journal of environmental sciences 135 (2024) 353–366 Available online at www.sciencedirect.com www.elsevier.com/locate/jes Research Article The effect of slime accumulated in a long-term operating UASB using crude glycerol to treat S-rich wastewater Xudong Zhou 1,2, Eva Fernández-Palacios 2, Antoni D. Dorado 3, Javier Lafuente 2, Xavier Gamisans 3, David Gabriel 2,∗ 1 KeyLaboratory of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China 2 GENOCOV Research Group, Department of Chemical, Biological and Environmental Engineering, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 3 Department of Mining, Industrial and ICT Engineering, Universitat Politècnica de Catalunya, Avinguda de les Bases de Manresa 61-73, 08242 Manresa, Spain a r t i c l e i n f o a b s t r a c t Article history: An up-flow anaerobic sludge blanket (UASB) reactor targeting sulfate reduction was oper- Received 26 September 2022 ated under a constant TOC/S-SO4 2− ratio of 1.5 ± 0.3 g C/g S for 639 days using crude glycerol Revised 15 November 2022 as carbon source. A filamentous and fluffy flocculant material, namely slime-like substances Accepted 15 November 2022 (SLS), was gradually accumulated in the bioreactor after the cease of methanogenic activity. Available online 24 November 2022 The accumulation of SLS was followed by a decrease in the removal efficiencies and a de- terioration in the performance. Selected characteristics of SLS were investigated to explore Keywords: the causes of its formation and the effect of SLS on the UASB performance. Results showed Sludge flotation that glycerol fermentation and sulfate reduction processes taking place in the reactor were Granular sludge mainly accomplished in the bottom part of the UASB reactor, as the sludge concentration in Slime-like substances the bottom was higher. The accumulation of SLS in the UASB reactor caused sludge flota- Glycerol fermentation tion that further led to biomass washout, which decreased the sulfate and glycerol removal Sulfate reduction efficiencies. Batch activity tests performed with granular sludge (GS), slime-covered gran- Removal efficiency ular sludge (SCGS) and SLS showed that there was no difference between GS and SLS in the mechanism of glycerol fermentation and sulfate reduction. However, the specific sul- fate reduction rate of GS was higher than that of SLS, while SLS showed a higher glycerol fermentation rate than that of GS. The different rates in GS and SLS were attributed to the higher relative abundances of fermentative microorganisms found in SLS and higher rela- tive abundances of sulfate reducing bacteria (SRB) found in GS. © 2023 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) ∗ Corresponding author. E-mail: david.gabriel@uab.cat (D. Gabriel). https://doi.org/10.1016/j.jes.2022.11.011 1001-0742/© 2023 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) 354 journal of environmental sciences 135 (2024) 353–366 addition, an optimum COD/S-SO4 2− ratio may also accelerate Introduction the adaptation period. Fernández-Palacios et al. (2019) inves- tigated different sulfate and organic loading rates in a sulfi- Despite anaerobic digestion is a well-known process in dogenic UASB reactor. It took 30 days after the inoculation of wastewater treatment to remove different types of organic the reactor to reach over 80% of sulfate RE at COD/S-SO4 2- ra- pollutants, anaerobic biotechnology is also an alternative for tio of 3.8 g O2 /g S-SO4 2− , while only 16 days after the inocula- sulfate-rich wastewater treatment. The success of the sul- tion, sulfate RE reached an 80% at COD/S-SO4 2- ratio of 5.4 g fate reduction process has been proven using a variety of car- O2 /g S-SO4 2- and an organic loading rate of 15.8 kg O2 /m3 /day bon sources, such as methanol (Vallero et al., 2003), ethanol (Fernández-Palacios et al., 2021). Temperature shocks could (Wu et al., 2018), butanol (Sarti and Zaiat, 2011), or crude glyc- also be a strategy to speed up the process of sulfidogenesis erol (Fernández-Palacios et al., 2019). Following the sulfate outcompetition over methanogenesis (Jung et al., 2019). reduction to sulfide, recovering valuable metals from metal- In addition, sludge granulation is an indispensable and sulfides precipitation has been applied in full-scale installa- critical factor for the robust treatment of wastewater. Gran- tions by Paques company (Huisman et al., 2006). Further par- ular biomass makes a significant contribution in maintain- tial oxidation of sulfide to elemental sulfur as a value-added ing the dynamic balance and the stability of reactors oper- product to valorize S-rich effluents has also been reported re- ation (Lu et al., 2015a). However, some studies found that cently (Mora et al., 2020a). non-granular particles were formed during sulfate reduction In order to further extend the application of valorizing processes. For example, Vallero et al. (2003) reported the for- sulfate-rich effluents, it is necessary to improve the robust- mation of thin fluffy flocculent particles in the treatment ness of biological processes by stabilizing the bioreactor, op- of synthetic wastewater containing methanol and sulfate in timizing operating conditions and avoiding inhibitory com- an UASB reactor inoculated with GS. Similarly, granules were pounds accumulation. Management strategies that are insuf- disintegrated and filamentous and fluffy particles aggregated ficiently evaluated may exacerbate risks, which further re- in an expanded GS bed reactor for sulfate reduction using sults in unexpected process failure and economic losses from methanol (Weijma et al., 2000). Weijma et al. (2000) reported their commercial application (Westerholm et al., 2018). Sev- negative effects of sulfate reduction on granulation. How- eral problems have been identified in the long-term opera- ever, there is a lack of previous studies in the characteriza- tion of UASB reactors. For example, fat and oil attached onto tion of fluffy flocculent particles, their formation and poten- the biomass or the high up-flow velocity has been reported tial effects on bioreactor performance as often they are not to lead to sludge flotation and the washout of biomass, thus positive. decreasing the COD removal efficiency (RE) and leading to When acetate, cheese whey, pig slurry, crude glycerol and process failure (Jeganathan et al., 2006; Rizvi et al., 2015). vinasses were selected as carbon sources for