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Effect of feed pH on reactor performance and production of soluble microbial products (SMPs) in a submerged anaerobic membrane bioreactor
Accepted Manuscript Effect of feed pH on Reactor Performance and Production of Soluble Microbial Products (SMPs) in a Submerged Anaerobic Membrane Bioreactor C. Kunacheva, Y.N.A. Soh, D.C. Stuckey PII: DOI: Reference:
S1385-8947(17)30355-8 http://dx.doi.org/10.1016/j.cej.2017.03.018 CEJ 16614
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
1 February 2017 6 March 2017 7 March 2017
Please cite this article as: C. Kunacheva, Y.N.A. Soh, D.C. Stuckey, Effect of feed pH on Reactor Performance and Production of Soluble Microbial Products (SMPs) in a Submerged Anaerobic Membrane Bioreactor, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.03.018
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Effect of feed pH on Reactor Performance and Production of Soluble Microbial Products (SMPs) in a Submerged Anaerobic Membrane Bioreactor
C. Kunacheva*, Y. N. A. Soh* and D. C. Stuckey**
* Advanced Environmental Biotechnology Centre, Nanyang Environment & Water Research Institute, Nanyang Technological University, 1 Cleantech Loop, CleanTech One, Singapore 637141, Singapore (E-mail: [email protected]; [email protected]) ** Department of Chemical Engineering, Imperial College London, London SW7 2AZ, UK (E-mail: [email protected])
Abstract The production of soluble microbial products (SMPs) and colloids in a submerged anaerobic membrane bioreactor (SAMBR) under different feed pHs (pH 5 and pH 11) was evaluated, and they were found to have a significant effect on SAMBR performance. Fluctuations in pH inside the SAMBR affected cell metabolism and/or enhanced cell lysis in the reactor, but the system recovered within 24 h for the pH 5 shock, and 8 h for the pH 11 shock. Carbohydrates (30k – 200kDa) were found at very high concentrations with the pH 5 shock, while higher concentrations of “protein-like” compounds (1,500kDa – 0.2μm) were found with the pH 11 shock. The pH shocks affected membrane fouling primarily through increased colloids, which formed a charged concentration polarization layer on the membrane surface. Larger colloids (1-5μm) related to “protein-like” compounds
caused more membrane fouling than smaller ones. Alkanes, alkenes, esters, alcohols, phenols, nitrogen-compounds and sulfur compounds were the major groups of compounds identified in the effluent and supernatant samples. The membrane-fouling layer (“dynamic membrane”) is an important factor in the removal of low MW compounds in the SAMBR, and changes in pH inside the SAMBR had a significant effect on effluent quality.
Keywords Anaerobic; biological treatment; colloids; pH; soluble microbial products (SMP)
1. INTRODUCTION Soluble microbial products (SMPs) are defined as the soluble organics created by biological metabolism (catabolism and cell lysis) in biological processes that are not known intermediates such as volatile fatty acids (VFAs), or incoming feed [1]. Their presence affects the performance of most biological treatment systems in terms of chemical oxygen demand (COD) removal, and causes the fouling of membranes. SMP production is complex and its sources are still unclear; it has been noted over the years in both aerobic and anaerobic biological processes that around 2% of the incoming feed COD is present in the effluent as SMPs [2]. However, under transient conditions including nutrient limitations, toxicants, or when the feed flow or composition is changed radically, the effluent SMPs can be as high as 17% of the influent COD [1, 3]. There is a strong need to understand the effect of a variety of factors on SMP production in biological processes in order to improve the design and operation of wastewater treatment plants by reducing SMP production. The effect of the hydraulic retention time (HRT), organic loading rate (OLR), and sludge retention time (SRT) on SMP production has been investigated, and shows that SMP production increases at shorter HRT, longer SRT, and higher feed strength [4-7]. SMP production is also sensitive to
substrate concentration, and Xie et al. found that total SMP production increased by 71% with a threefold increase in feed strength [8]. Under environmental stresses such as low pH, high salinity, low temperature, and nutrient deficiency, SMPs are produced as a response to these stresses. Moreover, a shift in the molecular weight (MW) distribution towards the production of higher MW SMPs was observed in most cases [4, 9-11]. The literature [12, 13] also reported that SMP and VFA production was enhanced under nutrient deficient conditions. Due to these increased VFAs, the percentage of SMPs to effluent COD was reduced to 45% when it was under steady-state; however, the SMP concentration increased to 37% of the incoming feed compared with 3% under steady-state conditions [12]. A similar trend occurred at low pH (<6.5) as a result of no alkalinity in the feed, and the normalized SMP production was statistically higher than during normal operation pH (6.8 – 7.2) in an anaerobic continuous stirred tank reactor (CSTR). An average 34% increase in SMP production was observed, which was possibly the result of enhanced cell lysis, as well as VFA accumulation [5].
Therefore, it is important to evaluate the production and composition of SMPs in a submerged anaerobic membrane bioreactor (SAMBR) in order to understand what these compounds are, and how they are produced; this is fundamental for developing methods to control membrane fouling. A pH shock is one factor that can affect the performance of the reactor when it receives industrial wastewater, and rapid changes in pH may create more SMPs. Currently there has been no study identifying the SMPs produced under pH shocks in a SAMBR. Hence, the objectives of this study were to evaluate the performance of a SAMBR, and to characterise the SMPs produced under different pHs shock in the feed (pH 5, pH 7 and pH 11).
2. MATERIALS AND METHODS 2.1 Reagents and chemicals
Methanol (LC-MS grade) was purchased from Sigma-Aldrich (Singapore). Acetone, chloroform, dichloromethane, and n-hexane (GC-MS grade or equivalent) were purchased from Merck (Singapore). Other solvents such as diethyl ether, ethyl acetate, n-heptane, methyl tert-butyl ether and toluene were of chromatographic grade and purchased from Fisher (Singapore). Formic, acetic, propionic, iso-butyric, butyric, iso-valeric and valeric acid (analytical grade) and the alkane standard mixture (C10 - C40, all even, 50 mg/L each) were purchased from Sigma-Aldrich (Singapore). Deionised water was obtained from a MilliQ water treatment system (Millipore Advantage A10).
2.2 Submerged Anaerobic Membrane Bioreactor (SAMBR) The SAMBRs were made from polymethyl methacrylate (Plexiglas®), and had a working volume of 3 L. A microfiltration flatsheet membrane (chlorinated polyethylene) from the Kubota Corp., Japan was used. The surface area of the membrane was 0.116 m2 with a nominal pore size of 0.2 μm, and the membrane flux was set at 15 litres per square meter per hour (LMH) at 35°C. Transmembrane pressure (TMP) was 2.95 kPa at the start of the experiment. The sludge inoculum was obtained from an anaerobic digester in a wastewater treatment plant in Singapore. Recycle biogas was pumped through a stainless steel tube diffuser to generate coarse bubbles in order to mix the biomass in the reactor, and clean the membrane surface to reduce fouling, and the gas flow rate was controlled at 8 L/min (4.14 m3/m2.h). The SAMBRs were operated at an HRT of 6 h and an SRT of 200 days, and the mixed liquor volatile suspended solids (MLVSS) were 6,000 mg/L. The reactor was placed in a water bath at 35±1°C, and continuously fed with a synthetic feed (466±35mg COD/L) comprised of glucose, peptone, meat extract, essential nutrients, and bicarbonate buffer (1,000 mg/L). Figure 1 shows a diagram of the experimental set-up used for this study.
To simulate the pH shock, and to understand the effect of pH on reactor performance, the pH in the feed was adjusted to 5, 7 and 11. The first experiment was started at pH 7, then changed to pH 5 for 4 h, and finally changed back to pH 7. The second experiment was also run in similar way; it was started at pH 7, then changed to pH 11 for 4 h. However, the performance of the SAMBR did not change after 4 h so we prolonged the pH 11 feed for 24 h, and then changed it back to pH 7 in order to see the effects on the reactor.
2.3 Analysis of general parameters Separation of the Colloids Supernatant was collected from inside the SAMBR and centrifuged at 4,000 rpm for 10 min. Separation of the colloids was conducted using a cellulose acetate membrane (Advantec, Japan) with different pore sizes (5 μm, 1 μm, 0.2 μm, and 100 kDa) in a stirred dead-end cell with 47 mm diameter filters [4]. The cell was connected to nitrogen gas tank to provide the constant pressure to the cell. The pressure was set at different level based on the pore size of the membrane. A 400 mL sample was passed through the 5 μm membrane, and 100 mL of the filtrate collected for analysis. The remaining filtrate (300 mL) was further filtered using a 1 μm membrane, and this sequential filtration was then continued using 0.2 μm and 100 kDa filters. The retentate fraction after each filtration step was analysed for total organic carbon (TOC), total nitrogen (TN) and carbohydrate, and the MW distribution calculated in terms of mass percentage (w/w) by performing a material balance.
