Ref # S2SMALL-04741
Azevedo, L. S.1, Castro, I. M. P.1, Santos, L. A.1, Leal, C. D.1, Araújo, J. C.1, Chernicharo, C. A. L.1*
1Federal University of Minas Gerais, Department of Sanitary and Environmental Engineering, Av. Antônio Carlos 6.627, Campus Pampulha, 31.270901 Belo Horizonte -MG - Brazil.
* Presenting Author: calemos@desa.ufmg.br
Abstract: Two phototrophic bioreactors were investigated as an alternative to post-treat effluent from an UASB reactor treating domestic sewage, aiming at sulphide oxidation to elemental sulphur and complementary organic matter removal. The bioreactors were operated at different hydraulic retention times and presence or absence (control) of packing material for biomass retention. Greater sulphide removal efficiencies were achieved in both reactors for HRT of 6 h, 75% and 92% for the control reactor and packed reactor, respectively. Higher organic matter (COD) and solids (TSS) removal were observed in the packed reactor, able to produce a final effluent with low COD and TSS concentrations (100 mgCOD.L-1 and 30 mgTSS.L-1). Denaturing gradient gel electrophoresis results revealed that purple and green biomass developed in both reactors and comprised a diverse bacterial community with sequences related to phototrophic sulphur bacteria such asThiocapsaand Chlorobaculum. Elemental Sulphur formation was detected and the potential for Sulphur recovery was observed. The phototrophic bioreactors were able to improve the quality of the anaerobic effluent and higher efficiencies related to the packed reactor were attributed to the presence of the packing material and higher cell retention time.
Keywords:post-treatment, phototrophic bioreactors, sulphide oxidation, UASB reactor.
INTRODUCTION
UASB reactors have been widely adopted for sewage treatment in many warm climate countries. Despite several advantages attributed to the system, some limitations still remain such as mal-odour emission and the need of a post-treatment unit for additional organic matter and nutrient removal (i.e. nitrogen, phosphorous and sulphur), in order to reduce the discharge impact of effluents on receiving waters (Chernicharo et al. 2015). Odour emission in anaerobic-based sewage treatment plants is mainly related to the release of dissolved sulphide present in the bulk liquid, especially in the outlet structures where turbulent conditions prevail (Souza et al. 2012). Along with its characteristic offensive odour, H2S has several negative effects such as toxicity, corrosivity and flammability (USEPA 2010).
Strategies for sulphide control involve either inhibition of H2S production or elimination of the produced gas (Garcia de Lomas et al. 2005). Sulphide oxidation may occur by biological or chemical pathways, however, biological process has major advantages such as low cost and high efficiency, achieving less than 1 mg.L-1 of sulphide concentration in the effluent (Henshaw et al. 1999). Biological sulphide oxidation in liquid or gas streams may occour through the activity of phototrophic or chemolithotrophic bacteria, under aerobic, anoxic or anaerobic conditions. Sulphide-oxidizing bacteria are grouped according to the used sulphur compound, colour and environmental conditions. Aerobic sulphide oxidation is performed by colorless sulphur bacteria while anaerobic oxidation occurs by means of anoxygenic photosynthesis, performed by green and purple bacteria that use light as energy source in anoxic environments (Madigan 2010). Although advantages of biological process have been reported, the scale up of phototrophic reactors is not straight forward due to the requirement of transparent material to allow light entrance (Janssen et al.1999).
Previous studies have investigated the biological oxidation of sulphide to elemental sulphur (Vannini et al.2008; Fajardo et al. 2012; Liu et al. 2015), however, information regarding the removal of dissolved sulphide using real anaerobic effluent and bacteria naturally present in it are scarce. In this study, two phototrophic bioreactors were investigated as an alternative to post-treat effluent from an UASB reactor treating domestic sewage, aiming at sulphide oxidation to elemental sulphur and complementary organic matter removal Partial sulphide oxidation to elemental sulphur is of particular interest due the possibility of elemental sulphur recovery. The phototrophic bioreactors were designed with a shape similar to the settler compartment of a UASB reactor and operated under conditions comparable to real scale.