sulfate reduc- Lu et al. (2015b) also described that the lack of consump- tion process, crude glycerol showed better results and it was tion of excess extracellular polymeric substances produced the most promising carbon source to reduce sulfate compared and the biogas generated in an UASB reactor treating starch- with others tested carbon sources (Mora et al., 2018). Crude rich wastewater resulted in the flotation of sludge that was glycerol is an inexpensive by-product mainly produced in the not able to settle, which eventually led to sludge washout biodiesel production industry. Approximately, 0.1 kg of crude and the clogging of the outlet of the reactor. The required glycerol is produced per kg of biodiesel (Kumar et al., 2019). As cleaning procedures due to clogged outlets or the reinocula- a way to valorize this waste product from the biodiesel indus- tion of biomass due to washout increase operating costs. Fur- try, crude glycerol has been applied in many processes such as thermore, when treating some specific wastewater (such as the bioconversion of glycerol to biodiesel (Chen et al., 2018), methanol-containing wastewater) in UASB reactors, the dis- methane production and hydrogen production in anaerobic integration of GS increased the washout of sludge, which not digestion (Baba et al., 2013), composting (Fehmberger et al., only reduced the quality of the outlet and decreased the COD 2020) and sulfate reduction (Mora et al., 2020a, 2020b). How- RE, but also increased the risk of failure (Lu et al., 2015a). ever, in a previous study (Fernández-Palacios et al., 2021), Therefore, long-term evaluation of the stability performance the long-term performance of an UASB to treat sulfate-rich of reactors for sulfate-rich effluents treatment is needed. wastewater using crude glycerol as a carbon source lead to the Many studies have already reported several factors affect- decrease of sulfate and organic compounds removal efficien- ing the anaerobic treatment of sulfate-rich wastewaters dur- cies, accompanied by the accumulation of a sticky flocculent ing the long-term operation, including the carbon source type, SLS that surrounded the granular biomass. The present work temperature, pH, sulfate loading rate and carbon to sulfur aims at characterizing such SLS that accumulated in the UASB (C/S) ratio (Lopes et al., 2007; Mora et al., 2020b; Shin et al., reactor, exploring the causes of its formation and the effect 1996; Vallero et al., 2004). Anaerobic methanogenic sludge that this substance can have on the stability of the UASB per- can be adapted to sulfidogenic conditions in order to achieve formance over long-term periods. Batch activity experiments the treatment of sulfate-rich wastewaters (Lens et al., 2002). were set up to determine the difference between GS and SLS However, it can take a long time for SRB to outcompete in the rates and the mechanism of sulfate reduction and or- methanogens. In order to adapt to the sulfate reduction condi- ganic compounds fermentation. SLS was characterized from a tions, previous studies showed some alternatives to speed up physical-chemical point of view including the analysis of mi- the start-up of the process such as adding a pure culture of SRB crobial diversity, scanning electron microscopy (SEM), fourier (Omil et al., 1997b) or using SRB enriched-biomass to target transform infrared spectroscopy (FTIR) and fatty acid methyl specific organic compounds (Kaksonen and Puhakka, 2007). In esters (FAMEs). journal of environmental sciences 135 (2024) 353–366 355 (when SLS covered GS) and on day 431 (when a large amount 1. Materials and methods of SLS had already accumulated in the reactor), as shown in Appendix A Fig. S2. Table 1 summarizes the operating condi- 1.1. Experimental setup of the sulfidogenic UASB reactor tions of activity tests performed with different carbon sources. The sludge collected from UASB1 on day 315 was separated A 2.5 L UASB reactor was used for the treatment of sulfate-rich into GS and slime-covered granular sludge (SCGS) (Appendix A wastewater using crude glycerol as carbon source to produce Fig. S3). The SCGS was the sludge taken directly from the reac- sulfide. Six sampling ports located along the UASB reactor (at tor without any treatment, which contained GS covered with 7, 20, 30, 45, 54 and 72 cm from the bottom to the top of the SLS, whereas GS was granular sludge that had been cleaned to UASB), named UASB1 to UASB6, respectively, were used to col- remove SLS. The cleaning process is presented in Appendix A lect samples along the height of the reactor during its long- Supplementary data. 150 mL of mineral medium were fed to term operation. A schematic diagram of the process is pre- both GS and SCGS, and acetate and propionate were added as sented in Appendix A Fig. S1. The UASB reactor was operated carbon sources. The initial concentrations were set to main- under constant conditions for 639 days, and the sulfate inlet tain the same conditions as the TOC/S ratio used along the concentration was set to 250 mg S-SO4 2− /L, while the crude long-term UASB operation. glycerol to sulfate ratio (TOC/sulfate) was steadily maintained Due to the low concentration of GS at UASB1, where almost at 1.5 ± 0.3 g C/g S. Detailed operating conditions of the UASB all the biomass was completely surrounded by SLS (Appendix reactor are presented in Appendix A Table S1. GS to inoculate A Fig. S2C) on day 431, the sludge collected by that time was the reactor was obtained from a full-scale anaerobic UASB di- from UASB6. The sludge collected from UASB6 on day 431 was gester from a pulp and paper recycling industry (UIPSA, Spain). divided into GS and SLS. The GS and SLS were fed with 150 mL The initial volatile suspended solids (VSS) concentration of of mineral medium and pure glycerol (99%, Panreac, Spain) as GS was 27.1 g VSS/L. However, the UASB reactor accumulated the carbon source. viscous, flocculent SLS along the operation, which are shown Once the sludge, mineral medium and carbon sources were in Appendix A Fig. S2. UASB dimensions, equipment, mineral added to the serum bottles, the gas phase of the bottles was medium composition and crude glycerol characteristics are refilled with N2 . Then bottles were immediately covered with further detailed in Fernández-Palacios et al. (2021). rubber stoppers and aluminum caps and incubated at 35 ± 1°C and 150 r/min in a constant temperature shaker (NB-T205, N- 1.2. Assessment of stratification in the UASB reactor Biotek). All batch tests were carried out in duplicate. The stratification of the UASB reactor was assessed by col- lecting samples from six sampling ports of the UASB reactor 1.4. Analytical methods under two different situations: on day 198, when the reactor was properly operating under sulfidogenic conditions, and on Sulfur compounds analyzed included sulfate, thiosulfate and day 575, when a large amount of SLS had already accumu- TDS. TDS was analyzed off-line by a sulfide selective elec- lated in the reactor and sulfate RE were dramatically reduced. trode (9616BNWP, Thermo Scientific, USA) connected to a The analysis of the inlet, outlet and the sampling ports of the benchtop meter (Symphony, VWR, USA). Before TDS measure- reactor included monitoring of sulfate, thiosulfate, total dis- ments, samples were diluted one-to-two for UASB samples solved sulfide (TDS), volatile fatty acids (VFAs), glycerol, total and one-to-twenty for activity tests samples with a sulfide organic carbon (TOC), total inorganic carbon (TIC) and total antioxidant buffer (SAOB), which composition is described by carbon (TC). The calculating process of volumetric rates and Mora et al. (2020a). Sulfate and thiosulfate were analyzed by mean cell residence time (MCRT) are presented in Appendix ion chromatography (ICS-2000 system, Dionex, USA) with a A supplementary methods. The sludge particle size was as- suppressed conductivity detector using an IonPac AS18-HC sessed at different bed heights of the UASB reactor (UASB1, column (4 × 250 mm, Dionex, USA). Prior to the analysis of UASB2 and UASB3) on days 50, 149 and 230, where the parti- sulfate and thiosulfate, samples were bubbled with nitrogen cle size distribution (PSD) and the median diameter of sludge to avoid chemical oxidation of sulfide and diluted to one fifth particles (D(0.5)) were detected. D(0.5) was chosen, because the with ultrapure water. median is relatively unaffected by skewed distributions or ex- Prior to the analysis of carbon compounds, samples were treme scores at end of distribution, compared to the mean also bubbled with nitrogen. Volatile fatty acids (VFAs) and (Field, 2013). alcohols (ethanol, 1,3-propanediol, n-butanol, 2,3-butanediol) were measured by high-performance liquid chromatography 1.3. Batch tests (HPLC, Ultimate 3000, Dionex, USA) equipped with an ICSep ICE-CPREGEL 87H3 column (7.8 mm × 150 mm) and a variable Since VFAs (mainly acetate and propionate) are reported as wavelength detector at 210 nm with a 6 mmol/L H2 SO4 mo- the most common intermediate products of glycerol degra- bile phase at a flow rate of 0.5 mL/min. Samples for VFAs and dation (Bertolino et al., 2014; Dinkel et al., 2010; Mora et al., alcohols were not diluted. 2020b; Zhou et al., 2022); acetate, propionate and glycerol were Total organic carbon (TOC), total inorganic carbon (TIC) and used as carbon sources to study the sulfate reduction process total carbon (TC) were determined in a TOC analyzer (multi in batch tests. In order to study whether the decrease of the N/C 2100S, analytikjena, Germany) equipped with a furnace sulfate RE in the UASB reactor was due to the presence of at the catalytic high-temperature of 850°C. Samples for TOC SLS, sludge was collected from the UASB reactor on day 315 analysis were diluted to one third with ultrapure water. 356 journal of environmental sciences 135 (2024) 353–366 Table 1 – Conditions of batch activity tests using glycerol and VFAs as carbon source. Sampling Carbon TOC Sulfate TOC/S Biomass day Source (mg C/L) (mg S/L) (g C/g S) (g VSS/L) 315 Acetate 326 248 1.31 0.28 ± 0.08a 0.48 ± 0.06b 315 Propionate 328 248 1.32 0.35 ± 0.05a 0.86 ± 0.11b 431 Glycerol 324 240 1.35 0.39 ± 0.13a 0.38 ± 0.03c a Granular sludge b Slime-covered granular sludge c Slime-like substance The particle size of sludge was evaluated by a laser diffrac- 7.3 ± 1.6 kg C/m3 /day for 639 days. The gas production rate tion testing instrument (Mastersizer 2000, Malvern Panalyt- in the UASB and the sulfate, TOC and glycerol RE are pro- ical, USA). The sludge samples were taken from different vided in Fig. 1 to illustrate the UASB performance. Further, heights of the UASB reactor and measured in triplicates. more complete details of the experimental performance re- VSS were analyzed according to Standard Methods sults of the UASB reactor can be found elsewhere (Fernández- (APHA, 2012) to calculate UASB biomass concentration and Palacios et al., 2021). In brief, the operation was divided into the specific rates obtained during the batch tests. four periods: stage I, from day 0 to day 16, corresponding to the start-up and GS adaptation period; stage II, from day 16 to day 1.5. 16S rRNA gene amplification for GS and SLS 100, corresponding to a period with a stable sulfate reduction with methane production; stage III, from day 100 to day 280, Identification of microbial populations was performed us- under stable sulfate reduction without methane production; ing Illumina platform on different samples. Sludge was col- and stage IV, from day 280 to day 639, when a decline in the lected from UASB1 and UASB6 on day 538 of the opera- sulfate RE and glycerol RE occurred. After the adaptation pe- tion. SCGS samples were firstly centrifuged at 4000 r/min riod, stable sulfate RE higher than 83% were achieved on stage for 10 min and, the supernatant was discarded. GS and SLS II and stage III and it decreased to 38.8% on stage IV. In terms were separated and manually transferred to a new falcon of carbon, over 97% of glycerol RE was observed on stage I, II tube where distilled water was added. The centrifugation and III, but it decreased to 64% on stage IV. It can be observed process was repeated 2 to 4 times until GS and SLS were that the TOC RE was positively correlated with the gas produc- separated in the falcon tubes. Then, GS and SLS were sep- tion. When the gas production rate was higher than 60 mL/hr, arated by plastic pipettes. After separation, samples were the TOC RE was higher than 86% on stage I and II. A progres- cleaned with 1XPBS (7.2 mmol/L Na2 HPO4 ·12H2 O, 2.8 mmol/L sive decrease in the gas production was accompanied by the NaH2 PO4 ·2H2 O and 0.13 mmol/L NaCl), and centrifuged at decrease of TOC RE on stage II and III. The pH in the outlet of 14000 r/min during 5 min (3 times). PowerSoilTM DNA iso- the UASB reactor was maintained at 7.0-7.3 (Appendix A Fig. lation kit (MoBio Laboratories, USA) was used to extract the S4). genomic DNA from both samples. The quantity and quality During stages II and III, a progressive degranulation of the of the extracted DNA were assessed by using a NanoDrop sludge was observed when methanogenic activity stopped. 