General Parameters All samples were filtered through 0.45µm glass fibre filters to separate residual biomass, and then analysed in duplicate for VFAs and COD. The amount of SMPs was estimated by subtracting the COD due to intermediate VFAs and residual substrate from the soluble effluent COD (and also not
including methane in the dissolved phase), as is accepted practise. The measurement of pH was accurate to within ±0.01 units using a pH meter (Mettler-Toledo). Total suspended solids (TSS), MLVSS and COD were measured as described in Standard Methods APHA [14]. VFAs were measured using a Shimadzu high-performance liquid chromatograph (HPLC, SPD-20AD) with a UV diode array detector (DAD, SPD-M20A) at 210 nm using an Aminex® HPX-87H (300×7.8mm) column. Analysis time was 25 min for each sample operating under isocratic and isothermal conditions using 0.005M H2SO4 as the mobile phase at a flowrate 0.8 mL/min at 55oC [15]. A total of seven VFAs including formic acid, acetic acid, propionic acid, iso-butyric acid, butyric acid, isovaleric acid and valeric acid were quantified using this method. Coefficients of variation (COV=SD/average value) for all VFAs were below ±6%. The composition of biogas (methane, oxygen, nitrogen, and carbon dioxide) from the SAMBR was determined using a Shimadzu GC2010plus gas chromatograph with a thermal conductivity detector (TCD) (COV below ±3%). A select permanent gases/CO2 (CP7429) column from Agilent was used for gas separation. Gas volume was measured with a gas-sampling bag using a gas pump with a flow meter.
Molecular weight distribution Samples were filtered using a 0.2μm syringe filter and then analysed for MW distribution using size exclusion chromatography (SEC). SEC was carried out using two columns (PolySep GFC-P1000 and 4000, Phenomenex (United States)) connected in series, using UV-DAD and refractive index (RI) detectors (Shimadzu). EasiVial polymer standards (Agilent, U.S.A.) were used for MW calibration.
Monosaccharides analysis Monosaccharides were hydrolysed using trifluoroacetic acid (4M) at 100°C for 2h, and those released were derivatized by the alditol acetate method [16]; eight neutral sugars were analysed
using inositol as the internal standard. The detection and quantification was done using gas chromatography (GC-2010 plus, Shimadzu) on a 30m × 0.25mm × 0.25µm RTX®-5MS column (Restek, Bellefonte, PA, USA) coupled with a mass spectrometry (GC-MS-QP2010, Shimadzu).
2.4 Sample pre-treatment for identifying low MW compounds (MW <580) in SMPs Organic compounds were extracted using a combination of solid-phase extraction (SPE) and liquidliquid extraction (LLE) [17]. Collected samples were filtered through a 0.45 µm glass fibre filter to remove TSS, and the pH adjusted if necessary. SPE cartridges (Waters Oasis®HLB) were conditioned using 10 mL of LC/MS-grade methanol followed by 20 mL of ultrapure water, and then the sample (1 L for effluent and 100 mL for supernatant samples) was loaded onto two cartridges connected in series using a peristaltic pump (Watson-Marlow 120U) at a flowrate of 10 mL/min. Finally, the compounds were eluted with 2 mL of the selected solvents (methanol, acetone, dichloromethane, n-hexane) in sequence into individual glass sample vials. The eluent from each SPE cartridge was collected and further pre-treated using LLE. For LLE at room temperature a sample (100 mL for effluent and 10 mL for supernatant samples) was placed into a 1-L separating funnel and a mixed solvent (hexane:dichloromethane:chloroform, 1:1:1) used for extraction. The funnel was then sealed and agitated vigorously for one minute with periodic venting to release the pressure. The organic phase was allowed to separate from the aqueous phase for at least 1 min, or until the two layers had separated completely, and then the organic layer was recovered into a 500 mL conical flask; the extraction was repeated twice and the extracts combined. Any water in the combined solvent phase was removed using anhydrous sodium sulfate which was then filtered, and the filtrate collected in a dry Florentine flask; the solvent was then volatilised using a rotary evaporator. One millilitre of dichloromethane was then added to dissolve the extract for chromatographic analysis, and a 2μL aliquot was injected into the GC-MS system directly. Plasticware was avoided during the elution procedure since plastic in contact with solvent can cause
leaching of contaminants into our samples. Ultrapure water was used as the control to identify any contamination during pre-treatment.
2.5 Gas chromatography-mass spectrometry (GC-MS) Identification Eluted samples were then analysed using a GC-MS system (GCMS-QP2010ULTRA, Shimadzu); the sample (3 μL for acetone, dichloromethane, and n-hexane samples; 2 μL for methanol) was injected onto an RTX®-5MS (30 m × 0.25 mm ID, Restek) column for the separation of low to mid polarity compounds. Splitless injection was used at a controlled temperature of 280°C, and Helium was used as a carrier gas at a column flow rate of 1 mL/min. The total runtime per sample was 60 minutes, and the temperature program was: 50°C, hold 7 min, rate 7°C/min to 325°C, and hold for 14 min. The mass spectrometer was operated in the electron ionisation mode (EI) with the ion source temperature at 230°C. Mass spectra were acquired from m/z 30 to 580 after a 10 min solvent cut time. The chromatographic peaks were identified using the NIST11 library (National Institute of Standards and Technology, Gaithersburg, MD, USA, http://www.nist.gov/srd/ mslist.htm), and the compound was considered “identified” if the match percentage was higher than 80%. Compounds that had a match percentage below 80% were mentioned as unknown peaks. Similarity index, mass spectrum and retention index were all used as selection criteria for compound identification from the NIST library list of suggested compounds. Method blanks (deionised water) were run through the same pretreatment and analysis, while feed samples were also run to identify compounds in the feed. Finally, the plastic reactor and tubing were soaked in deionised water for a month to provide a blank for compounds potentially leaching from the reactor and system components; all these blanks were then subtracted from the SMP results to specifically identify microbially produced compounds.
Estimation of concentration Quantifying all the SMPs identified would be very time consuming based on individual compound calibrations, and hence a crude approximation was made to estimate the concentration of each SMP using alkanes as representative standards. This choice was based on the literature, availability, and cost; alkanes have different chain lengths (C10 – C40) which cover most of the volatility range of the RTX®-5MS column. The calibration curves consisted of five concentrations (0, 0.1, 0.25, 0.5, and 1 mg/L), and the correlation (R2) values were greater than 0.99 except for C38 and C40 (Table S1). Alkane standards quantified the unknown compounds based on their retention time, and this was done separately for each unknown using the closest alkane standard which was run in every batch of analyses. Blank solvents (methanol, acetone, dichloromethane, n-hexane) were also run in every batch, and the instrument identification limit (IDL) of the alkane standards was evaluated for each compound based on the maximum blank concentration, and a signal-to-noise ratio of 3.
2.6 Membrane fouling test To understand the effect of colloids produced during the pH shock on membrane fouling, a small reactor (50 mL) was designed with a similar configuration to the SAMBR, with gas sparging underneath the small membrane section (5 × 3 cm) to control membrane fouling. Filtered samples from the separation of the colloids (5 μm, 1 μm, 0.2 μm, and 100 kDa) were used as a feed for this reactor to evaluate which colloid size had the most effect on membrane fouling; in this case 5μm represented the colloids smaller than 5μm. A virgin piece of membrane was used in every test, and the TMP was recorded using a sensitive pressure sensor and a datalogger on the permeate side. This test was run for 24 h, and deionised water was also run as a blank to measure membrane resistance.
3. RESULTS AND DISCUSSION
3.1 SAMBR’s performance The performance of the SAMBR was excellent with 98±1% removal and methane gas production of 252±27 mL (CH4)/g.CODdegraded (only gas phase, 72% of theoretical value) when the reactor feed was “normal” (pH 7); the pH in the reactor was 6.83 at the beginning of the experiment. The performance of the SAMBR decreased when fed with a pH 5 feed for 4 h, and the pH inside the SAMBR gradually decreased to 6.52 at 4 h; the COD in the effluent also increased slightly from 7 to 17 mg/L (Figure 2). Aquino and Stuckey [5] also found that SMP production was higher than at normal pHs (6.8 – 7.2) in an anaerobic CSTR at low pHs (<6.5) as a result of no alkalinity addition to the feed. An average 34% increase in SMP production was observed which was possibly as a result of enhanced cell lysis, as well as VFA accumulation [5]. VFAs (acetate and butyrate) started to accumulate in the supernatant inside the SAMBR, and increased from not detected (ND) to 5 mgCOD/L and continued to increase to 9.8 mgCOD/L (at 8 h) even after the feed was changed back to pH 7. However, VFAs were not detected in the effluent indicating that the fouling layer on the membrane may remove/reject VFAs, which was similar to a previous study using a SAMBR to treat saline wastewater [18]. After the SAMBR received the pH 5 feed, the average particle size in the sludge decreased significantly from 16.9±0.2μm to 13.2±0.3μm.