Therefore, results obtained in this work may be used to optimize reactor operational conditions, in order to favour elemental sulphur formation and to improve the overall performance of the phototrophic bioreactors.
MATERIALS AND METHODS Experimental set-up
The experiments were performed in a system comprised by a pilot-scale UASB reactor built in fibreglass (340 L) followed by two Plexiglas bioreactors (30 L) operating in parallel, designed to oxidise dissolved sulphide present in the UASB reactor effluent. The phototrophic reactors were designed to be fed from the bottom (upflow mode), with a configuration similar to the settler compartment of UASB reactors: cone-shaped bottom (h=20cm) and cylindrical chamber (h=30cm), as shown in Figure 1 (a). The Plexiglas reactors were identical, except for the absence (R1-control) or presence (R2-packed) of packing material (Figure 1-b).
R1 R2
Figure 1. Schematic representation and dimensions of the (a) bioreactors and (b) packing material.
The set-up was installed at the Centre for Research and Training in Sanitation UFMG/COPASA, located at the Arrudas WWTP, the main treatment plant of Belo Horizonte city, Brazil (coordinates 19°53′42″ S and 43°52′42″ W, altitude 800 m). The pilot-scale UASB reactor was fed with an aliquot of the raw sewage that feeds the full-scale plant, after being submitted to preliminary treatment for solids and grit removal. The main characteristics of the typical urban wastewater were: pH = 7.5; COD = 516 mg.L-1; S2-= 0.5 mg.L-1; SO42-= 32 mg.L-1.
The bioreactors were monitored over 7 months under distinct operational conditions, according to the following hydraulic retention time (HRT): 6 h, 4 h and 2 h. Throughout the monitoring period, redox potential and pH inside the reactors ranged between 68 mV and -110 mV (Standard hydrogen electrode) and 6.8 - 7.1, respectively. At the end of each phase,
(b) (a)
H = 35 cm
Ø = 45 cm
the rectors were emptied and the packing material replaced. R2-packed was equipped with a perforated stainless-steel basket, in order to retain the polypropylene rings (Ø 45 mm, h= 35 mm, 86 m²/m³ specific surface – Bio Project, Brazil), used as packing material (Figure 1-b).
The adoption of packing material intended to provide higher retention of microorganisms naturally present in the UASB reactor effluent once the phototrophic reactors were not inoculated.
Physicochemical analysis
Sulphate (SO42-), total suspended solids (TSS) and chemical oxygen demand (COD) were measured according to the standard methods (APHA, 2012). Sulphide was measured according to Plas et al. (1992) and elemental sulphur (S0) by high performance liquid chromatography (HPLC) using a PRP-1 reverse phase HPLC column (dimensions: 15 cmL × 4.1 mmID), as described by Henshaw et al. (1998). Analyses were performed twice a week except for TSS and S0, which were analyzed once a week.
Analysis of the microbial community
At the end of each phase, samples were collected from the microbial layer (attached and non-attached biomass) and sludge (bottom of the reactors) aiming to allow the comparison of bacterial diversity for all tested HRT. Cells were taken from several polypropylene rings located at different heights to provide a representative sample. Samples were centrifuged three times (4000 rpm for 10 minutes) and washed with phosphate buffer saline solution (1x PBS, NaCl, Na2HPO4, NaH2PO4, pH = 7.2-7.4). The supernatant was discharged and the pellets were stored at -20°C. DNA was extracted using PowerSoil DNA Isolation Kit (MO BIO Laboratories,E.U.A.). Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) was performed using the primer set 1055F and 1392R with a GC clamp according to Ferris et al. (1996). DGGE was performed at 60°C in 0.5×TAE buffer at 80 V for 16,5h using a DCode system (Bio-Rad Universal Mutation Detection, (Hercules, CA, USA) comprising 8% polyacrylamide gel with a 50–65% gradient of urea formamide denaturant. Gels were stained with SYBR Gold solution (Life Technologies) and visualized under UV transillumination. Specific gel bands were excised, re-amplified, purified, and sequenced using a genomic service (Macrogen Inc., Seoul, Korea). Sequences were compared with that from Ribossomal Database Project(https://rdp.cme.msu.edu/classifier/classifier.jsp) and National Center for Biotechnology Information (NCBI) database. DGGE patterns were analysed using BioNumerics software 6.6 and the Shannon Index (Gafan et al. 2005) was employed to assess bacterial biodiversity in biomass and sludge under the tested conditions.
