52
Spatial growth activity patterns in non-spherical fermentative granular
53
incubated biofilms with 15NH4Cl as growth activity marker. This enabled determining species-specific growth activity patterns across the biofilm as a potential explanation for the non-spherical growth of these fermentative granular biofilms.
Results
Three organisms were isolated from caproic acid-producing granular biofilms, and full-length 16S rRNA gene sequences were obtained for development of FISH-probes. Reclassification of 16S rRNA gene amplicon sequences from the granular biofilm with these new sequences showed the three organisms represented the three most dominant OTU in the system. Accounting for over half of the granular biofilm community (54.3±9.6%), these three species (one Olsenella, two Ruminococcaceae) are representative of the key catalytic organisms in the system. These three organisms showed no clear species-based stratification in the granular biofilm (Figure 1.1). All observed granules had an active layer of 150-500 µm with an average granule diameter of approx. 3.0 mm . Deeper layers showed inactive or dying cells (indicated by DAPI-staining but not FISH-staining) and eventually even lack of DAPI-staining, indicating the presence of a biologically inert core.
We used nanoSIMS to analyze 15N-incorporation – representing growth activity – in cells along transects starting from the convex vs. planar periphery, representing respectively slow and fast product diffusion (Figure 1.2). Average label incorporation at the edge of the granule was similar, but, appears to be more sustained with increasing depth in the granule at the planar periphery (Figure 1.2A) than at the convex periphery (Figure 1.2B). This could indicate a higher total growth activity at the planar periphery, i.e. the thin edge of the granule, an observation in support of our working hypothesis.
The combination of FISH and nanoSIMS was used to investigate species-specific growth activity patterns. A first key observation is that the average 15N-incorporation of organisms targeted by FISH (i.e.
Olsenella, Ruminococcaceae) was consistently higher than that of organisms only stained with a general Bacterial probe. This confirms the relevance of the FISH-probes to investigate the questions at hand.
Second, our initial hypothesis suggested the activity of Olsenella would decrease faster than that of Ruminococcaceae due to caproic acid toxicity. However, this part of our working hypothesis appears to not hold true, as Olsenella-classified cells were equally or more active than Ruminococcaceae with increasing depth.
Discussion
We investigated the mechanisms driving the lenticular morphology of caproic acid-producing granular biofilms using a combination of FISH and nanoSIMS. We hypothesized that caproic acid toxicity would lead to (i) stratification of lactic acid and caproic acid producing Bacteria (respectively Olsenella and Ruminococcaceae) due to differential caproic acid tolerance, and (ii) higher overall growth activity at the planar edge of the granule than at the convex edge due to faster outward diffusion of caproic acid, leading to further growth along the plane of the lenticular biofilm.
The first part of this hypothesis does not appear to hold true. FISH-visualization of the granular biofilm community structure showed no clear species-based stratification, but only an activity gradient. This is further confirmed by FISH-nanoSIMS for species-specific activity analysis, where no differentiation could be observed between Olsenella and Ruminococcaceae.
The second part of this hypothesis – focusing on the lenticular morphology – could still hold true based on higher growth activity deeper into the granule, despite similar growth activities at the periphery of the biofilm. Future research should confirm whether this pattern is due to enhanced caproic acid diffusion at the planar periphery of the biofilm, or whether other mechanisms underlie this pattern.
Ultimately, this study is the first report of the spatial organization of fermentative granular biofilms, but more importantly, aimed to investigate the unique non-spherical morphology of these biofilms. The data
54
presented indicate differential growth patterns across the granular biofilm could cause this lenticular morphology.
Figure 1.1 Fluorescence microscopy image of a representative lenticular granule section. The tearing and lack of FISH-stain at the bottom of the image indicates breakage of the granule. Colur code: Blue = DAPI, Red = all Bacteria (Amann et al., 1990; Daims et al., 1999), Green = Ruminococcaceae, Yellow = Olsenella
Figure 1.2 Comparison of isotope-incorporation determined by nanoSIMS along transects from planar periphery (Panel A) and convex periphery (Panel B) for all cells (Averaged) or for cell types determined by FISH. Lines represent average for each group. Isotope label amounted to 12% 15NH4Cl.