1000 Spectrophotometer (Thermo Fisher Scientific, USA) and Sludge particle size distribution over the operation period is finally, DNA samples were preserved at -20°C for further analy- shown in Appendix A Fig. S5. The particle size of the inocu- sis. Illumina MiSeq platform was used to performed amplicon lated sludge below 200 μm accounted for 74% of the inoculated sequencing 16S rRNA genes of both samples by the “Genomic sludge. Compared with inoculated sludge, the proportion of 0- and Bioinformatics service” of Universitat Autònoma de 200 μm sludge in UASB1, UASB2 and UASB3 decreased on day Barcelona amplifying the V3-V4 hyper variable region with the 50 and day 149, accounting for 49.6% ± 4.5% in average. How- universal primers by Illumina (5’-CCTACGGGNGGCWGCAG- ever, the proportion of particles smaller than 200 μm in UASB1, 3’) and (5’-GACTACHVGGGTATCTAATCC-3’) selected from UASB2 and UASB3 increased to 70.6% ± 3.2% on day 230. Also, Klindworth et al. (2013). the proportion of sludge greater than 1000 μm increased from The analytical methods of SEM, FT-IR, and FAMEs are 2.1% in the inoculum sludge to over 7.3% in UASB1, UASB2 shown in Appendix A Supplementary data. and UASB3 on days 50 and 149. However, on day 230, the pro- portion of particles larger than 1000 μm were reduced to 1.6% 2. Results (in UASB2) and 1.7% (in UASB3), respectively. Results of D(0.5) revealed that granular size increased from day 50 to day 149 2.1. Stratification and long-term performance of the as the inoculum sludge D(0.5) was 105 μm, while the sludge UASB reactor D(0.5) in UASB1, UASB2 and UASB3 were higher than 150 μm on days 50 and 149 (Appendix A Fig. S6). However, the D(0.5) The UASB reactor was operated under a constant sulfate load- of the sludge measured at different heights of the UASB re- ing rate of 5.0 ± 0.6 kg S/m3 /day and an organic loading rate of actor decreased on day 230 of the operation. Results of D(0.5) journal of environmental sciences 135 (2024) 353–366 357 Fig. 1 – Gas production rate (a) and removal efficiency (b) of the sulfidogenic UASB reactor along 639 days. Fig. 2 – Variation of biomass in the UASB reactor (a), biomass loss (b), mean cell residence time (c) and biomass concentration in UASB versus sulfate removal efficiency and sulfate removal capacity (d). and particle size distribution revealed that the granule size of was between 20 and 30 g/L before day 400 and dropped to 4.3 the sludge increased on days 50 and 149 in UASB1, UASB2 and g/L on day 639. The biomass concentration in UASB6 was 0 UASB3, but decreased on day 230 when no biogas was pro- g/L before day 300. Due to flotation, part of the sludge was re- duced. tained at UASB6, where biomass concentration was 21.6 g/L When the reactor was operated under sulfidogenic condi- on day 390 and dropped to 8.7 g/L on day 639. The average tions with low biogas production on stage Ⅲ, flocculent SLS biomass concentration in the UASB reactor gradually dropped gradually surrounded and covered GS (Appendix A Fig. S2b), from 48.5 g/L to 14.9 g/L during 639 days operation. The TSS later forming aggregates and attaching to the reactor wall (Ap- concentration in the UASB reactor showed the same trend as pendix A Fig. S2c, S2d). Compared with GS, SLS was viscous VSS (data not shown). The VSS/TSS ratio ranged from 74% to and fluffy, which caused severe sludge flotation and biomass 87% during the whole operation, which shows that there was washout. As can be observed in Appendix A Fig. S2b, part of the almost no accumulation of non-volatile suspended solids in sludge floated to the gas-liquid-solid separator and remained the UASB reactor indicating that the main component of SLS suspended on the top of the reactor after 316 days of operation accumulated in the reactor were volatile solids. and later on (Appendix A Fig. S2c, S2d). Fig. 2b shows the washout of biomass from the reactor. Biomass concentration in the UASB reactor and washout of The biomass loss fluctuated in the range of 0.04 to 2.4 g/L be- biomass are shown in Fig. 2. As shown in Fig. 2b, the biomass fore day 200. High sludge concentration losses were caused concentration in UASB1 showed a decline from 58.6 g/L on day by an excess of biomass provided to the reactor in the inocu- 78 to 22.1 g/L on day 639. The biomass concentration in UASB3 lation stage coupled to the movement and raising of granules 358 journal of environmental sciences 135 (2024) 353–366 Fig. 3 – UASB sulfur and carbon concentration profile on day 198 (a, c) and on day 575 (b, d) of the long-term operation using crude glycerol as carbon source. a and b show sulfur species. c and d show carbon species. TDS represents total dissolved sulfide. caused by methane bubbles produced, thus resulting in sludge ties of SLS that accumulated in the reactor, GS and SCGS col- washout. When there was no methane production in the re- lected from UASB1 on day 315 were investigated by batch ac- actor, the bed become a static bed, leading to a reduced sludge tivity tests using acetate as the carbon source to assess the in- loss that remained stable between 0.1 to 0.6 g/L after day 260. fluence of SLS on the mechanism of sulfate reduction (Fig. 4). The MCRT was below 65 day during the period of methane pro- Fig. 4 shows that there were no changes in the acetate and duction while the sludge remained longer in the reactor dur- sulfate concentrations in batch tests performed with GS and ing the period of non-methane production (days 200 to 300), SCGS, which further confirmed that after 315 days of opera- as shown in Fig. 2c. The MCRT was maintained at 50 day af- tion, acetoclastic methanogens and acetotrophic SRB were not ter 400 days of operation. Fig. 2d shows that the biomass in present in the UASB reactor. the reactor correlated with sulfate RE (%) and sulfate removal Fig. 5 shows the time course of sulfate reduction with GS capacity (kg S/m3 /day). and SCGS using propionate as carbon source. GS and SCGS Stratification of the UASB reactor was investigated under both reduced sulfate to sulfide, while acetate and inorganic sulfidogenic conditions. Fig. 3 shows the profiles of