RESULTS AND DISCUSSION Biological sulphide oxidation
Dissolved sulphide and elemental sulphur concentrations in the effluent of the UASB reactor and phototrophic bioreactors are shown in Figure 2. Dissolved sulphide values observed in the UASB reactor effluent, 5 - 11 mgS2-.L-1, were similar to those obtained by Souza et al.
(2012), 7 - 11 mgS2-.L-1, and higher than those observed by Garcia et al., (2017), 2 - 3 mgS 2-.L-1. These studies investigated anaerobic reactors treating domestic wastewater from the same contribution region.
Sulphide median concentration in the UASB reactor effluent was higher for the operational period at 2h HRT, 11,3 mgS2-.L-1, as compared to the previous phases, HRT of 6 and 4 h (5.4 and 6.6 mgS2-.L-1). Due to this huge variation between phases, removal efficiencies instead of absolute concentrations values were used to compare the results. Higher sulphide removal
efficiencies were observed for both bioreactors for HRT of 6h, 75% for R1-control and 92%
R2-packed. Garcia et al.(2017) reported sulphide removal efficiencies between 50 - 60%, for the same HRT. This lower efficiency could be related to median sulphide concentration in the UASB reactor effluent, 2 mgS2-.L-1, 63% lower than the one determined in this study, 5.4 mgS2-.L-1. Sulphide effluent concentrations of the phototrophic bioreactors were 1.2, 2.0 and 4.9 mgS2-.L-1 for R1-control, and 0.5, 3.1 and 3.4 mgS2-.L-1, for HRT of 6, 4 and 2 h,
Figure 2.(I) Dissolved sulphide and (II) Elemental sulphur concentrations in the effluent of the UASB reactor and phototrophic bioreactors. R1-control, R2-packed with polypropylene rings.
Among the studied conditions, higher sulphate concentrations were observed in R1-control effluent, pointing that the absence of packing material favoured sulphide oxidation to sulphate, with median concentrations of 40.2, 27.0 and 28.1 mgSO42-.L-1 for 6, 4 and 2h of HRT, respectively. Although R2-packed effluent showed higher sulphide oxidation under 6 and 2 h HRT conditions, the same did not occur regarding sulphate formation, with median concentrations of 28.0 and 24.1 mgSO42-.L-1, possibly due the greater formation of elemental sulphur provided by the use of packing material.
Since biologically produced elemental sulphur has a white or yellow-pale colour (Janssen et al. 1999), the formation of this compound was observed from the white precipitate present in the effluent of the phototrophic reactors and confirmed by high performance liquid chromatography. The white precipitate observed in the R2-packed effluent corresponding to extracellular sulphur is presented in Figure 3 (I-II), whereas intracellular stored sulphur is presented in Figure 3-III.
Regarding elemental sulphur formation, higher concentrations were observed in R2-packed effluent for all tested HRT, even when higher sulphide removal efficiency was associated to R1-control (see Figure 2-II). Elemental sulphur concentrations in the R1-control effluent were 2.0, 3.6 and 5.6 mgS0.L-1, while higher concentrations were observed in the R2-packed effluent, 3.8, 3.6 and 7.3 mgS0.L-1, for HRT of 6, 4 and 2h, respectively. The greatest sulphur formation was related to R2-packed, suggesting that the presence of polypropylene rings provided longer cellular retention time and favoured elemental sulphur formation.