References
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Deverux R, Stahl DA. 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol.
56:1919–1925.
Carvajal-Arroyo JM, Candry P, Andersen SJ, Props R, Seviour T, Ganigué R, Rabaey K. 2019. Granular fermentation enables high rate caproic acid production from solid-free thin stillage. Green Chem. 21:1330–1339.
55
Daims H, Bruhl A, Amann R, Schleifer K, Wagner M. 1999. The Domain-specific Probe EUB338 is Insufficient for the Detection of all Bacteria : Development and Evaluation of a more Comprehensive Probe Set. Syst. Appl. Microbiol.
22:434–444.
Jeon BS, Kim BC, Um Y, Sang BI. 2010. Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp.
BS-1. Appl. Microbiol. Biotechnol. 88:1161–1167.
Zhu X, Zhou Y, Wang Y, Wu T, Li X, Li D, Tao Y. 2017. Production of high-concentration n-caproic acid from lactate through fermentation using a newly isolated Ruminococcaceae bacterium CPB6. Biotechnol. Biofuels 10:1–12.
Presenting Author
Dr. Candry Pieter Post-doctoral researcher
Department of Civil & Environmental Engineering, University of Washington, Seattle, WA, USA
Is the presenting author an IWA Young Water Professional? No (i.e. an IWA member under 35 years of age)
Bio: Dr. Candry is a young researcher focusing on metabolic interactions between microbial functional guilds in engineered and natural settings. In his Ph.D., he investigated the kinetics and microbial ecology of chain elongation systems in both suspended biomass systems and biofilm systems. In his current post-doctoral research position, he investigates the feedback loops between climate change and wetland microbiota, using biofilm-like hydrogels to construct synthetic soils.
56
A performance comparison of aerobic granular sludge and hybrid bio-granular activated carbon treating recalcitrant and toxic dissolved organic compounds under hypersaline conditions
Abdullah Ibrahim*, Yasawantha Hiripitiyage**, Edward F. Peltier**, Belinda S. M. Sturm**
*Department of Environmental Engineering, University of Mosul, Mosul, Iraq 41002.
**Department of Civil, Environmental, and Architectural Engineering, University of Kansas, Lawrence, KS, USA 66045
Keywords: AGS; BGAC; recalcitrant compounds Summary of key findings
The performance of aerobic granular sludge (AGS) and bio-garnular activated carbon (BGAC), as biofilm-based biological processes, was compared in this study. Organic load removal and the capacity of biofilm to accumulate organic matter were observed to investigate the performance of these
biosystems. The microbiome analysis proved that the biocommunity structure is a function of carbon source and operation conditions. BGAC system achieved the higher adsorption capacity compared to AGS. The integrated results from batch experiment and XPS analysis showed that the high adsorption capacity was associated with high removal efficiency. The detected intermediate derivative compounds suggest that the approach that used by microorganisms was to adsorb then biodegrade organic matter, or both processes can happen simultaneously.
Background and relevance
The increasing demand for fossil fuels has stimulated the generation of oil and gas wastewater, or produced water (PW) as a byproduct of this industry (Jimenez et al. 2018). Toxic and recalcitrant compounds are often found in PW can limit microbial metabolisms and further hinder conventional biological treatment process (i.e., activated sludge) (Butkovskyi et al. 2017). The presence of refractory organic compounds limits options for cost-effective treatment (Nie et al. 2020).
Biological treatment has been applied successfully to remove a wide rangr of contaminats from wastewater (Acharya et al. 2019, Ren et al. 2017, Silva et al. 2019, Sudmalis et al. 2018, Wang et al.