After the SAMBR was exposed to pH 5 for 4 h, the feed was changed back to pH 7, and the pH inside the SAMBR gradually increased to 6.81 in 24 h. The recovery from the pH shock was quite slow, and the effluent COD did not drop below 10 mg/L, even after 24 h. SEC was used to evaluate the MW distribution of the compounds produced, and inside the reactor the distribution changed dramatically; large MW compounds (MW>1,500 kDa) decreased significantly (Figure 4a), which were protein-like compounds (280 nm) (Figure 4b), while compounds around 200 kDa decreased in
size to about 50 kDa after the feed pH decreased to 5. Protein-like compounds were also detected with an increase in salinity in a SAMBR with a forward osmosis membrane for low-strength wastewater treatment in which Chen et al. indicated that protein-like substances in the form of tryptophan and tryptophan-like protein and humic acid-like substances increased; however, tyrosine and tyrosine-like proteins and fulvic acid like substances exhibited the opposite trend [9]. Figure 4b also shows a higher baseline after 16 min, suggesting that there were considerable low MW compounds produced during the pH 5 shock.
Figure 4c (RI detector) shows a huge peak between 30k and 130k indicating that carbohydrates were also formed at low pH. This result was confirmed by the analysis of monosaccharides (Figure S1); eight monosaccharides were measured inside the SAMBR. A previous study reported that three hexoses were the primary components of ECPs in mixed sludge and pure cultures, and glucose was also one of the main components of the amino sugars in bacterial cell walls [19]. After we changed the feed pH to 5, glucose and mannose increased about 3-5 times in the 10k-100k sample. In addition, rhamnose, which is a basic constituent of cell wall lipopolysaccharides, increased significantly from ND to 73 mg/L in the 100 kDa – 0.2μm sample, and ribose which is a product of ribonucleic acid (RNA) hydrolysis [20], also increased 4 times. Based on these results, bacterial cells and/or cellular debris were expected to be the main source of colloids and SMPs in the SAMBR during the pH 5 shock. Previous studies showed that the effect of a low pH leads to acidification of the cytoplasm, which is sufficient to inhibit microbial growth, but may also have other effects on cell metabolism [21]. However, microorganisms may survive in low pH conditions, and although their growth may have ceased, they may still be metabolically active. The energy requirements for a microorganism in a low pH environment are greater than that at optimal pH because it needs energy for proton pumping, with protons being pumped out of the cell to maintain an optimum cellular environment. In a high pH environment protons may need to be pumped into
the cell. If the pH is not balanced, the cell is unable to synthesize normal cellular components and is unable to divide and grow [21, 22].
Figure 3 shows the results from the pH 11 shock experiment. Initially we planned to run the pH shock for 4 h, which was identical to the last experiment (pH 5), however, the reactor performance did not change after 4 h so we prolonged the time to 24 h to determine if pH 11 had any effect on reactor performance. The pH in the reactor started at 7, but rapidly increased to 8.9 in 8 h, and then gradually increased to 9.1 in 24 h. Effluent COD started to increase after the SAMBR was fed with pH 11 feed for 6 h, and increased to 52 mg/L after 8 h, and finally reached a maximum of 414 mg/L after 26 h (2 h after the feed was changed back to pH 7). VFAs started to accumulate in the reactor after 8 h, and were detected in the effluent after 22 h, and acetate and propionate were the dominant VFAs. VFAs reached a maximum of 148 mgCOD/L at 26 h when the calculated SMP was at its highest value of 266 mgCOD/L (55% of the incoming feed COD). Formate, acetate and iso-butyrate were the major VFAs found in the effluent, but there was no propionate detected which was different to HRT shocks in our earlier work [23]. Literature shows that propionate was more rapidly degraded than acetate at pH 8.4 and above, and 6.0 and below by a methanogenic enrichment culture [24]. The lower pH would also have caused higher trace metal leaching from the sludge to the effluent which may have affected the methanogens due to a reduction in trace metal bioavailability [25, 26]. After the feed was changed back to normal (pH 7), the SAMBR recovered its original performance very quickly (within 8 h-pH inside reactor at 7.3). SEC showed that the MW distribution inside the reactor was the opposite from pH 5 (Figure 4), with the higher MW (<1,500 kDa) compounds increasing significantly comparing to the normal feed. These larger molecules were mainly protein-like compounds as the higher peaks also shown in the chromatogram were detected at 280 nm. Colloids and low MW SMPs changed significantly during the pH shocks, and this will be discussed in the following section.
3.2 Effects of feed pH on colloids Colloids inside the reactor were analysed to help understand the effect of pH shocks on membrane fouling. Figure 5 shows the TOC and TN of colloids inside the SAMBR at different sizes, which were separated using ultrafiltration membranes. After the pH 5 shock, compounds which were smaller than 0.2 μm decreased in concordance with the results from SEC, while a significant increase in TOC and TN occurred in the range of 0.2 μm – 1 μm. This indicated that some large proteins/peptides may have increased in the colloids, possibly from cell lysis. Figure 5b shows the colloids after the pH 11 shock; a similar trend to the results from SEC was also found in that compounds smaller than 0.2 μm increased after the pH shock. The results also show that colloids in the range of 1 μm – 5 μm increased significantly after the feed pH changed to 11.
A membrane fouling experiment was set up to evaluate the effect of different sizes of colloids on membrane fouling using a small reactor that simulated large SAMBR reactor operation; Figure 6 shows the results from the membrane fouling test. The test was started using the supernatant from a normal feed (pH 7), with a flux of 20 LMH to speed up the fouling process (compared with 15 LMH in the SAMBR), and a blank (deionised water) to obtain the baseline. Colloids < 5 μm fouled the membrane rapidly, but reached stable conditions after about 600 min, while the TMP was around 12.5 kPa throughout the experiment; colloids in the size range between 1 μm to 5 μm caused the most fouling in the SAMBR. Colloids from the pH 5 shock showed a slower fouling rate, however, the TMP kept increasing and did not reach stable conditions even after 24 h. The smaller compounds (<100 kDa) caused more fouling than the colloids under normal conditions (pH 7), and there was little effect on fouling by colloids from 100 kDa to 0.2 μm, and SEC also showed very low concentrations in this range (sample filtered by a 0.2 μm membrane in SEC analysis).
Colloids from the pH 11 shock caused the membrane to foul badly immediately after starting the small rig; the TMP increased rapidly to >30 kPa within 100 min which was caused by the 0.2 μm to 5 μm colloids. However, there was a drop to 23 kPa over time (Figure 6) which may be due to the thicker fouling layer being scoured from the membrane surface, and SEC results showed that compounds in the range 100 kDa to 0.2 μm increased after the pH increased to 11. This had a major impact on membrane fouling, and was most likely due to an increase in protein-like compounds during the pH change. Zhou et al. also found that protein-like compounds were present more in the supernatant and foulant compared with polysaccharides. The ratio of protein-like solutes to carbohydrates in the foulant was as high as 3.1, indicating that protein-like material preferred to attach more to the membrane [27]. A higher portion of protein-like compounds in the supernatant would favor the formation of a fouling layer because of its higher stickiness [28]. Studies with E. coli have shown that cells will move away from unstable pH environments by sensing the decrease/increase in pH using a transmembrane transducer (a single protein) [21]. This protein is induced under low/high pHs, and allows for the detection of pH changes. Protein synthesis is also necessary to return the cytoplasmic pH to normal [29], and this may have occurred in our sample.
3.3 Effects of feed pH on low MW compounds (<580 Da) Compound identification in SMPs was performed using SPE and LLE coupled with GC-MS analysis, and more than 50 compounds were identified in the effluent samples. Alkanes, alkenes, esters, alcohols, phenols, nitrogen-compounds and sulfur compounds were the major groups of compounds identified in the effluent and supernatant samples. Alkanes, alkenes and esters have been found in the literature [30-32], and alkanes and alkenes were found to be synthesized by S. lutea through substrate metabolism that utilizes fatty acids [33]. In a recent study, Shi et al. also found that a laccase enzyme from the endophyte Pantoea ananatis Sd-1 could degrade lignin and produce metabolites such as alkanes and acids [34], similar to what we found in our study. A study
on volatile compounds released by 50 bacterial strains also revealed that bacteria can produce esters, alcohols, nitrogen-compounds and sulfur compounds [30]. Micromonospora aurantiaca, which are naturally present in both soil and water, can produce a large diversity of volatile fatty acids and fatty acid methyl esters [35], while further studies found that N-compounds, acids and esters are precursors for disinfection by–products [36, 37]. Liu et al. reported that the disinfection by-products with chlorination include trihalomethane, haloacetic acids, halopropanones, haloacetonitriles and trihalonitromethanes [36]; alcohols, esters and acids are possible to chlorinate, and become trihalomethane and haloacetic acids. Haloacetonitriles and trihalonitromethanes are both nitrogenous disinfection by-products that can be attributed to N-compounds in SMPs. The full lists of the compounds identified are in a supplementary section (Table S1, S2, S3 and S4).