sulfur and carbon were produced. The specific rates of propionate con- carbon species on days 198, when the reactor was properly op- sumption and sulfate reduction of GS and SCGS are shown in erating under sulfidogenic conditions, and on day 575, when Table 2. The propionate consumption rate and sulfate reduc- UASB performance was deficient. The sulfate RE on day 198 tion rate of GS were 68% and 66% higher than those of SCGS, was 96.2% (Fig. 3a) and decreased to 20.2% on day 575 (Fig. 3b), respectively. while the glycerol RE was 100% on day 198 (Fig. 3c) and de- creased as well to 60% on day 575 (Fig. 3d). Fig. 3 also shows 2.2.2. GS and SLS tests feeding glycerol the rates calculated with the data obtained in the UASB strat- In order to analyze the difference between GS and SLS and to ification sampling events. The glycerol consumption rate and assess the influence of SLS on glycerol fermentation, sludge sulfate reduction rate from UASB inlet to UASB1 were the high- withdrawn from UASB6 on day 431 was handled. Fig. 6 shows est, compared with the rest of the sampling points of the UASB the activity test using glycerol as the carbon source. GS re- reactor. On day 198, sulfate reduction and glycerol degrada- duced sulfate throughout the test (Fig. 6a). Instead, SLS re- tion were mainly completed at the bottom part of the UASB quired an adaptation period as it started to reduce sulfate only reactor (UASB inlet to UASB2). However, sulfate reduction and after 24 hr (Fig. 6b). Glycerol was fermented both by GS and glycerol degradation occurred along the whole UASB reactor SLS leading to ethanol, 1,3-propanediol, formate, acetate and on day 575 as the local activity of biomass was reduced with propionate production in both cases followed by ethanol, 1,3- respect to the period of proper performance. propanediol, formate and propionate consumption accompa- nied by sulfate reduction and coupled to acetate and inor- 2.2. Anaerobic activity tests of GS, SCGS, and SLS ganic carbon accumulation throughout the test. However, the rates of accumulation and depletion of each compound in the 2.2.1. GS and SCGS tests feeding acetate and propionate tests with GS and SLS were different. Essentially, a much faster Acetate and propionate, often reported as main products of fermentation rate was observed for SLS with respect to GS, glycerol degradation, were selected as electron donors for pre- while a faster use of electron donors was observed for GS than liminary sulfate reduction tests. In order to study the proper- for SLS. Table 3 shows the specific rates calculated for glyc- journal of environmental sciences 135 (2024) 353–366 359 Fig. 4 – Activity tests of sludge obtained from UASB1 on day 315 feeding acetate and sulfate using GS (a, c) and SCGS (b, d). (a) and (b) show sulfur species, while (c) and (d) show carbon species where TDS and TIC correspond to total dissolved sulphide and total inorganic carbon, respectively. Fig. 5 – Activity tests of sludge obtained from UASB1 on day 315 feeding propionate and sulfate using GS (a, c) and SCGS (b, d). (a) and (b) show sulfur species, (c) and (d) show carbon species. TDS represents total dissolved sulfide. TIC represents total inorganic carbon. 360 journal of environmental sciences 135 (2024) 353–366 Table 2 – Specific rates calculated for the sludge collected from UASB1 on day 315 feeding propionate and sulfate. Sample Time Propionate consumption rate Sulfate reduction rate (hr) (mg C/hr/g VSS) (mg S/hr/g VSS) GS 6-120 6.9 ± 1.3 4.8 ± 0.6 SCGS 6-97 4.1 ± 0.8 2.9 ± 0.7 Fig. 6 – Activity tests of sludge obtained from UASB6 on day 431 feeding glycerol and sulfate using GS (a, c) and SLS (b, d). (a) and (b) show sulfur species, (c) and (d) show carbon species. TDS represents total dissolved sulfide. TIC represents total inorganic carbon. Table 3 – Specific rates calculated for the sludge collected from UASB6 on day 431 feeding glycerol and sulfate. Sample Time Glycerol consumption rate Sulfate reduction rate (hr) (mg C/hr/g VSS) (mg S/hr/g VSS) GS 0-23 33.1 ± 12.2 6.3 ± 3.4 23-62 0 9.1 ± 1.7 SLS 0-11 70.1 ± 8.0 0.5 ± 0.5 24-73 0 7.3 ± 2.7 erol consumption and sulfate reduction using GS and SLS in the most abundant genus detected in the samples of GS and the tests. During the degradation of glycerol, the glycerol con- SLS on day 538 of operation using Illumina 16S rRNA sequenc- sumption rate of SLS was 2.1 times that of GS, and the sul- ing. Propionispora and Dysgonomonas were the most abundant fate reduction rate of GS was 1.2 times higher than that of genus detected on day 358 in both samples. The relative abun- SLS when the carbon source required for sulfate reduction was dance of genus Propionispora increased from 15.2% in GS to sufficient. 21.5% in SLS, respectively. On the other hand, Dysgonomonas was most abundant in GS with a relative abundance of 13.2%, whereas the relative abundance of this genus was 9.9% in SLS. 2.3. Biological and physical-chemical characterization of Enterobacter, Klebsiella, and Helicobacter genera raised their rel- GS and SLS ative abundances in SLS if compared to GS, from 2.2% to 8.3%; from 2.1% to 8.5% and lastly from 0.3% to 6.4% respectively. The differences in microbial communities of GS and SLS were Desulfobulbus decreased its relative abundance from 11.6% in also studied. Fig. 7 compares the relative abundances (%) of journal of environmental sciences 135 (2024) 353–366 361 Fig. 7 – Relative abundance of genus detected in granular sludge (GS) and slime-like substance (SLS) on day 538. GS to 2.5% in SLS, while Desulfovibrio decreased from 10.8% in pKa of acetic and propionic acids, 4.75 and 4.87, respec- GS to 7.5% in SLS. tively. The microbial diversity dynamics during the long-term Samples of SLS were collected for SEM analysis (Appendix operation of the UASB reactor was studied by Fernández- A Fig. S7). Biofilm growth and a wide variety of microorgan- Palacios et al. (2021). The relative abundance of methanogens, isms were observed in these samples. In some images, many including Methanosaeta, Methanobacteria, Methanomicrobiales, fiber-like strings that are much larger than microbes can be showed a downward trend. Methanogens were hardly de- also seen around the microbes. tected after 230 days operation, which confirmed the wash- The FTIR spectra obtained from the lyophilized SLS and GS out of methanogens. In contrast, a remarkable increase in the is presented in Appendix A Figs. S8 and S9. Appendix A Fig. S8a relative abundance of SRB was observed. Illumina sequencing shows the region from 4000 to 3000 cm−1 , where O-H stretch- results were consistent with the results of methane produc- ing can be seen. Absorbance at wavenumbers below 3000 cm−1 tion and VFAs accumulation, proving that methanogenic GS shows C-H groups that are sp3 hybridized, such as saturated progressively switched to sulfidogenic sludge performing high fats or CH3 groups (Appendix A Fig. S8b). Different bands can sulfate RE. be seen between 1700-1600 cm−1 (Appendix A Fig. S8c) that in- The pH and sulfide concentration play a major role in the dicate different secondary protein structures (α-helix, β-sheet, competition between SRB and methanogens (O’Flaherty et al., β-turn and random coil), which named the amide I bands. 