg sample Decrease
S2 + buffer 50.96 ± 0.70 % -
CS + buffer 90.80 ± 0.51 -
CS + ChABCase 0 ± 0 100
S2 + DNAse 49.26 ± 0.62 3.34
S2 + ChABCase 37.55 ± 2.93 26.31
S2 + DNAse+ChABCase 36.46 ± 1.83 28.45
Discussion
The EPS extracted from the acidophilic biofilm was analysed for anionic molecules. From the data presented it is shown that the anionic molecules are most probably sulfated polysaccharides that are sGAG-like. Sulfated polysaccharides are assumed as sulfated groups are a stronger acidic group and the BlyscanTM kit (Biocolor, UK) is developed to precipitate sulfated polymers. . In addition to the characterization, analysis of mycobacterial genomes showed the presence of sulfatases and sulfotransferases which indicated the capability of producing sulfated polymers (Mougous et al., 2002). The genome analysis, if available, is a powerful tool that can help further elucidation of the presence and role of extracellular anionic polymers (Van Vliet et al., 2020). Multiple instances of sGAG-like polymers have been discussed in previous studies (Bourven et al., 2015; Lin et al., 2018).
The biofilms in these studies all experienced different conditions but still sGAG-like polymers were produced. Therefore sGAG-like polymers must either fulfil a similar role in all of the biofilm or could fill multiple roles.
Biofilms mostly consist of communities of varying microorganisms which can thus contain different polymers with similar functions (Seviour et al., 2019). An important factor is the high
negative charge of sGAG which contributes to their ability to bind water or cations and thus influences biofilm stability (Varki, A., 2017; Gagliano et al., 2018). Especially in acidophilic environments highly acidic polymers like sGAG might be needed to maintain the negative charged species in the EPS. Weak acidic groups like carboxylic acids become protonated and thus lose their charge (Boleij et al., 2020; Felz et al., 2020).
Mycobacterium are known to have an extensive extracellular envelope, and can protect the mycobacterium from acidic conditions and desiccation (Vincent et al., 2018). This diverse
extracellular layer contains glycosylated proteins. This is a possible location for the sGAG-like polymers found in the EPS. The sGAG-like polymer could possibly be produced to bind water in the extracellular envelope.