Identification of recalcitrant compounds in a pilot-scale AB system: an adsorption (A) stage followed by a biological (B) stage to treat municipal wastewater

This manuscript presents a comparison of the A-stage and B-stage sludges in terms of anaerobic biodegradability and low molecular weight compounds present in the supernatant using Gas Chromatography–Mass Spectrometry (GC–MS). The GC–MS analysis of A-stage and B-stage supernatants identified respectively 43 and 19 organic compounds consisting mainly of aromatics (27.9% and 21%), alcohols (25.6% and 15%) and acids (30.2% and 15%). The methane potential was found to be 349 ± 1 mL CH4/g VS and 238 ± 12 mL CH4/g VS, respectively. After anaerobic digestion of these sludges, a greater proportion of aromatics (42% and 58%) and a lower proportion of acids (10% and 10%) and alcohols (16% and 10%) was observed

The production and biotransformational changes of soluble microbial products (SMPs) and its effects on anaerobic wastewater treatment

Effluent quality has been an important factor in the regulatory requirement for discharge of effluent from wastewater treatment plants. Characterization of effluents from both aerobic and anaerobic processes has shown that soluble microbial products (SMP) produced by microorganisms during biological treatment constitute the major proportion of the residual chemical oxygen demand (COD), and this can be up to 100%. Over the past twenty years, advancements in SMP analysis have allowed for their chemical characterization with increasing specificity, however, these advances are still not widely used in the study of anaerobic wastewater treatment systems. Hence, this study aims to examine: the biotransformational changes in soluble microbial products (SMPs), and the trends in their formation and disappearance during anaerobic wastewater treatment; the effects of feed macronutrients on the SMPs produced and their effects on membrane fouling, and; finally, evaluating SMP production in full- scale wastewater treatment plants. An anaerobic baffled reactor (ABR) was used because this type of reactor “splits” the biological reactions (trophic groups) into their constituent parts, as opposed to a continuously stirred tank reactor (CSTR), and hence enables us to monitor the production and degradation/ biotransformation of the SMPs down the reactor over time. The samples collected were extracted by sequential solid phase extraction (SPE) and liquid-liquid extraction (LLE), followed by untargeted analysis using gas chromatography coupled mass spectrometry (GC-MS) and liquid chromatography coupled tandem quadruopole time-of-flight (LC-Q-ToF). The first part of the study on the biotransformational changes of SMPs found the net production of alkanes and alkenes during the pseudo steady-state operation of an anaerobic baffled reactor set at a 24-hour hydraulic retention time (HRT). Previous work reported that the ‘recalcitrant’ phthalate esters are degradable, and a literature search found earlier studies proving that they are products of marine algae. This again showed that such ‘recalcitrant’ microbial products are degradable but are dependent on reactor time (HRT) and conditions (solids retention time-SRT). In terms of the impact of the change in carbon and nitrogen source (macronutrients) in the bioreactor feed on the production of SMPs, the results obtained do not show any obvious changes in the physical parameters routinely monitored for reactor performance (such as COD removal, and volatile fatty acid - VFA production). However, the chemical analysis using chromatographic methods coupled to mass spectrometry revealed underlying biochemical changes over a period of 24 hours after the change in the macronutrients in the feed. One significant finding is that a switch from an organic to an inorganic nitrogen source induced an inhibitory effect on glucose metabolism shown by an increase in the quantity of aliphatic aldehydes produced, which are reaction intermediates of the glucose metabolic pathway, and the ‘disappearance’ of alkanes. A considerable amount of work in the past which has examined fouling in membrane bioreactors were conducted in aerobic systems, but limited information has been available on anaerobic systems. The foulants were characterised in the past using liquid chromatography coupled oraganic carbon detection and organic nitrogen detection (LC-OCD-OND) and excitation- emission matrix (EEM), and these only showed the presence of polysaccharides and proteins. This lack of information led to the detailed chemical characterization of membrane foulants in this study, which has not been reported before. The untargeted scanning using LC-Q-ToF of the extracted membrane foulants found that the compounds detected were in the range of 200 – 799 Da, other than colloids and larger sized particles. A majority of compounds in the foulant were identified as fatty acids and their conjugates, and steroids and their derivatives. Finally, we reported on the characterisation of SMPs in the samples collected at various points of the full-scale industrial wastewater treatment plant, which is an area with limited information in the literature. The chemical characterisation showed some degree of similarity to the SMPs detected during the transient states of the lab-scale bioreactor operating on synthetic feed, while there was a small number of similar compounds primarily comprised of long- chain alkanes and phthalates.Doctor of Philosoph

Micro-level evaluation of organic compounds transformation in anaerobic digestion under feast and famine conditions assisted by iron-based materials - revealing the true mechanism of AD enhancement