2018b). However, the presence of refractory and toxic organic compounds in wastewater can pose a tremendous threat to biological process. (Kim and Ihm 2011, Ramos et al. 2015). The high salinity of many produced waters provides an additional source of stress on biological treatment components (Nie et al. 2020). For these reasons, implementation of conventional activated sludge (CAS) for petroleum wastewater treatment is typically unreliable s (Felz et al. 2020, Gao et al. 2020).
Biofilm-based techniques have shown promise as an alternative treatment approach to CAS systems that provide additional protection against toxic and shock loadings to the embedded microorganisms (Wang et al. 2018a, Yusoff et al. 2018, Zhao et al. 2019). Substrates mass-diffusion transport through biofilm improves the ability of microorganisms to withstand toxic and other potential shocks (Corsino et al.
2015, He et al. 2020, Ofman et al. 2020). AGS, as a self-immobilized biofilm technique, is a new technology that has seen rapid growth for municipal and industrial wastewater treatment within the last decade (Wu et al. 2020). The granule structure controls the flux of organic compounds to the biomass and helps to retain slower growing microorganisms, resulting in a robust system with multiple potential biodegradation pathways (Corsino et al. 2017, Fang et al. 2018, Li et al. 2017, Ou et al. 2018, Ramos et al. 2015).
Hybrid biofilm techniques have also been applied recently to further improve the performance of self-immobilized systems (Wang et al. 2018b). The use of hybrid biofilm techniques, including integration of granular activated carbon (GAC) as an adsorptive biofilm surface (BGAC), can further improve
treatment performance by incorporating both biological and physico-chemical processes to remove
57
organics (Wang et al. 2018b, Zhang et al. 2013). The high adsorption capacity of GAC can act as a short-term sink for organic compounds. reducing toxicity and protecting against toxic shocks (Puyol et al.
2015). Over time, the biodegradation of refractory organic compounds is achieved due to close proximity of the attached biomass to adsorbed organic matter and nutrients (Puyol et al. 2015, Wang et al. 2018b).
The BGAC design can also reduce the amount of GAC required compared to packed biofilm reactors and improve the settling properties of the biomass (Loukidou and Zouboulis 2001, Wang et al. 2018a, Wang et al. 2018b).
The performance of two different types of biofilm based aerobic reactors was examined in this study for their ability to remove influent aromatics and prevent the formation of toxic by-products. A mixture of aromatic copmounds was supplied to represent te most commonly found aromatic compounds in PW.
The ability of biofilms to adsorp aromatic compounds was investigated by running batch studies and then analyzied using X-ray photoelectron spectroscopy (XPS) technique. Biocommunities in AGS abd BAC were also monitored to investigate the impact of adding of aromatic compounds on microbial
community.
Results
BGAC reactor showed higher performance in terms of aromatic compounds removal, approximately 99
% of parent aromatic compounds (benzyl alcohol, phenol and o-Cresol) were removed. The
concentration of catechol, a bioproduct of phenol and benzyl alcohol biodegradation, in effluent was approximately 0.4 ± 0.1 mg L-1. The overall removal eefeciency of aromatic compounds was
approximately 93% in AGS reactor. Although the both reactors were seeded with same microbial at the beginning of operation phase, the microbiome analysis showed that the microbial communities was very different by the end of operating phase (Figure 1). The XPS analysis showed that the biosorption process was tookplace in both biofilms, but the following biodegradation process was slower in GAS biofilm (Figure 2).
Discussion
The performance of BGAC reactor was higher than AGS reactor in terms of aromatic copmounds removal efficiency. The potential reason behind that can be attributed to integrated physico-chemical and biological processes on the surface of granular activated carbon. The microbiome analysis showed that the microbial communities were shifted in both reactors as the carbon source changed . This finding indicates that the growning conditions play a curocial role to create favorible conditions for retaining selected group of microorganisms. It has also showed that carbon source is another factor that select for specific microorganisms within the biofilm. The XPS analysis showed that the peaks positions shifted and the area under curves changed as the operation phases switched from feast to famine (Figure 1). This observation indicates that the carbon form within the biofilm was converted from one form to another as a result of bioactivy of microorganisms that impeded in biofilm.