It is possible to observe, from Figure 2-II, that elemental sulphur was produced in the UASB reactor, probably in the settling compartment. Green and purple colours were observed in the phototrophic reactors possibly due the existence of naturally occurring sulphur bacteria in the UASB reactor effluent. Therefore, considering sun light penetration into the upper part of the reactor (uncovered settling compartment) it is possible to assume that the elemental sulphur in the UASB reactor effluent is related to sulphur bacteria activity, located in the reactor scum
layer, naturally formed and maintained on the surface of the settler compartment of the UASB reactor.
I II III
Figure 3.(I) Settling test of R2-packed effluent (HRT = 6h) after 30 minutes, with white solids at the bottom of the Imhoff cone; (II) white precipitate present in the R2-packed effluent; (III) microscopic image of purple bacteria in R2-packed biomass (HRT = 4h), emphasizing intracellular elemental sulphur.
Overall performance of the system
The overall performance of the system for the parameters COD and TSS is depicted in Figure 4. Regarding COD, higher median removal efficiency was observed for R2-packed, about 40% for HRT of 6 and 4h, as compared to around 28% observed for R1-control for HRT of 4 h. R1-control median effluent COD concentrations were 113.2, 122.3 and 120.7 mgCOD.L-1, whereas lower concentrations were observed for R2-packed, 91.5, 96.0 and 104.4 mgCOD.L
-1, for HRT of 6, 4 and 2h, respectively.
Figure 4. Concentrations of (I) COD and (II) TSS in UASB and phototrophic reactors effluent at different operational conditions (TDH = 6, 4 and 2 h).
Considering TSS, lower concentrations were observed in the R2-packed effluent for HRT of 6h and 4h, 22.8 and 26.4 mgTSS.L-1, while 40.5 and 44.7 mgSST.L-1were observed in the R1-control effluent. For the lower HRT (2h), the TSS concentration was around 29 mgSST.L
-1for both bioreactors, indicating that the higher flowrate induced the loss of biomass attached to the polypropylene rings. For this operational condition the presence of packing material did not contribute to increase the solids retention capacity of R2-packed.
Higher COD removal efficiencies and TSS retention were observed in the R2-packed, which was able to produce a final effluent with low COD and TSS concentrations, close to 100 mgCOD.L-1and 30 mgTSS.L-1, therefore able to comply with restrictive discharge standards.
Effect of packing material on microbial diversity
Bacterial community diversity of biomass and sludge was evaluated at the end of each phase by PCR-DGGE technique. The comparison of DGGE profiles was performed by the dendrogram showed in Figure 5, which reports the similarity between the profiles. DNA extraction of biomass was not successful and for this reason it was not included in the following stages. DGGE results revealed that bacterial community was diversified in the bioreactors, between, different operational conditions, and three major and distinct clusters were identified according to the tested HRT (showing 57-71% similarity). Except for HRT of 6h, with 74,3% of similarity between biomass, the profiles of microbial diversity observed for the others HRT were different.
The lowest similarity of biomass was observed for HRT of 2 h, 46%. Regarding sludge bacterial composition, R1-control and R2-packed showed lower similarities for 6 and 4 h HRT, 57 and 65%, respectively. The highest similarity observed in the dendrogram were related to sludge samples under 2h HRT conditions. The difference between the profiles of the reactors suggests that the presence of the packing material in R2-packed and absence in control affected both sludge and biomass bacterial community. Sludge and biomass of R1-control presented similarity between 52- 71%, while the samples of R2-packed showed lower similarity, 46-57%. Among the values observed for sludge and biomass, lowest similarity was observed to R2-packed (46%) for HRT of 2h, indicating that the presence of the packing material favoured distinct bacterial compositions between sludge and biomass.