Conductive materials have been applied to assist syntrophic metabolism in anaerobic digestion. However, their role in the transformation of organic compounds, particularly recalcitrant compounds, has not been revealed. In this study, iron-based materials - magnetite nanoparticles and Fe2+- were employed to explore their effects on the transformation of different organic matters in anaerobic system. Prompted methane production rates and quantity in iron-based materials groups were found due to the improved solubilization of organic particles, enhanced degradation of recalcitrant compounds, and maintained microbial activity under substrate-limited conditions. Specifically, the proportion of the reducing functional groups (C-C/H or CC) and O/C ratio were always significantly lower in iron-based materials supplemented groups (Fe groups) compared to Control group, despite hydrolysis was greatly enhanced in Fe groups. The greater dehydrogenation oxidation was confirmed in the presence of iron-based materials. The remaining humic-like substances (HS), a typical type of recalcitrant compound, was about 2.5 times higher in Control group (221.2 ± 5.3 mg/L-C) compared to Fe groups after 30 days degradation. By tracking the aromaticity of HS and individual compounds at molecular level, this study reveals that iron-based materials were more effective in stimulating the degradation of aliphatic moieties than the aromatic moieties of recalcitrant compounds. When readily biodegradable substrates were limited, Fe groups continued methane generation by using recalcitrant compounds (e.g. thiethylperazine and fluvoxamino acid) as carbon source, and the microbial activity was maintained according to higher relative abundance of protonated nitrogen and continuous methanogenesis activity at starvation phase.Published versio

Soluble microbial products (SMPs) in the effluent from a submerged anaerobic membrane bioreactor (SAMBR) under different HRTs and transient loading conditions

This study investigated the performance of a submerged anaerobic membrane bioreactor (SAMBR) fed with synthetic wastewater (544 ± 22 mgCOD/L) operating at different hydraulic retention times (HRTs-12 h, 8 h, 6 h, 4 h, 2 h, and 1 h) at both steady state, and under transient load conditions (2 and 1 h), and the SMPs produced under these conditions. COD removal at decreasing HRTs (12 h, 8 h, 6 h, 4 h, and 2 h) was high (>94%), but decreased to 80% when operating at 1 h HRT. VFAs accumulated when the HRT was decreased to 2 h and 1 h, accounting for 69% and 89% of the effluent COD, respectively. Effluent SMPs accounted for an average of 14 ± 2 mgCOD/L at steady state, but this fluctuated more during transient conditions (12 ± 6 mgCOD/L). The COD equivalent of dissolved methane in the effluent was 17% at 4 h HRT, exceeding the saturation value of methane. Low MW compounds were identified using gas chromatography–mass spectrometry (GC–MS), with solid phase extraction (SPE) as the pre-treatment. 120 compounds were identified in the effluent at steady state, and were alkanes (39), alkenes (3), esters (11), alcohols (7), nitrogenated compounds (11), phenols (11), and others (9). Increases in cyclooctasulfur, N-butyl-benzenesulfonamide, alkanes, 1-naphthalenol, camphor, 2-methylphenol, and (Z)-9-octadecenamide were also found during transient conditions, and these compounds were not found in the feed; hence it is possible that these compounds were produced by microorganism as by-products from substrate utilization

Identification of soluble microbial products (SMPs) from the fermentation and methanogenic phases of anaerobic digestion

The production and transformation of Soluble Microbial Products (SMPs) in biological treatment systems is complex, and their genesis and reasons for production are still unclear. SMPs are important since they constitute the main fraction of effluent COD (both aerobic and anaerobic), and hence are the main precursors for disinfection by-products (DBPs). In addition, they are a key component of fouling in membrane bioreactors. Hence, it is important to identify the chemical composition of SMPs, determine their origin, and understand what system parameters influence their production so we can possibly develop strategies to control their production. This study focuses on the production and identification of SMPs in an anaerobic batch process being fed a synthetic feed. To further understand the origins of SMPs, and how they are produced, we analysed the processes of fermentation and methanogenesis independently which has never been done in detail before. SMP concentration, molecular weight distribution and carbohydrate analyses were used to estimate the amount of SMPs in the supernatants. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-Time-of-Flight mass spectrometry (LC-ESI-Q-ToF) were used to identify many of the SMPs which have relative masses up to 2 kDa. Our results showed that fermentation released much higher SMP concentrations compared to methanogenesis, especially in the range of 70 k-1000 k Da and 106-1500 Da. Alkanes, alkenes, alcohols, acids, and nitrogen-compounds were the major group of compounds identified in the supernatant of both fermentation and methanogenesis, and 71% of the compounds identified were found in both phases of digestion. Results from LC-ESI-Q-ToF analysis identified components of the cell membrane, such as phosphatidylglycerol, phosphatidylethanolamine and phosphatidylserine, as well as other compounds such as flavonoids, acylglycerol, terpene and terpenoids, benzenoid, glyceride, steroid and steroid derivatives.National Research Foundation (NRF)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

Transformation of dissolved organic matters produced from alkaline-ultrasonic sludge pretreatment in anaerobic digestion : from macro to micro