58
Figure 1: Microbial community structure for AGS &BGAC reactors.
Figure 2: XPS wide survey scans of carbon atom at feast and famine phases for Bio-GAC and AGS.
References
Acharya, K., Werner, D., Dolfing, J., Meynet, P., Shamas Tabraiz, Baluja, M.Q., Petropoulos, E., Mrozik, W. and Davenport, R.J. (2019) The experimental determination of reliable biodegradation rates for mono-aromatics towards evaluating QSBR models. Water Research 160, 278-287.
Corsino, S.F., Campo, R., Bella, G.D., Michele Torregrossa a and Viviani, G. (2015) Cultivation of granular sludge with hypersaline oily wastewater. International Biodeterioration & Biodegradation 105, 192-202.
AGS BGAC
282 284
286 288
290
Relative intensity
Binding energy (eV) Bio-GAC_Feast
282 284
286 288
290
Relative intensity
Binding energy (eV) Bio-GAC_Famine
282 284
286 288
290
Relative intensity
Binding energy (eV) AGS_Feast
282 284
286 288
290
Relative intensity
Binding energy (eV) AGS_Famine
BGAC_ Famine BGAC_ Feast
59
Corsino, S.F., Capodici, M., Torregrossa, M. and Viviani, G. (2017) Physical properties and Extracellular Polymeric Substances pattern of aerobic granular sludge treating hypersaline wastewater. Bioresource Technology 229, 152–
159.
Fang, F., Yang, M.-M., Wang, H., Yan, P., Chen, Y.-P. and Guo, J.-S. (2018) Effect of high salinity inwastewater on surface properties of anammox granular sludge. Chemosphere 210, 366-375.
Felz, S., Neu, T.R., Loosdrecht, M.C.M.v. and Lin, Y. (2020) Aerobic granular sludge contains Hyaluronic acid-like and sulfated glycosaminoglycans-like polymers. Water Research 169, 115291.
Gao, S., He, Q. and Wang, H. (2020) Research on the aerobic granular sludge under alkalinity in sequencing batch reactors: Removal efficiency, metagenomic and key microbes. Bioresource Technology 296, 122280.
He, Q., Song, J., Zhang, W., Gao, S., Wang, H. and Yu, J. (2020) Enhanced simultaneous nitrification, denitrification and phosphorus removal through mixed carbon source by aerobic granular sludge. journal od Hazardous Materials 382, 121043.
Jimenez, S., Mic, M.M., Arnaldos, M., Medina, F. and Contreras, S. (2018) State of the art of produced water treatment, Chemosphere 192, 186-208.
Kim, K.-H. and Ihm, S.-K. (2011) Heterogeneous catalytic wet air oxidation of refractory organic pollutants in industrial wastewaters: A review. Journal of Hazardous Materials 186, 16-34.
Li, X., Luo, J., Guo, G., Mackey, H.R., Hao, T. and Chen, G. (2017) Seawater-based wastewater accelerates development of aerobic granular sludge: A laboratory proof-of-concept. Water Research 115, 210-219.
Loukidou, M.X. and Zouboulis, A.I. (2001) Comparison of two biological treatment processes using attached- growth biomass for sanitary landfill leachate treatment. Environmental Pollution 111(2), 273-281.
Nie, H., Nie, M., Diwu, Z., Wang, L., Yan, H., Lin, Y., Zhang, B. and Wang, Y. (2020) Biological treatment of high salinity and low pH produced water in oilfield with immobilized cells of P. aeruginosa NY3 in a pilot-scale. Journal of Hazardous Materials 381, 121232.
Ofman, P., Struk-Sokołowska, J., Skoczko, I. and Wiater, J. (2020) Alternated biodegradation of naphthalene (NAP), acenaphthylene (ACY) and acenaphthene (ACE) in an aerobic granular sludge reactor (GSBR). Journal of
Hazardous Materials 383, 121184.