Figure 7 shows the groups of compounds identified in the effluent of the SAMBR under different pH feeds; it is clear that alkanes and alkenes were produced more with the pH 5 feed compared to pH 7 (normal conditions). Groups of bacteria appear to be able to degrade alkanes under anaerobic conditions [38], however, a pH shock may disrupt or kill bacteria in the SAMBR, and that is why we found more alkanes and alkenes when the pH dropped inside the SAMBR. Nitrogen compounds and phenols were not found in pH 5 and pH 7 samples, but they were found in the effluent from a pH 11 feed. However, esters, which were found in high percentages at pH 5 and pH 7, were not found in the pH 11 sample. To adapt to low pH environments, cells try to control the pH gradient across its membrane to generate a proton motive force, and hence produce energy. One study found that E. coli expresses carboxylase enzymes, especially at low pHs [21]. The function of the enzymes is to increase the external pH, and contribute to induced acid tolerance in some situations [39]. Lysine decarboxylase is an amino acid protein that contributes to control the pH by converting exogenously supplied lysine to cadaverine, an alkaline product that neutralizes the acidic environment when secreted from the cell [29, 39]. We did not detected lysine in this study;
however, it was found in previous work where UPLC-Q-TOF-MS was used for identification. Tipthara et al reported that lysine acetate was found in a supernatant of an anaerobic reactor after being fed glucose [40].
Figure 8 shows the chromatograms of N-butyl-benzenesulfonamide and cyclooctasulfur. N-butylbenzenesulfonamide, a type of plasticiser was detected at very high concentrations; this compound has also been found in a batch anaerobic stirred tank reactor [2], in a landfill leachate [41], and also in SAMBRs operating at low temperature (20°C) [42] and low HRT (1 h) [23]. Elleuch et al. [43] found that Streptomyces sp. TN262 can produce this compound as a metabolite during large scale fermentation. Cyclooctasulfur, which is a central intermediate in the biotic or abiotic oxidation of sulfides [44], was also found in extremely high concentrations during the pH shocks, and a similar trend was also found in a SAMBR under high transient load [23]. The cause of an increase in this compound is still unclear at present, but may be due to cell leakage of intermediate metabolites. However, cyclooctasulfur was found to be degraded by deltaproteobacteria such as Desulfobulbaceae and Desulfuromonadales [45].
Concentrations of the compounds identified were estimated using a series of alkane standards. Figure 9 shows the rejection of low MW compounds in a SAMBR under different pH feeds; the results show that the membrane-fouling layer was very effective in COD removal, and could remove 84% of the low MW compounds from the SAMBR supernatant. Concentrations of low MW compounds increased after the feed pH decreased to 5 (pH 6.2 in the reactor); however, the removal rate also increased to 94%. In contrast, percentage removal decreased greatly to only 50% when the SAMBR was fed with pH 11 feed (pH 9.1 in the reactor). This may indicate that the membranefouling layer can remove/reject the compounds by “charge” and/or “concentration polarisation”. An interesting study looked at the rejection of pharmaceuticals in nanofiltration membrane filtration;
Botton et al. reported that the presence of the negatively charged biofouling induced accumulation of positively charged pharmaceuticals within the fouling layer, which probably also hindered back diffusion [46]. This caused a reduction in rejection efficiency of positively charged solutes but did not affect to rejection of neutral and negatively charged pharmaceuticals. Another study on the rejection of pharmaceuticals showed that negatively charged colloids enhanced the concentration polarization and charge effects on the membrane surface, and increased the rejection of some negatively charged pharmaceuticals by 15%. However, the charged colloids also reduced the rejection of some positively charged pharmaceuticals by more than 20% [47]. Chang et al. found that the characteristics of the membrane played an important role in solute rejection; high solute removals were attributed to the adsorption and/or sieving onto the cakes. Consequently, the difference in solute rejection efficiency between hydrophilic and hydrophobic membranes was mainly due to the degree of sieving and/or adsorption onto the cakes deposited on the membrane, and only partly due to adsorption in membrane pores and the surfaces [48]. However, most of these studies have been done on surface water for water treatment, and a further study on the rejection of various types of charged SMP solutes by the fouling layer in a SAMBR would be a very interesting approach to understand how different types of SMPs are rejected.
4. CONCLUSIONS The results showed that pH shocks (pH 5 and pH 11) had a significant effect on SAMBR performance. Changes in pH inside the SAMBR caused disruption of cellular metabolism and/or enhanced cell lysis. However, the system recovered very quickly within 24 h (pH 5 shock) and 8 h (pH 11 shock). Carbohydrates (30 k – 200 kDa) were found at higher concentrations under the pH 5 shock, while larger MW (1,500 kDa – 0.2 μm) protein-like compounds were found under the pH 11 shock. pH shocks had varying effects on membrane fouling by colloids, which may be due to
“charge” and “concentration polarization” effects on the membrane fouling layer . Larger colloids (1-5 μm), which are related to protein-like compounds, caused more fouling on the membrane than smaller ones. More than 50 low MW compounds were identified in the effluent samples. Alkanes, alkenes, esters, alcohols, phenols, nitrogen-compounds and sulfur compounds were the major groups of compounds identified in both the effluent and supernatant samples. The membranefouling layer (dynamic membrane) is an important factor in the removal of low MW compounds in the SAMBR, and pH changes inside the SAMBR had a significant effect on effluent quality.
ACKNOWLEDGEMENT This research work was supported by the Singapore National Research Foundation under its Environmental & Water Technologies Strategic Research Programme, and administered by the Environment & Water Industry Programme Office (EWI) of the PUB.
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Figure 1
Gas$sampling$bag$ Gas$out$
Pressure$$ Recycle$$ sensor$ pump$
Effluent$$ pump$
pH&ORP$meter$
Recycle$line$
Gas$out$for$recircula8on$
Feed$
Baffle$
Effluent$
Membrane$ module$
Flow$
Feed$pump$
540 mm
Gas$flow$$ meter$
Gas$pump$ Spacing between membrane and the wall is 5 mm
Feed$tank$
Gas$in$
32 mm
Figure 1. Diagram of the SAMBR.
Effluent$tank$
Figure 2
pH 5
8.0 7.5
45
CODeff
40
Supernatant 10
6.5
25 20
6.0
15 10
5.5
VFAs (mg/L)
30
COD (mg/L)
35
7.0 pH
12
50 pH
Effluent
8 6 4 2
5 5.0
0 0
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15
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ND
ND
ND
Time (h)
Figure 2. Effluent COD, pH and VFA concentrations during pH 5 shock in the feed.
Figure 3
pH 11
500
9.5
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9.0
pH
450
CODeff
400
VFA
350
8.5
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150
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0 0
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45
h Time (h)
Figure 3. Effluent COD, pH and VFA concentrations during pH 11 shock in the feed.
Figure 4
1,500k 20000
130k
30k
a) 210 nm
pH7 pH5
15000
uV
pH11 10000
5000
0 2500 5
b) 280 nm
10 pH 11
15 Time (min)
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uV
pH 7 1000 pH 5 500 0 1200 5
10
c) RID
15 Time (min)
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uV
800
pH 5
600 pH 11
400
pH 7
200 0 5
10
15 Time (min)
20
25
Figure 4. Size Exclusion Chromatography of the SAMBR reactor supernatant during changes in feed pH.
Figure 5
a) pH5 450 400
140
4h
120
,5
300
TN (mg/L)
TOC (mg/L)
350
160
0h
250 200 150
80 60 40
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20 1-5um 5um
0.2-1um 1um
100kDa 0.2um -0.2um
4h
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0h
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1um 0.2-1um
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b) pH11 160
350 300
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244hh
120 TN (mg/L)
TOC (mg/L)
250
0 h0 h
200 150
80 60 40
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20 5um 1-5um
1um 0.2-1um
100kDa 0.2um -0.2um
4 h 24 h
100
100
0
0 h0 h
0 100k <100kDa
5um 1-5um
Figure 5. TOC and TN concentrations of supernatant inside the SAMBR under feed pH5 (a) and pH11 (b).
Figure 6
30 Blank < 100kDa
25
100kDa - 0.2um 0.2um - 1um
20
TMP (kPa)
1um - 5um 15
10
5
0 0
500 1000 Time (min)
0
500 1000 Time (min)
0
500
1000
Time (min)
Figure 6. Flux and TMP experiment on the membrane fouling layers under different pHs.
Figure 7
Alkane
pH11
Alkene Ester Alcohol
pH5
Others Unknown N
pH7
Phenol 0%
20%
40%
60%
80%
100%
Figure 7. Identified compounds in the effluent of a SAMBR under different pH feeds.
Figure 8
Intensity
500000 450000 400000 350000 300000 250000 200000 150000 100000 50000 0
0h 4h 8h
Cyclooctasulfur
N-butyl benzenesulfonamide
28
29
30
31 Time (min)
32
33
34
35
Figure 8. Chromatogram of the low MW compounds (GC-MS) in the reactor supernatant before and after a change in the pH of the feed.
Figure 9
0.40 Supernatant
Effluent
0.35
Concentration (mg/L)
0.30 0.25
50%
0.20 94%
0.15 84%
0.10 0.05 0.00 pH7
pH5
pH11
Figure 9. Concentration of low MW SMP (MW < 580 Da) and percentage removals of supernatants and effluents under different feed pHs.