74.3
Figure 5. Dendrogram based on the DGGE profiles of the bacterial community in biomass and sludge samples collected at different operational conditions (HRT = 6, 4 and 2h).
The similarity in the dendrogram was confirmed by the Shannon diversity index, used to estimate bacterial diversity of biomass (attached and non-attached) and sludge (bottom of the reactors) samples ranged from 1.05 to 1.29, indicating that the decrease of HRT did not expressively affect the microbial diversity. Concerning the sludge index, higher values were observed, 1.18-1.29, when compared to biomass index, 1.05-1.20. Although similar, it is possible to note that both biomass and sludge presented higher diversity for HRT of 6h, except for sludge under 4h HRT condition.
Microbial community in the phototrophic reactors determined by sequencing
Sequences similar to several bacteria genera were identified by sequencing the strongest bands in the DGGE gel. DNA sequences similar to Anaerolineaceae (likely Anaerolinea genus) were identified in the sludge of R2-packed (6h and 2h HRT) and R1-control (4h). This genus consists of fermentative hydrolytic bacteria, possible associated to the degradation of organic matter present in the UASB reactor effluent (Huget al.,2013).
Sequences related to Rhodopseudomonas and Rhodocyclus, purple non-sulphur bacteria, were identified in the biomass of R1-control (6h HRT) and R2-packed (2h HRT) and in the sludge of R1-control (4h HRT). Rhodopseudomonas palustris, a phototrophic bacteria with versatile metabolism and capable of organic matter degradation (Madigan, 2010), was identified in the biomass of R2-packed (2h HRT). DNA sequences similar to Spirulina genus, a strong band present in all the samples from both reactors, is related to filamentous cyanobacteria.
Phototrophic sulphur bacteria and cyanobacteria may coexist, due to sulphur bacteria oxygen tolerance (Stal 1995).
Sequences similar to sulphur bacteria, Thiocapsa, Chlorobaculum, Sulfurimonas and Sulfuricella, were identified in sludge and biomass of R1-control (6h HRT). Purple Sulphur bacteria from Thiocapsa genus perform anoxygenic photosynthesis, using H2S as an electron donor to produce intracellular elemental sulphur. Purple bacteria and intracellular sulphur were observed (Figure 3-III), corroborating that sulphur bacteria occurred at different HRT.
Chlorobaculum sequences, which is a green sulphur bacteria, were identified in the biomass of R1-control for HRT of 4h. The main advantage attributed to green bacteria is related to extracellular elemental sulphur formation (Madigan, 2010). Although Chlorobaculum genus was identified specifically in one band from R1-control, the occurrence of extracellular elemental sulphur in the phototrophic reactors effluent was verified throughout monitoring.
Even though the sequencing of all DGGE bands was not accomplished, the presence of phototrophic sulphur bacteria was verified in most of the DGGE profiles (bands in similar position were sequenced); thus suggesting that phototrophic sulphur bacteria occurred in all operational conditions.
CONCLUSIONS
The results demonstrated that the phototrophic bioreactors were able to improve the quality of the effluent from the UASB reactor, regarding suspended solids removal/retention, COD removal, biological sulphide oxidation. The higher efficiencies related to R2-packed are likely due to the presence of the packing material and higher cell retention time. Higher sulphide removal efficiencies were obtained for the HRT of 6 h (R1-control: 78% and R2-packed:
92%) although higher elemental sulphur production was obtained for the lowest HRT (2 h), with median effluent concentrations of 5.5 and 7.3 mgS0.L-1, respectively for R1-control and packed. Higher COD removal efficiencies and TSS retention were observed in the R2-packed reactor, which was able to produce a final effluent with low COD and TSS concentrations, (100 mgCOD.L-1and 30 mgTSS.L-1), therefore able to comply with restrictive discharge standards. Microbial diversity in the phototrophic reactors was diverse and similar.