1998). The growth rate of methanogens was higher than that Amide II bands are shown in Appendix A Fig. S8d, which is in- of SRB at pH < 7.0, and opposite results were found at pH > dicative of proteins. The region from 1200 to 1100 cm−1 shows 7.5. The pH in the inlet of the UASB reactor was over 7.5 dur- the indicative of C-O-C binding, typically as sugars bonds (Ap- ing this study (Appendix A Fig. S4). The toxicity of sulfide is pendix A Fig. S9). The absorbance at 1150 cm−1 was 0.16 and caused by undissociated sulfide molecules that permeate the 0.07 for SLS and GS, respectively. cell membrane (Kaksonen and Puhakka, 2007). The inhibitory The same UASB reactor was operated under different sul- effect of H2 S on methanogens was greater than that on SRB fate and organic loading rates in previous works (Fernández- (Sarti and Zaiat, 2011). Methane production by methanogenic Palacios et al., 2019; Fernández-Palacios, 2020). After the long- archaea can be limited also by LCFAs, specifically palmitic acid term operation varying COD/S-SO4 2− ratios, SLS was also ob- (Deaver et al., 2020; Silva et al., 2016). Palmitic acid was iden- served. Results of FAMEs analysis of SLS samples extracted tified as the main component among the LCFAs in the UASB from that operation after 490 days are presented in Appendix reactor (Appendix A Table S2). Palmitic acid has a toxic effect A Table S2. Palmitic acid was the main LCFAs identified and on methanogenic archaea (Silva et al., 2016), and the adsorp- quantified compound in SLS from samples collected at differ- tion of LCFAs onto microbial biomass can lead to mass transfer ent heights of the UASB reactor. Other interesting compounds limitation of substrates (Pereira et al., 2005), which might be a that were detected in the samples but were not quantified potential factor for the inhibition of methanogenic activity. were methyl esters and wax esters. The overall glycerol RE of the UASB reactor was over 90% with high sulfate RE (over 83%) before day 280. However, both the overall glycerol RE and sulfate RE started to de- 3. Discussion cline after 280 days of operation. The decline of granules size was observed after the cease of methanogenic activ- A progressive decrease in methane production was accom- ity. Wu et al. (2016) found that granule size positively corre- panied by the decrease of TOC RE at a TOC/S-SO4 2− ra- lated with biogas production rate. Acetotrophic methanogens tio of 1.5 ± 0.3 g C/g S when crude glycerol was fed in played a key role in granulation (Hulshoff Pol et al., 2004), the UASB reactor. At the same time, VFAs (mainly acetate, and the lack of methane production resulted in poor sludge propionate) progressively accumulated on stage III and IV granulation (Mora et al., 2020b). Previous studies found that (Appendix A Fig. S4). However, the concentration of the granules strength was reduced upon sulfidogenic operation non-ionic forms of VFAs (acetic acid and propionic mainly) of anaerobic reactors and the diameter of GS decreased were residual under the pH of operation considering the (Kobayashi et al., 2015; Omil et al., 1997a), which might ex- 362 journal of environmental sciences 135 (2024) 353–366 plain the observation of degranulation when methanogenic GS, SCGS and SLS were also investigated by batch activ- activity ceased. Weijma et al. (2000) found the disintegration ity tests to identify the influence of SLS on the mechanism of sludge in an expanded GS bed reactor treating methanol of sulfate reduction and glycerol degradation. When propi- during the period of low biogas production, and reported that onate was used as carbon source to reduce sulfate, SRB con- sludge particles were gradually covered by fluffy cotton-like verted propionate to acetate and inorganic carbon. The mech- material. Similarly, in the present study, when no biogas was anism of sulfate reduction with propionate observed herein produced in the UASB reactor, decrease in granuleś size was is consistent with previous studies (Liamleam and Annach- also observed followed by SLS accumulation. hatre, 2007; Muyzer and Stams, 2008). Since the same prod- As it can be seen in SEM images, the SLS presented a ucts were observed in the activity test performed with GS and white cotton-like aspect. The formation of SLS may be at- SCGS using propionate to reduce sulfate as shown in Fig. 5, tributed to the accumulation of intermediate metabolites in it can be concluded that the SLS presented the same mecha- extracellular polymeric substances, such as fats, oils, greases, nism of sulfate reduction. However, the specific sulfate reduc- or polysaccharides (Jeganathan et al., 2006; Lu et al., 2015b). tion rate was higher in the case of GS than in that of SCGS, The formation of SLS may also be due to the continuous ac- indicating that SLS may affect the mass transfer of the sub- cumulation of other organic carbon substances in crude glyc- strates, resulting in a decrease of the specific sulfate reduction erol that were not completely degraded by GS, thus encir- rate. cling the GS. It has been previously reported that crude glyc- In the activity tests performed with GS and SLS with glyc- erol contains FAMEs, free fatty acids, glycerides and LCFAs erol as the carbon source to reduce sulfate, both GS and SLS (Hu et al., 2012; Viana et al., 2012). LCFAs can form a layer on fermented glycerol into formate, acetate, propionate, ethanol, the surface of biomass and prevent the substrate from enter- 1,3-propanediol and inorganic carbon. In addition, the inter- ing the biomass, thus leading to limited substrate diffusion mediate products of