Ou, D., Li, H., Li, W., Wu, X., Wang, Y.-q. and Liu, Y.-d. (2018) Salt-tolerance aerobic granular sludge: Formation and microbial community characteristics. Bioresource Technology 249, 132-138.
Puyol, D., Monsalvo, V.M., Sanchis, S., Sanz, J.L., Mohedano, A.F. and Rodriguez, J.J. (2015) Comparison of bioaugmented EGSB and GAC–FBB reactors and their combination with aerobic SBR for the abatement of chlorophenols. Chemical Engineering Journal 259, 277-285.
Ramos, C., Suárez-Ojeda, M.E. and Carrera, J. (2015) Long-term impact of salinity on the performance and microbial population of an aerobic granular reactor treating a high-strength aromatic wastewater. Bioresource Technology 198, 844–851.
Ren, L.-F., Chen, R., Zhang, X., Shao, J. and He, Y. (2017) Phenol biodegradation and microbial community dynamics in extractive membrane bioreactor (EMBR) for phenol-laden saline wastewater. Bioresource Technology 244, 1121-1128.
Silva, N.C.G., Macedo, A.C.d., Pinheiro, Á.D.T. and Rocha, M.V.P. (2019) Phenol biodegradation by Candida tropicalis ATCC 750 immobilized on cashew apple bagasse. Journal of Environmental Chemical Engineering 7, 103076.
Sudmalis, D., Silva, P.D., Temmink, H., Bijmans, M.M. and Pereira, M.A. (2018) Biological treatment of produced water coupled with recovery of neutral lipids. Water Research 147, 33-42.
Wang, B., Zhao, M., Guo, Y., Peng, Y. and Yuanb, Y. (2018a) Long-term partial nitritation and microbial characteristics in treating low C/N ratio domestic wastewater. Environmental Science Water Research &
Technology.
Wang, F., Zhou, L. and Zhao, J. (2018b) The performance of biocarrier containing zinc nanoparticles in biofilm reactor for treating textile wastewater. Process Biochemistry 74, 125-131.
Wu, X., Li, H., Lei, L., Ren, J., Li, W. and Liu, Y. (2020) Tolerance to short-term saline shocks by aerobic granular sludge. Chemosphere 243, 125370.
Yusoff, N.A., Ong, S.-A., Ho, L.-N., Wong, Y.-S., Saad, F.N.M., Khalik, W.F. and Lee, S.-L. (2018) Theoretical development of biofilm in hybrid growth sequencing batch reactor (HG-SBR) for degradation of phenol. Desalination and Water Treatment 107, 100-108.
60
Zhang, W., Ding, W. and Ying, a.W. (2013) Biological Activated Carbon Treatment for Removing BTEX from Groundwater. journal of environmental Engineering 139(10), 1246-1254.
Zhao, Y., Liu, D., Huang, W., Yang, Y., Ji, M., Nghiem, L.D., Trinh, Q.T. and Tran, N.H. (2019) Insights into biofilm carriers for biological wastewater treatment processes: Current state-of-the-art, challenges, and opportunities.
Bioresource Technology 288, 121619.
Presenting Author
Dr. Abdullah Ibrahim Lecturer
Department of Environmental Engineering, University of Mosul, Mosul, Iraq Is the presenting author an IWA Young Water Professional? Y/N N
Bio: I have received my M.Sc. Environmental Engineering from Mosul University, Iraq in 2004 then I worked as a faculty member in Civil Engineering department 2005-2013. As well as, I was one of funders and faculty members of Environmental Engineering department 2011-2013.
In 2014, I have joined Kansas University, USA as a PhD student in Environmental
Engineering field. I received my PhD in Spring 2020 in the field of biological treatment of gas and oilfield produced water with emphasis on manipulation of biofilm technology to treat wastewater under harsh conditions.