Sulphur bacteria were detected and identified, demonstrating the occurrence of biological sulphide oxidation in the reactors. Elemental Sulphur formation and the potential for Sulphur recovery were observed.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the support obtained from the following Brazilian institutions: Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq;
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES; Financiadora de Estudos e Projetos – Finep; Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG; Instituto Nacional de Ciência e Tecnologia em Estações Sustentáveis de Tratamento de Esgoto – INCT ETEs Sustentáveis (INCT Sustainable Sewage Treatment Plants). We also acknowledge the support from Copasa.
REFERENCES
Chernicharo C. A. L., Van Lier J. B., Noyola A. & Ribeiro T. B. 2015 Anaerobic sewage treatment:
state of the art, constraints and challenges. Environ Sci Biotechnol, 14, 649–679.
Fajardo C., Corral-mosquera A., Campos J. L. & Méndez R. 2012. Autotrophic denitrification with sulphide in a sequencing batch reactor. Journal of Environmental Management, 113, 552-556.
Ferris M. J., Muyzer G & Ward D. M. 1996 Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microb., 62, 340–346.
Gafan G.P., Lucas V.S., Roberts G.J., Petrie A., Wilson M. & Spratt D.A. 2005 Statistical analyses of complex denaturing gradient gel electrophoresis profiles. J. Clin. Microbiol., 43, 3972–3978.
Garcia de Lomas J., Corzo A., Gonzalez J. M., Andrades J.A., Iglesias, E. & Montero, M. J. 2005 Nitrate promotes biological oxidation of sulfide in wastewater: experiment at plant-scale.
Biotechnology and Bioengineering,93(4), 801–811.
Garcia G. P. P., Diniz R. C. O., Bicalho S. K., Franco V., Pereira A. D., Brandt E. F., Etchebehere C., Chernicharo C. A. L., Araujo J. C. 2017 Microbial community and sulphur behaviour in phototrophic reactores treating UASB effluent under different operational conditions. International Biodeterioration and Biodegradation, 119, 486-498.
Henshaw P. F., Bewtra J. K. & Biswas N. 1998 Hydrogen sulphide conversion to elemental sulphur in a suspended-growth continuous stirred tank reactor using Chlorobium limicola. Water Res., 32(6), 1769- 1778.
Hug L. A., Castelle C. J., Wrighton K. C., Thomas, B. C., Sharon, I., Frischkorn, K. R., Williams K.
H., Tringe S. G., Banfield, J. F. 2013 Community genomic analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and indicate in sediment carbon cycling. Microbiome, 1(22), 1-17.
Janssen A. J. H., Lettinga G. & Keiser A. 1999 Removal of hydrogen sulfide from wastewater and waste gases by biological conversion to elemental sulfur. Colloidal and interfacial aspects of biologically produced sulphur particles. Elsevier,151, 389-397.
Liu C., Zhao D., Yan L., Wang A., Gu Y. & Lee D. 2015 Elemental sulfur formation and nitrogen removal from wastewaters by autotrophic denitrifiers and anammox bacteria. Bioresource Technology, 191, 332–336.
Madigan M. T., Martinko J. M. & Parker, J. 2010 Brock biology of microorganisms. Prentice-Hall, 3rd ed., 1155 p.
Plas C., Harant H., Danner H., Jelinek E., Wimmer, K., Holubar P. & Braun, R. 1992 Ratio of biological and chemical oxidation during the aerobic elimination of sulphide by colourless Sulphur sulphur bacteria. Applied Microbiology and Biotechnology,36(6), 817-822.
Souza C. L.; Chernicharo C. A. L. & Melo, G. C. B. 2012 Methane and hydrogen sulfide emissions in
Souza C. L.; Chernicharo C. A. L. & Melo, G. C. B. 2012 Methane and hydrogen sulfide emissions in