glycerol were consumed with sulfate re- into the granules (Viana et al., 2012). In addition, LCFAs can duction observed. This indicates that SLS is also capable of fer- attach the biomass and cause its flotation (Viana et al., 2012). menting glycerol and reducing sulfate. When comparing the Hwu et al. (1998) found that treatment of LCFAs-containing specific rates of glycerol fermentation between GS and SLS, wastewater in an UASB lead to methanogenesis inhibition and SLS was more efficient in degrading glycerol than GS. The spe- granular sludge flotation. LCFAs accumulated in the UASB re- cific substrate utilization activity of GS and flocculant sludge actor after long-term operation under sulfidogenic conditions was compared in UASB reactors processing wastewater from in our work. Sludge flotation is a limiting factor for anaero- food industries (Oleszkiewicz and Romanek, 1989). They found bic treatment of wastewater with higher loading rates because that the flocculant sludge formed during the operation of an this phenomenon can lead to a severe biomass washout and UASB reactor had a higher specific activity for the degrada- system performance deterioration (Jeganathan et al., 2006). As tion of complex polymers than the GS in anaerobic processes, can be observed in Fig. 2d, the decrease in biomass concentra- which is similar to the results found herein. tion is accompanied by a decrease in sulfate RE and sulfate Fig. 6a and b show that sulfate reduction was not related removal capacity, which indicated that sludge flotation lead to glycerol fermentation. Sulfate was reduced accompanied to the washout of biomass and the drop of the reactor perfor- by the degradation intermediate products of glycerol degra- mance. This may explain the gradual decline of sulfate RE and dation. This confirmed that the carbon sources used for sul- glycerol RE at stage IV of the long-term UASB operation. fate reduction were formate, ethanol and 1,3-propanediol, The evaluation of UASB reactor stratification can help to in which the mechanism of sulfate reduction using glycerol understand the effect of the sludge at different heights on sul- as the carbon source is consistent with our previous work fate reduction and degradation of organic compounds. It is ob- (Zhou et al., 2022). When comparing the specific rates of sul- served that sulfate reduction and glycerol degradation were fate reduction between GS and SLS, SLS was found to be less mainly completed at the bottom of the UASB reactor, which efficient in reducing sulfate than GS. In addition, as can be was due to the high biomass concentration at this height of seen in Fig. 6d, even if there was sufficient carbon source (for- the UASB reactor (UASB inlet to UASB2). The biomass at the mate, ethanol and 1,3-propanediol) to reduce sulfate, SLS still bottom of the sludge bed (UASB inlet to UASB2) accounted needed time to adapt and perform the sulfate reduction pro- for over 70% of the overall VSS of the reactor before 300 days cess, which may be caused by mass transfer limitation of sul- of UASB operation. The biomass from UASB inlet to UASB2 fate. Compared with GS in the UASB reactor, GS covered by also accounted for 57% and 78% on day 390 and day 639, re- SLS may cause sulfate to be discharged from the UASB reac- spectively. Results are consistent with previous studies, which tor before entering the cell due to mass transfer limitation, found that the higher methanogenic activity was observed at which may lead to a decrease in the sulfate reduction effi- the bottom of the sludge bed in an UASB-anaerobic membrane ciency. Therefore, a significant reason for the reduced sulfate bioreactor treating municipal wastewater because the con- RE in the UASB reactor may also be that the SLS limited the centration of VSS increased along with the decreasing height mass transfer of sulfate, in addition to biomass washout from of the sludge bed (Mahmoud et al., 2004; Ozgun et al., 2019). the UASB reactor. Similarly to what was observed by Ozgun et al. (2019), the high- The difference of specific rates between GS and SLS were est specific methanogenic activity was observed at the bottom also caused by the bacteria community differences. A wide part of the sludge bed, as the more active biomass remained range of microorganisms have been observed to ferment glyc- near the inlet of the reactor, while the inactive biomass moved erol, including Dysgonomonas (Moscoviz et al., 2018), Klebsiella towards the top of the sludge bed or suspended on the top of (Cheng et al., 2007), Propionibacterium (Himmi et al., 2000) and the reactor. Propionispora (Abou-Zeid et al., 2004). Obligate anaerobic Dys- journal of environmental sciences 135 (2024) 353–366 363 gonomonas genus is able to convert glycerol to 1,3-propanediol. covered GS, SLS first contacted LCFAs and sulfide during glyc- Propionibacterium and Propionispora were reported to convert erol fermentation and sulfate reduction. Thus, compared to crude glycerol to propionate and acetate (Abou-Zeid et al., GS, SLS had higher exposure to sulfide and LCFAs, which may 2004; Biebl et al., 2000; Himmi et al., 2000). Anaerobic Propionis- explain higher protein contents in SLS. pora genus typically degrades organic carbon compounds in Overall, different methods (SEM, FAMEs, Illumina sequenc- acidogenesis rather than acetogenesis. Enterobacter and Kleb- ing and FT-IR) used for SLS analysis showed that SLS was the siella, facultative anaerobes, are able to produce hydrogen mixture of biomass and organic/inorganic compounds com- and acetate through the fermentation of organic substrates ing from the impurities contained in crude glycerol and the (Hung et al., 2011; Joubert and Britz, 1987). Helicobacter is con- formation of other intermediate products. With all the in- sidered to be a microaerophile capable of oxidizing a variety of formation acquired, the formation of SLS was attributed to organic acids, such as formate, lactate, succinate and pyruvate a combination of factors along the long-term operation, in- (Joubert and Britz, 1987). In this work, the relative abundances cluding sulfide production, the cease of methanogenic activity, of fermentative microorganisms in GS (33.0%) were lower than washout of methanogens, static sludge bed, impurities com- that in SLS (54.6%), which agrees with the lower glycerol fer- pounds contained in crude glycerol and the formation of other mentation rate in GS and