Highlights
pH shocks (5 and 11) over 4 and 24 h had a significant effect on SAMBR performance
SAMBR recovered very quickly from pH shocks (5, 11) within 24 and 8 h, respectively
shocks had varying effects on fouling by 1-5 μm colloids due to charge/polarization
membrane fouling layer (dynamic membrane) important in removing of low MW compounds
S1385-8947(17)30355-8 http://dx.doi.org/10.1016/j.cej.2017.03.018 CEJ 16614
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
1 February 2017 6 March 2017 7 March 2017
Please cite this article as: C. Kunacheva, Y.N.A. Soh, D.C. Stuckey, Effect of feed pH on Reactor Performance and Production of Soluble Microbial Products (SMPs) in a Submerged Anaerobic Membrane Bioreactor, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.03.018
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Effect of feed pH on Reactor Performance and Production of Soluble Microbial Products (SMPs) in a Submerged Anaerobic Membrane Bioreactor
C. Kunacheva*, Y. N. A. Soh* and D. C. Stuckey**
* Advanced Environmental Biotechnology Centre, Nanyang Environment & Water Research Institute, Nanyang Technological University, 1 Cleantech Loop, CleanTech One, Singapore 637141, Singapore (E-mail: [email protected]; [email protected]) ** Department of Chemical Engineering, Imperial College London, London SW7 2AZ, UK (E-mail: [email protected])
Abstract The production of soluble microbial products (SMPs) and colloids in a submerged anaerobic membrane bioreactor (SAMBR) under different feed pHs (pH 5 and pH 11) was evaluated, and they were found to have a significant effect on SAMBR performance. Fluctuations in pH inside the SAMBR affected cell metabolism and/or enhanced cell lysis in the reactor, but the system recovered within 24 h for the pH 5 shock, and 8 h for the pH 11 shock. Carbohydrates (30k – 200kDa) were found at very high concentrations with the pH 5 shock, while higher concentrations of “protein-like” compounds (1,500kDa – 0.2μm) were found with the pH 11 shock. The pH shocks affected membrane fouling primarily through increased colloids, which formed a charged concentration polarization layer on the membrane surface. Larger colloids (1-5μm) related to “protein-like” compounds
caused more membrane fouling than smaller ones. Alkanes, alkenes, esters, alcohols, phenols, nitrogen-compounds and sulfur compounds were the major groups of compounds identified in the effluent and supernatant samples. The membrane-fouling layer (“dynamic membrane”) is an important factor in the removal of low MW compounds in the SAMBR, and changes in pH inside the SAMBR had a significant effect on effluent quality.
Keywords Anaerobic; biological treatment; colloids; pH; soluble microbial products (SMP)
1. INTRODUCTION Soluble microbial products (SMPs) are defined as the soluble organics created by biological metabolism (catabolism and cell lysis) in biological processes that are not known intermediates such as volatile fatty acids (VFAs), or incoming feed [1]. Their presence affects the performance of most biological treatment systems in terms of chemical oxygen demand (COD) removal, and causes the fouling of membranes. SMP production is complex and its sources are still unclear; it has been noted over the years in both aerobic and anaerobic biological processes that around 2% of the incoming feed COD is present in the effluent as SMPs [2]. However, under transient conditions including nutrient limitations, toxicants, or when the feed flow or composition is changed radically, the effluent SMPs can be as high as 17% of the influent COD [1, 3]. There is a strong need to understand the effect of a variety of factors on SMP production in biological processes in order to improve the design and operation of wastewater treatment plants by reducing SMP production. The effect of the hydraulic retention time (HRT), organic loading rate (OLR), and sludge retention time (SRT) on SMP production has been investigated, and shows that SMP production increases at shorter HRT, longer SRT, and higher feed strength [4-7]. SMP production is also sensitive to
substrate concentration, and Xie et al. found that total SMP production increased by 71% with a threefold increase in feed strength [8]. Under environmental stresses such as low pH, high salinity, low temperature, and nutrient deficiency, SMPs are produced as a response to these stresses. Moreover, a shift in the molecular weight (MW) distribution towards the production of higher MW SMPs was observed in most cases [4, 9-11]. The literature [12, 13] also reported that SMP and VFA production was enhanced under nutrient deficient conditions. Due to these increased VFAs, the percentage of SMPs to effluent COD was reduced to 45% when it was under steady-state; however, the SMP concentration increased to 37% of the incoming feed compared with 3% under steady-state conditions [12]. A similar trend occurred at low pH (<6.5) as a result of no alkalinity in the feed, and the normalized SMP production was statistically higher than during normal operation pH (6.8 – 7.2) in an anaerobic continuous stirred tank reactor (CSTR). An average 34% increase in SMP production was observed, which was possibly the result of enhanced cell lysis, as well as VFA accumulation [5].
Therefore, it is important to evaluate the production and composition of SMPs in a submerged anaerobic membrane bioreactor (SAMBR) in order to understand what these compounds are, and how they are produced; this is fundamental for developing methods to control membrane fouling. A pH shock is one factor that can affect the performance of the reactor when it receives industrial wastewater, and rapid changes in pH may create more SMPs. Currently there has been no study identifying the SMPs produced under pH shocks in a SAMBR. Hence, the objectives of this study were to evaluate the performance of a SAMBR, and to characterise the SMPs produced under different pHs shock in the feed (pH 5, pH 7 and pH 11).
2. MATERIALS AND METHODS 2.1 Reagents and chemicals
Methanol (LC-MS grade) was purchased from Sigma-Aldrich (Singapore). Acetone, chloroform, dichloromethane, and n-hexane (GC-MS grade or equivalent) were purchased from Merck (Singapore). Other solvents such as diethyl ether, ethyl acetate, n-heptane, methyl tert-butyl ether and toluene were of chromatographic grade and purchased from Fisher (Singapore). Formic, acetic, propionic, iso-butyric, butyric, iso-valeric and valeric acid (analytical grade) and the alkane standard mixture (C10 - C40, all even, 50 mg/L each) were purchased from Sigma-Aldrich (Singapore). Deionised water was obtained from a MilliQ water treatment system (Millipore Advantage A10).
2.2 Submerged Anaerobic Membrane Bioreactor (SAMBR) The SAMBRs were made from polymethyl methacrylate (Plexiglas®), and had a working volume of 3 L. A microfiltration flatsheet membrane (chlorinated polyethylene) from the Kubota Corp., Japan was used. The surface area of the membrane was 0.116 m2 with a nominal pore size of 0.2 μm, and the membrane flux was set at 15 litres per square meter per hour (LMH) at 35°C. Transmembrane pressure (TMP) was 2.95 kPa at the start of the experiment. The sludge inoculum was obtained from an anaerobic digester in a wastewater treatment plant in Singapore. Recycle biogas was pumped through a stainless steel tube diffuser to generate coarse bubbles in order to mix the biomass in the reactor, and clean the membrane surface to reduce fouling, and the gas flow rate was controlled at 8 L/min (4.14 m3/m2.h). The SAMBRs were operated at an HRT of 6 h and an SRT of 200 days, and the mixed liquor volatile suspended solids (MLVSS) were 6,000 mg/L. The reactor was placed in a water bath at 35±1°C, and continuously fed with a synthetic feed (466±35mg COD/L) comprised of glucose, peptone, meat extract, essential nutrients, and bicarbonate buffer (1,000 mg/L). Figure 1 shows a diagram of the experimental set-up used for this study.
To simulate the pH shock, and to understand the effect of pH on reactor performance, the pH in the feed was adjusted to 5, 7 and 11. The first experiment was started at pH 7, then changed to pH 5 for 4 h, and finally changed back to pH 7. The second experiment was also run in similar way; it was started at pH 7, then changed to pH 11 for 4 h. However, the performance of the SAMBR did not change after 4 h so we prolonged the pH 11 feed for 24 h, and then changed it back to pH 7 in order to see the effects on the reactor.
2.3 Analysis of general parameters Separation of the Colloids Supernatant was collected from inside the SAMBR and centrifuged at 4,000 rpm for 10 min. Separation of the colloids was conducted using a cellulose acetate membrane (Advantec, Japan) with different pore sizes (5 μm, 1 μm, 0.2 μm, and 100 kDa) in a stirred dead-end cell with 47 mm diameter filters [4]. The cell was connected to nitrogen gas tank to provide the constant pressure to the cell. The pressure was set at different level based on the pore size of the membrane. A 400 mL sample was passed through the 5 μm membrane, and 100 mL of the filtrate collected for analysis. The remaining filtrate (300 mL) was further filtered using a 1 μm membrane, and this sequential filtration was then continued using 0.2 μm and 100 kDa filters. The retentate fraction after each filtration step was analysed for total organic carbon (TOC), total nitrogen (TN) and carbohydrate, and the MW distribution calculated in terms of mass percentage (w/w) by performing a material balance.