higher fermentation rate in SLS. intermediates. Desulfovibrio is a genus of SRB reported to oxidize organic As a by-product of the biodiesel industry, there are clear ad- carbon compounds (including lactate, ethanol, malate, pyru- vantages in the use of crude glycerol as electron donor such as vate, succinate) to acetate for sulfate reduction (Odom and its large COD content, its availability and low cost among oth- Peck, 1981; Qatibi et al., 1991; Wu et al., 2018). Desulfobulbus ers. However, the presence of impurities may hinder its appli- genus can oxidize pyruvate, propionate and ethanol to ac- cation unless alternatives to face long-term operation issues etate (Bak and Pfennig, 1991; Bertolino et al., 2012; Muyzer and are found. Oleszkiewicz and Romanek (1989) reported that the Stams, 2008; Zeng et al., 2019). The main sulfate-reducer gen- addition of calcium and phosphate promoted sticky, floccu- era found herein were Desulfobulbus and Desulfovibrio, both re- lant sludge formation, while granular biomass was formed in ported as incomplete oxidizing SRB, which explains the ac- the treatment of wastewater supplemented with ferric ions cumulation of acetate in the batch activity tests. Compared and traces of nickel and cobalt. The use of traces of metallic to SLS, higher relative abundances of Desulfobulbus and Desul- elements can promote granulation and avoid sludge disinte- fovibrio in GS agree with the higher sulfate reduction rate gration and washout of biomass. Increasing the shear force in found in GS, meaning that the populations of SRB were prob- the reactor could be another strategy for preventing granules ably mainly growing in the core of the granules, what can also flotation (Chen et al., 2014) and crude glycerol by-products ac- explain the decrease of sulfate RE when granules turned into cumulation. The increase of shearing forces can be achieved slime-covered granules. by adding an internal recirculation pump to increase the up- Batch activity tests and 16s rRNA gene analysis of the mi- flow velocity as well as the use of intermittent pulses of nitro- crobial communities revealed that GS played a more impor- gen from the bottom of the reactor to also avoid bed packing. tant role in sulfate reduction than SLS since GS contained However, further research is needed in order to assess the vi- more sulfate reducers (mainly Desulfobulbus and Desulfovibrio). ability of such alternatives. Despite the fact that the perfor- In terms of fermentation, glycerol fermentation was not af- mance deterioration was mainly attributed to the washout fected by SLS. Conversely, the fermentation rate of glycerol of biomass due to SLS accumulation and consequent sludge in SLS was higher than that of GS. The analysis of microbial flotation, still other factors not considered in the present work diversity revealed that high relative abundances of fermen- such as the change of the metabolic functions of the micro- tative microorganisms was identified in SLS. This shows that bial cultures linked to the SLS accumulation should be inves- the reason for the decrease of the glycerol RE in UASB reactor tigated. was not related to the degradation mechanism of glycerol by the SLS, but because the SLS triggered the washout of biomass from the UASB reactor. FTIR spectra showed no significant differences between GS 4. Conclusions and SLS samples. The biggest difference of peaks between GS and SLS was around at 1635 and 1030 cm−1 . The ratio The lack of biogas production in a sulfate-reducing UASB lead between the absorbance at 1635 cm−1 over the absorbance to a reduction in the particle size of granular biomass and to at 1030 cm−1 shows the relatively quantity of protein. The the accumulation of a SLS due to the presence of organic com- ratio was 1.1 for SLS and 0.8 for GS, which indicates that pounds and other impurities present in crude glycerol. SLS ac- SLS contained relatively more proteins than GS. Proteins are cumulation also led to sludge flotation, which resulted in an fundamental components of biomolecules. The soluble pro- increased washout of sludge. Subsequently, the sulfate RE and tein content generally increases after exposure to pollutants. glycerol RE in the reactor decreased, and the system perfor- In order to reduce toxic effects, microbes can be induced mance deteriorated. Sulfate removal capacity and glycerol re- to synthesize defensive proteins under the stress of haz- moval capacity at different heights of the UASB reactor corre- ardous substances (Imlay, 2008; Sabatini et al., 2009). Specif- lated well with the sludge concentration. In the sulfate reduc- ically, Li et al. (2022) showed that excessive extracellular poly- tion processes with propionate and glycerol as carbon sources meric substances (EPS) production, especially the protein- in batch tests, results confirmed that SLS did not change the like substances, was an effective strategy for reducing certain mechanism of sulfate reduction and glycerol degradation but nanoparticles toxicity in anaerobic granular sludge. Since SLS decreased the rate of sulfate reduction. 364 journal of environmental sciences 135 (2024) 353–366 Klebsiella pneumoniae. Process Biochem. 42, 740–744. Declaration of Competing Interest doi:10.1016/j.procbio.2007.01.001. Deaver, J.A., Diviesti, K.I., Soni, M.N., Campbell, B.J., Finneran, K.T., The authors declare that they have no known competing fi- Popat, S.C., 2020. Palmitic acid accumulation limits methane nancial interests or personal relationships that could have ap- production in anaerobic co-digestion of fats, oils and grease peared to influence the work reported in this paper. with municipal wastewater sludge. Chem. Eng. J. 396, 125235. doi:10.1016/j.cej.2020.125235. Dinkel, V.G., Frechen, F.B., Dinkel, A.V., Smirnov, Y.Y., Kalyuzhnyi, S.V., 2010. Kinetics of anaerobic biodegradation of Acknowledgments glycerol by sulfate-reducing bacteria. Appl. Biochem. Microbiol. 46, 712–718. doi:10.1134/s0003683810070069. Authors acknowledge the Spanish Government, through the Fehmberger, C., Dos Santos, F.T., Aloisio, C.M., Hermes, E., project RTI2018-099362-B-C21 MINECO/FEDER, EU, for the Zenatti, D.C., Bautitz, I.R., 2020. Effectiveness of incorporation of crude glycerin as a source of labile carbon in the financial support provided to perform this research. The composting of poultry production residues. J. Clean. 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