General Parameters All samples were filtered through 0.45µm glass fibre filters to separate residual biomass, and then analysed in duplicate for VFAs and COD. The amount of SMPs was estimated by subtracting the COD due to intermediate VFAs and residual substrate from the soluble effluent COD (and also not
including methane in the dissolved phase), as is accepted practise. The measurement of pH was accurate to within ±0.01 units using a pH meter (Mettler-Toledo). Total suspended solids (TSS), MLVSS and COD were measured as described in Standard Methods APHA [14]. VFAs were measured using a Shimadzu high-performance liquid chromatograph (HPLC, SPD-20AD) with a UV diode array detector (DAD, SPD-M20A) at 210 nm using an Aminex® HPX-87H (300×7.8mm) column. Analysis time was 25 min for each sample operating under isocratic and isothermal conditions using 0.005M H2SO4 as the mobile phase at a flowrate 0.8 mL/min at 55oC [15]. A total of seven VFAs including formic acid, acetic acid, propionic acid, iso-butyric acid, butyric acid, isovaleric acid and valeric acid were quantified using this method. Coefficients of variation (COV=SD/average value) for all VFAs were below ±6%. The composition of biogas (methane, oxygen, nitrogen, and carbon dioxide) from the SAMBR was determined using a Shimadzu GC2010plus gas chromatograph with a thermal conductivity detector (TCD) (COV below ±3%). A select permanent gases/CO2 (CP7429) column from Agilent was used for gas separation. Gas volume was measured with a gas-sampling bag using a gas pump with a flow meter.
Molecular weight distribution Samples were filtered using a 0.2μm syringe filter and then analysed for MW distribution using size exclusion chromatography (SEC). SEC was carried out using two columns (PolySep GFC-P1000 and 4000, Phenomenex (United States)) connected in series, using UV-DAD and refractive index (RI) detectors (Shimadzu). EasiVial polymer standards (Agilent, U.S.A.) were used for MW calibration.
Monosaccharides analysis Monosaccharides were hydrolysed using trifluoroacetic acid (4M) at 100°C for 2h, and those released were derivatized by the alditol acetate method [16]; eight neutral sugars were analysed
using inositol as the internal standard. The detection and quantification was done using gas chromatography (GC-2010 plus, Shimadzu) on a 30m × 0.25mm × 0.25µm RTX®-5MS column (Restek, Bellefonte, PA, USA) coupled with a mass spectrometry (GC-MS-QP2010, Shimadzu).
2.4 Sample pre-treatment for identifying low MW compounds (MW <580) in SMPs Organic compounds were extracted using a combination of solid-phase extraction (SPE) and liquidliquid extraction (LLE) [17]. Collected samples were filtered through a 0.45 µm glass fibre filter to remove TSS, and the pH adjusted if necessary. SPE cartridges (Waters Oasis®HLB) were conditioned using 10 mL of LC/MS-grade methanol followed by 20 mL of ultrapure water, and then the sample (1 L for effluent and 100 mL for supernatant samples) was loaded onto two cartridges connected in series using a peristaltic pump (Watson-Marlow 120U) at a flowrate of 10 mL/min. Finally, the compounds were eluted with 2 mL of the selected solvents (methanol, acetone, dichloromethane, n-hexane) in sequence into individual glass sample vials. The eluent from each SPE cartridge was collected and further pre-treated using LLE. For LLE at room temperature a sample (100 mL for effluent and 10 mL for supernatant samples) was placed into a 1-L separating funnel and a mixed solvent (hexane:dichloromethane:chloroform, 1:1:1) used for extraction. The funnel was then sealed and agitated vigorously for one minute with periodic venting to release the pressure. The organic phase was allowed to separate from the aqueous phase for at least 1 min, or until the two layers had separated completely, and then the organic layer was recovered into a 500 mL conical flask; the extraction was repeated twice and the extracts combined. Any water in the combined solvent phase was removed using anhydrous sodium sulfate which was then filtered, and the filtrate collected in a dry Florentine flask; the solvent was then volatilised using a rotary evaporator. One millilitre of dichloromethane was then added to dissolve the extract for chromatographic analysis, and a 2μL aliquot was injected into the GC-MS system directly. Plasticware was avoided during the elution procedure since plastic in contact with solvent can cause
leaching of contaminants into our samples. Ultrapure water was used as the control to identify any contamination during pre-treatment.
2.5 Gas chromatography-mass spectrometry (GC-MS) Identification Eluted samples were then analysed using a GC-MS system (GCMS-QP2010ULTRA, Shimadzu); the sample (3 μL for acetone, dichloromethane, and n-hexane samples; 2 μL for methanol) was injected onto an RTX®-5MS (30 m × 0.25 mm ID, Restek) column for the separation of low to mid polarity compounds. Splitless injection was used at a controlled temperature of 280°C, and Helium was used as a carrier gas at a column flow rate of 1 mL/min. The total runtime per sample was 60 minutes, and the temperature program was: 50°C, hold 7 min, rate 7°C/min to 325°C, and hold for 14 min. The mass spectrometer was operated in the electron ionisation mode (EI) with the ion source temperature at 230°C. Mass spectra were acquired from m/z 30 to 580 after a 10 min solvent cut time. The chromatographic peaks were identified using the NIST11 library (National Institute of Standards and Technology, Gaithersburg, MD, USA, http://www.nist.gov/srd/ mslist.htm), and the compound was considered “identified” if the match percentage was higher than 80%. Compounds that had a match percentage below 80% were mentioned as unknown peaks. Similarity index, mass spectrum and retention index were all used as selection criteria for compound identification from the NIST library list of suggested compounds. Method blanks (deionised water) were run through the same pretreatment and analysis, while feed samples were also run to identify compounds in the feed. Finally, the plastic reactor and tubing were soaked in deionised water for a month to provide a blank for compounds potentially leaching from the reactor and system components; all these blanks were then subtracted from the SMP results to specifically identify microbially produced compounds.
Estimation of concentration Quantifying all the SMPs identified would be very time consuming based on individual compound calibrations, and hence a crude approximation was made to estimate the concentration of each SMP using alkanes as representative standards. This choice was based on the literature, availability, and cost; alkanes have different chain lengths (C10 – C40) which cover most of the volatility range of the RTX®-5MS column. The calibration curves consisted of five concentrations (0, 0.1, 0.25, 0.5, and 1 mg/L), and the correlation (R2) values were greater than 0.99 except for C38 and C40 (Table S1). Alkane standards quantified the unknown compounds based on their retention time, and this was done separately for each unknown using the closest alkane standard which was run in every batch of analyses. Blank solvents (methanol, acetone, dichloromethane, n-hexane) were also run in every batch, and the instrument identification limit (IDL) of the alkane standards was evaluated for each compound based on the maximum blank concentration, and a signal-to-noise ratio of 3.
2.6 Membrane fouling test To understand the effect of colloids produced during the pH shock on membrane fouling, a small reactor (50 mL) was designed with a similar configuration to the SAMBR, with gas sparging underneath the small membrane section (5 × 3 cm) to control membrane fouling. Filtered samples from the separation of the colloids (5 μm, 1 μm, 0.2 μm, and 100 kDa) were used as a feed for this reactor to evaluate which colloid size had the most effect on membrane fouling; in this case 5μm represented the colloids smaller than 5μm. A virgin piece of membrane was used in every test, and the TMP was recorded using a sensitive pressure sensor and a datalogger on the permeate side. This test was run for 24 h, and deionised water was also run as a blank to measure membrane resistance.
3. RESULTS AND DISCUSSION
3.1 SAMBR’s performance The performance of the SAMBR was excellent with 98±1% removal and methane gas production of 252±27 mL (CH4)/g.CODdegraded (only gas phase, 72% of theoretical value) when the reactor feed was “normal” (pH 7); the pH in the reactor was 6.83 at the beginning of the experiment. The performance of the SAMBR decreased when fed with a pH 5 feed for 4 h, and the pH inside the SAMBR gradually decreased to 6.52 at 4 h; the COD in the effluent also increased slightly from 7 to 17 mg/L (Figure 2). Aquino and Stuckey [5] also found that SMP production was higher than at normal pHs (6.8 – 7.2) in an anaerobic CSTR at low pHs (<6.5) as a result of no alkalinity addition to the feed. An average 34% increase in SMP production was observed which was possibly as a result of enhanced cell lysis, as well as VFA accumulation [5]. VFAs (acetate and butyrate) started to accumulate in the supernatant inside the SAMBR, and increased from not detected (ND) to 5 mgCOD/L and continued to increase to 9.8 mgCOD/L (at 8 h) even after the feed was changed back to pH 7. However, VFAs were not detected in the effluent indicating that the fouling layer on the membrane may remove/reject VFAs, which was similar to a previous study using a SAMBR to treat saline wastewater [18]. After the SAMBR received the pH 5 feed, the average particle size in the sludge decreased significantly from 16.9±0.2μm to 13.2±0.3μm.
After the SAMBR was exposed to pH 5 for 4 h, the feed was changed back to pH 7, and the pH inside the SAMBR gradually increased to 6.81 in 24 h. The recovery from the pH shock was quite slow, and the effluent COD did not drop below 10 mg/L, even after 24 h. SEC was used to evaluate the MW distribution of the compounds produced, and inside the reactor the distribution changed dramatically; large MW compounds (MW>1,500 kDa) decreased significantly (Figure 4a), which were protein-like compounds (280 nm) (Figure 4b), while compounds around 200 kDa decreased in
size to about 50 kDa after the feed pH decreased to 5. Protein-like compounds were also detected with an increase in salinity in a SAMBR with a forward osmosis membrane for low-strength wastewater treatment in which Chen et al. indicated that protein-like substances in the form of tryptophan and tryptophan-like protein and humic acid-like substances increased; however, tyrosine and tyrosine-like proteins and fulvic acid like substances exhibited the opposite trend [9]. Figure 4b also shows a higher baseline after 16 min, suggesting that there were considerable low MW compounds produced during the pH 5 shock.
Figure 4c (RI detector) shows a huge peak between 30k and 130k indicating that carbohydrates were also formed at low pH. This result was confirmed by the analysis of monosaccharides (Figure S1); eight monosaccharides were measured inside the SAMBR. A previous study reported that three hexoses were the primary components of ECPs in mixed sludge and pure cultures, and glucose was also one of the main components of the amino sugars in bacterial cell walls [19]. After we changed the feed pH to 5, glucose and mannose increased about 3-5 times in the 10k-100k sample. In addition, rhamnose, which is a basic constituent of cell wall lipopolysaccharides, increased significantly from ND to 73 mg/L in the 100 kDa – 0.2μm sample, and ribose which is a product of ribonucleic acid (RNA) hydrolysis [20], also increased 4 times. Based on these results, bacterial cells and/or cellular debris were expected to be the main source of colloids and SMPs in the SAMBR during the pH 5 shock. Previous studies showed that the effect of a low pH leads to acidification of the cytoplasm, which is sufficient to inhibit microbial growth, but may also have other effects on cell metabolism [21]. However, microorganisms may survive in low pH conditions, and although their growth may have ceased, they may still be metabolically active. The energy requirements for a microorganism in a low pH environment are greater than that at optimal pH because it needs energy for proton pumping, with protons being pumped out of the cell to maintain an optimum cellular environment. In a high pH environment protons may need to be pumped into
the cell. If the pH is not balanced, the cell is unable to synthesize normal cellular components and is unable to divide and grow [21, 22].
Figure 3 shows the results from the pH 11 shock experiment. Initially we planned to run the pH shock for 4 h, which was identical to the last experiment (pH 5), however, the reactor performance did not change after 4 h so we prolonged the time to 24 h to determine if pH 11 had any effect on reactor performance. The pH in the reactor started at 7, but rapidly increased to 8.9 in 8 h, and then gradually increased to 9.1 in 24 h. Effluent COD started to increase after the SAMBR was fed with pH 11 feed for 6 h, and increased to 52 mg/L after 8 h, and finally reached a maximum of 414 mg/L after 26 h (2 h after the feed was changed back to pH 7). VFAs started to accumulate in the reactor after 8 h, and were detected in the effluent after 22 h, and acetate and propionate were the dominant VFAs. VFAs reached a maximum of 148 mgCOD/L at 26 h when the calculated SMP was at its highest value of 266 mgCOD/L (55% of the incoming feed COD). Formate, acetate and iso-butyrate were the major VFAs found in the effluent, but there was no propionate detected which was different to HRT shocks in our earlier work [23]. Literature shows that propionate was more rapidly degraded than acetate at pH 8.4 and above, and 6.0 and below by a methanogenic enrichment culture [24]. The lower pH would also have caused higher trace metal leaching from the sludge to the effluent which may have affected the methanogens due to a reduction in trace metal bioavailability [25, 26]. After the feed was changed back to normal (pH 7), the SAMBR recovered its original performance very quickly (within 8 h-pH inside reactor at 7.3). SEC showed that the MW distribution inside the reactor was the opposite from pH 5 (Figure 4), with the higher MW (<1,500 kDa) compounds increasing significantly comparing to the normal feed. These larger molecules were mainly protein-like compounds as the higher peaks also shown in the chromatogram were detected at 280 nm. Colloids and low MW SMPs changed significantly during the pH shocks, and this will be discussed in the following section.
3.2 Effects of feed pH on colloids Colloids inside the reactor were analysed to help understand the effect of pH shocks on membrane fouling. Figure 5 shows the TOC and TN of colloids inside the SAMBR at different sizes, which were separated using ultrafiltration membranes. After the pH 5 shock, compounds which were smaller than 0.2 μm decreased in concordance with the results from SEC, while a significant increase in TOC and TN occurred in the range of 0.2 μm – 1 μm. This indicated that some large proteins/peptides may have increased in the colloids, possibly from cell lysis. Figure 5b shows the colloids after the pH 11 shock; a similar trend to the results from SEC was also found in that compounds smaller than 0.2 μm increased after the pH shock. The results also show that colloids in the range of 1 μm – 5 μm increased significantly after the feed pH changed to 11.
A membrane fouling experiment was set up to evaluate the effect of different sizes of colloids on membrane fouling using a small reactor that simulated large SAMBR reactor operation; Figure 6 shows the results from the membrane fouling test. The test was started using the supernatant from a normal feed (pH 7), with a flux of 20 LMH to speed up the fouling process (compared with 15 LMH in the SAMBR), and a blank (deionised water) to obtain the baseline. Colloids < 5 μm fouled the membrane rapidly, but reached stable conditions after about 600 min, while the TMP was around 12.5 kPa throughout the experiment; colloids in the size range between 1 μm to 5 μm caused the most fouling in the SAMBR. Colloids from the pH 5 shock showed a slower fouling rate, however, the TMP kept increasing and did not reach stable conditions even after 24 h. The smaller compounds (<100 kDa) caused more fouling than the colloids under normal conditions (pH 7), and there was little effect on fouling by colloids from 100 kDa to 0.2 μm, and SEC also showed very low concentrations in this range (sample filtered by a 0.2 μm membrane in SEC analysis).
Colloids from the pH 11 shock caused the membrane to foul badly immediately after starting the small rig; the TMP increased rapidly to >30 kPa within 100 min which was caused by the 0.2 μm to 5 μm colloids. However, there was a drop to 23 kPa over time (Figure 6) which may be due to the thicker fouling layer being scoured from the membrane surface, and SEC results showed that compounds in the range 100 kDa to 0.2 μm increased after the pH increased to 11. This had a major impact on membrane fouling, and was most likely due to an increase in protein-like compounds during the pH change. Zhou et al. also found that protein-like compounds were present more in the supernatant and foulant compared with polysaccharides. The ratio of protein-like solutes to carbohydrates in the foulant was as high as 3.1, indicating that protein-like material preferred to attach more to the membrane [27]. A higher portion of protein-like compounds in the supernatant would favor the formation of a fouling layer because of its higher stickiness [28]. Studies with E. coli have shown that cells will move away from unstable pH environments by sensing the decrease/increase in pH using a transmembrane transducer (a single protein) [21]. This protein is induced under low/high pHs, and allows for the detection of pH changes. Protein synthesis is also necessary to return the cytoplasmic pH to normal [29], and this may have occurred in our sample.
3.3 Effects of feed pH on low MW compounds (<580 Da) Compound identification in SMPs was performed using SPE and LLE coupled with GC-MS analysis, and more than 50 compounds were identified in the effluent samples. Alkanes, alkenes, esters, alcohols, phenols, nitrogen-compounds and sulfur compounds were the major groups of compounds identified in the effluent and supernatant samples. Alkanes, alkenes and esters have been found in the literature [30-32], and alkanes and alkenes were found to be synthesized by S. lutea through substrate metabolism that utilizes fatty acids [33]. In a recent study, Shi et al. also found that a laccase enzyme from the endophyte Pantoea ananatis Sd-1 could degrade lignin and produce metabolites such as alkanes and acids [34], similar to what we found in our study. A study
on volatile compounds released by 50 bacterial strains also revealed that bacteria can produce esters, alcohols, nitrogen-compounds and sulfur compounds [30]. Micromonospora aurantiaca, which are naturally present in both soil and water, can produce a large diversity of volatile fatty acids and fatty acid methyl esters [35], while further studies found that N-compounds, acids and esters are precursors for disinfection by–products [36, 37]. Liu et al. reported that the disinfection by-products with chlorination include trihalomethane, haloacetic acids, halopropanones, haloacetonitriles and trihalonitromethanes [36]; alcohols, esters and acids are possible to chlorinate, and become trihalomethane and haloacetic acids. Haloacetonitriles and trihalonitromethanes are both nitrogenous disinfection by-products that can be attributed to N-compounds in SMPs. The full lists of the compounds identified are in a supplementary section (Table S1, S2, S3 and S4).
Figure 7 shows the groups of compounds identified in the effluent of the SAMBR under different pH feeds; it is clear that alkanes and alkenes were produced more with the pH 5 feed compared to pH 7 (normal conditions). Groups of bacteria appear to be able to degrade alkanes under anaerobic conditions [38], however, a pH shock may disrupt or kill bacteria in the SAMBR, and that is why we found more alkanes and alkenes when the pH dropped inside the SAMBR. Nitrogen compounds and phenols were not found in pH 5 and pH 7 samples, but they were found in the effluent from a pH 11 feed. However, esters, which were found in high percentages at pH 5 and pH 7, were not found in the pH 11 sample. To adapt to low pH environments, cells try to control the pH gradient across its membrane to generate a proton motive force, and hence produce energy. One study found that E. coli expresses carboxylase enzymes, especially at low pHs [21]. The function of the enzymes is to increase the external pH, and contribute to induced acid tolerance in some situations [39]. Lysine decarboxylase is an amino acid protein that contributes to control the pH by converting exogenously supplied lysine to cadaverine, an alkaline product that neutralizes the acidic environment when secreted from the cell [29, 39]. We did not detected lysine in this study;
however, it was found in previous work where UPLC-Q-TOF-MS was used for identification. Tipthara et al reported that lysine acetate was found in a supernatant of an anaerobic reactor after being fed glucose [40].
Figure 8 shows the chromatograms of N-butyl-benzenesulfonamide and cyclooctasulfur. N-butylbenzenesulfonamide, a type of plasticiser was detected at very high concentrations; this compound has also been found in a batch anaerobic stirred tank reactor [2], in a landfill leachate [41], and also in SAMBRs operating at low temperature (20°C) [42] and low HRT (1 h) [23]. Elleuch et al. [43] found that Streptomyces sp. TN262 can produce this compound as a metabolite during large scale fermentation. Cyclooctasulfur, which is a central intermediate in the biotic or abiotic oxidation of sulfides [44], was also found in extremely high concentrations during the pH shocks, and a similar trend was also found in a SAMBR under high transient load [23]. The cause of an increase in this compound is still unclear at present, but may be due to cell leakage of intermediate metabolites. However, cyclooctasulfur was found to be degraded by deltaproteobacteria such as Desulfobulbaceae and Desulfuromonadales [45].
Concentrations of the compounds identified were estimated using a series of alkane standards. Figure 9 shows the rejection of low MW compounds in a SAMBR under different pH feeds; the results show that the membrane-fouling layer was very effective in COD removal, and could remove 84% of the low MW compounds from the SAMBR supernatant. Concentrations of low MW compounds increased after the feed pH decreased to 5 (pH 6.2 in the reactor); however, the removal rate also increased to 94%. In contrast, percentage removal decreased greatly to only 50% when the SAMBR was fed with pH 11 feed (pH 9.1 in the reactor). This may indicate that the membranefouling layer can remove/reject the compounds by “charge” and/or “concentration polarisation”. An interesting study looked at the rejection of pharmaceuticals in nanofiltration membrane filtration;
Botton et al. reported that the presence of the negatively charged biofouling induced accumulation of positively charged pharmaceuticals within the fouling layer, which probably also hindered back diffusion [46]. This caused a reduction in rejection efficiency of positively charged solutes but did not affect to rejection of neutral and negatively charged pharmaceuticals. Another study on the rejection of pharmaceuticals showed that negatively charged colloids enhanced the concentration polarization and charge effects on the membrane surface, and increased the rejection of some negatively charged pharmaceuticals by 15%. However, the charged colloids also reduced the rejection of some positively charged pharmaceuticals by more than 20% [47]. Chang et al. found that the characteristics of the membrane played an important role in solute rejection; high solute removals were attributed to the adsorption and/or sieving onto the cakes. Consequently, the difference in solute rejection efficiency between hydrophilic and hydrophobic membranes was mainly due to the degree of sieving and/or adsorption onto the cakes deposited on the membrane, and only partly due to adsorption in membrane pores and the surfaces [48]. However, most of these studies have been done on surface water for water treatment, and a further study on the rejection of various types of charged SMP solutes by the fouling layer in a SAMBR would be a very interesting approach to understand how different types of SMPs are rejected.
4. CONCLUSIONS The results showed that pH shocks (pH 5 and pH 11) had a significant effect on SAMBR performance. Changes in pH inside the SAMBR caused disruption of cellular metabolism and/or enhanced cell lysis. However, the system recovered very quickly within 24 h (pH 5 shock) and 8 h (pH 11 shock). Carbohydrates (30 k – 200 kDa) were found at higher concentrations under the pH 5 shock, while larger MW (1,500 kDa – 0.2 μm) protein-like compounds were found under the pH 11 shock. pH shocks had varying effects on membrane fouling by colloids, which may be due to
“charge” and “concentration polarization” effects on the membrane fouling layer . Larger colloids (1-5 μm), which are related to protein-like compounds, caused more fouling on the membrane than smaller ones. More than 50 low MW compounds were identified in the effluent samples. Alkanes, alkenes, esters, alcohols, phenols, nitrogen-compounds and sulfur compounds were the major groups of compounds identified in both the effluent and supernatant samples. The membranefouling layer (dynamic membrane) is an important factor in the removal of low MW compounds in the SAMBR, and pH changes inside the SAMBR had a significant effect on effluent quality.
ACKNOWLEDGEMENT This research work was supported by the Singapore National Research Foundation under its Environmental & Water Technologies Strategic Research Programme, and administered by the Environment & Water Industry Programme Office (EWI) of the PUB.
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Figure 1
Gas$sampling$bag$ Gas$out$
Pressure$$ Recycle$$ sensor$ pump$
Effluent$$ pump$
pH&ORP$meter$
Recycle$line$
Gas$out$for$recircula8on$
Feed$
Baffle$
Effluent$
Membrane$ module$
Flow$
Feed$pump$
540 mm
Gas$flow$$ meter$
Gas$pump$ Spacing between membrane and the wall is 5 mm
Feed$tank$
Gas$in$
32 mm
Figure 1. Diagram of the SAMBR.
Effluent$tank$
Figure 2
pH 5
8.0 7.5
45
CODeff
40
Supernatant 10
6.5
25 20
6.0
15 10
5.5
VFAs (mg/L)
30
COD (mg/L)
35
7.0 pH
12
50 pH
Effluent
8 6 4 2
5 5.0
0 0
5
10
15
20
25
0
ND
ND
ND
Time (h)
Figure 2. Effluent COD, pH and VFA concentrations during pH 5 shock in the feed.
Figure 3
pH 11
500
9.5
pH
9.0
pH
450
CODeff
400
VFA
350
8.5
300
8.0
250
7.5
200
COD (mg/L)
10.0
150
7.0
100
6.5
50
6.0
0 0
5
10
15
20
25
30
35
40
45
h Time (h)
Figure 3. Effluent COD, pH and VFA concentrations during pH 11 shock in the feed.
Figure 4
1,500k 20000
130k
30k
a) 210 nm
pH7 pH5
15000
uV
pH11 10000
5000
0 2500 5
b) 280 nm
10 pH 11
15 Time (min)
20
25
20
25
2000 1500
uV
pH 7 1000 pH 5 500 0 1200 5
10
c) RID
15 Time (min)
1000
uV
800
pH 5
600 pH 11
400
pH 7
200 0 5
10
15 Time (min)
20
25
Figure 4. Size Exclusion Chromatography of the SAMBR reactor supernatant during changes in feed pH.
Figure 5
a) pH5 450 400
140
4h
120
,5
300
TN (mg/L)
TOC (mg/L)
350
160
0h
250 200 150
80 60 40
50
20 1-5um 5um
0.2-1um 1um
100kDa 0.2um -0.2um
4h
100
100 0
0h
0 100k <100kDa
5um 1-5um
1um 0.2-1um
100kDa 0.2um -0.2um
100k <100kDa
1um 0.2-1um
100kDa 0.2um -0.2um
100k <100kDa
b) pH11 160
350 300
140
244hh
120 TN (mg/L)
TOC (mg/L)
250
0 h0 h
200 150
80 60 40
50
20 5um 1-5um
1um 0.2-1um
100kDa 0.2um -0.2um
4 h 24 h
100
100
0
0 h0 h
0 100k <100kDa
5um 1-5um
Figure 5. TOC and TN concentrations of supernatant inside the SAMBR under feed pH5 (a) and pH11 (b).
Figure 6
30 Blank < 100kDa
25
100kDa - 0.2um 0.2um - 1um
20
TMP (kPa)
1um - 5um 15
10
5
0 0
500 1000 Time (min)
0
500 1000 Time (min)
0
500
1000
Time (min)
Figure 6. Flux and TMP experiment on the membrane fouling layers under different pHs.
Figure 7
Alkane
pH11
Alkene Ester Alcohol
pH5
Others Unknown N
pH7
Phenol 0%
20%
40%
60%
80%
100%
Figure 7. Identified compounds in the effluent of a SAMBR under different pH feeds.
Figure 8
Intensity
500000 450000 400000 350000 300000 250000 200000 150000 100000 50000 0
0h 4h 8h
Cyclooctasulfur
N-butyl benzenesulfonamide
28
29
30
31 Time (min)
32
33
34
35
Figure 8. Chromatogram of the low MW compounds (GC-MS) in the reactor supernatant before and after a change in the pH of the feed.
Figure 9
0.40 Supernatant
Effluent
0.35
Concentration (mg/L)
0.30 0.25
50%
0.20 94%
0.15 84%
0.10 0.05 0.00 pH7
pH5
pH11
Figure 9. Concentration of low MW SMP (MW < 580 Da) and percentage removals of supernatants and effluents under different feed pHs.
Highlights
pH shocks (5 and 11) over 4 and 24 h had a significant effect on SAMBR performance
SAMBR recovered very quickly from pH shocks (5, 11) within 24 and 8 h, respectively
shocks had varying effects on fouling by 1-5 μm colloids due to charge/polarization
membrane fouling layer (dynamic membrane) important in removing of low MW compounds