Soluble organic compounds released by alkaline (ALK), ultrasonic (ULS) and combined alkaline-ultrasonic (ALK-ULS) pretreatment as well as their transformation in the anaerobic digestion systems were investigated. The maximum methane production of 197.1 ± 3.0 mL CH4/g tCODfeed was observed with ALK-ULS pretreated sludge (pH 12 and specific energy input of 24 kJ/g TS). The combined treatment likely enhanced the sludge solubilization and produced more low molecular weight (LMW) substances, which were beneficial to improve the biogas generation rate. However, such pretreatment released not only easily biodegradable substances but also more recalcitrants, such as humic substances (HS) and complex high molecular weight (HMW) proteins. Thus, more residual dissolved organic matters (DOMs) were detected after digestion, which may pose adverse effects on the downstream water treatment. Refractory HS and hydrophobic dissolved organic carbon (HO DOC) were the main components of the residual DOMs, which accounted up to 35.0% and 22.3% respectively. At the molecular level, a large amount of residual polycyclic steroid-like matters, alkanes and aromatics were identified. Specific higher MW residual compounds, e.g. polar metabolites (like dipeptide, benzene and substituted derivatives), and non-polar lipids (like diacylglycerols, long chain fatty acids, alkenes, flavonoids, sphingolipids, glycerolipids, glycerophospholipids and their derivatives) were also identified. The results indicate that further polishing steps should be considered to remove the remaining soluble recalcitrant compounds. This study helps to understand the insight of sludge treatment from macro to micro level.Accepted versio

Biotransformation of phosphorus in enhanced biological phosphorus removal sludge biochar

Biochar derived from enhanced biological phosphorus removal (EBPR) sludge could be a potential phosphorus (P) fertilizer. Soil microorganisms play a regulating role on the turnover of P in soil. When the EBPR sludge biochar is added to soil, it would inevitably interact with soil microorganisms. Thus, for the wise use of the EBPR sludge biochar, it is imperative to understand the interaction between the biochar and soil microorganisms. In this study, Pseudomonas putida (P. putida), a common soil microorganism, was applied to investigate the biotransformation of P in two EBPR sludge biochars. The results reveal that P released from biochar produced at 700 °C (E700) was more easily absorbed by P. putida than that released from biochar produced at 400 °C (E400). This is attributed to the higher polyphosphates (poly-P) content in E700 and poly-P has higher affinity to P. putida surface compared to orthophosphates. Furthermore, E400 has a negative effect on intracellular poly-P formation in P. putida, which is probably caused by the oxidative stress induced by the free radicals from E400. As intracellular poly-P plays a critical role on bacteria survival and their interaction with surrounding environment, high-temperature biochar (E700) in this case would be more suitable for soil remediation.Ministry of Education (MOE)This research was supported by Ministry of Education (MoE) Singapore under Tier 1 project Conductive biosystem - Enhanced biodegradation of recalcitrant compounds in industrial wastewater

Biotransformation of Soluble Microbial Products (SMPs) in an Anaerobic Baffled Reactor (ABR)

The change in SMP composition of an ABR operating at 24-hour HRT treating synthetic wastewater of medium-strength glucose feed was analysed using SPE-GC-MS. The results showed that alkanes and alkenes generally saw an increase down the compartments of the ABR. The number of esters identified on the first three sampling times when glucose feed was 2 gCOD/L, were only different by 2-3 compounds. On day 96 when the ABR was approaching a pseudo steady state, the number of detected ester compounds increased, with 8 out of 11 esters increasing in concentration by 5-6 times. A 1.5-3-fold increase in the number of alkane, alkene, ester, acid, aromatic and nitrogenous compounds, and other unidentified compounds, were observed when the feed concentration doubled. The possible reasons for the formation of the compounds is discussed, using established pathways from the well-researched areas in natural product synthesis and metabolism in plants, fungi, algae, and pure strains of microorganisms.Accepted versio

Lipopolysaccharide neutralizing peptide–porphyrin conjugates for effective photoinactivation and intracellular imaging of gram-negative bacteria strains

A simple and specific strategy based on the bioconjugation of a photosensitizer protophophyrin IX (PpIX) with a lipopolysaccharide (LPS) binding antimicrobial peptide YI13WF (YVLWKRKRKFCFI-Amide) has been developed for the effective fluorescent imaging and photodynamic inactivation of Gram-negative bacterial strains. The intracellular fluorescent imaging and photodynamic antimicrobial chemotherapy (PACT) studies supported our hypothesis that the PpIX-YI13WF conjugates could serve as efficient probes to image the bacterial strains and meanwhile indicated the potent activities against Gram-negative bacterial pathogens especially for those with antibiotics resistance when exposed to the white light irradiation. Compared to the monomeric PpIX-YI13WF conjugate, the dimeric conjugate indicated the stronger fluorescent imaging signals and higher photoinactivation toward the Gram-negative bacterial pathogens throughout the whole concentration range. In addition, the photodynamic bacterial inactivation also demonstrated more potent activity than the minimum inhibitory concentration (MIC) values of dimeric PpIX-YI13WF conjugate itself observed for E. coli DH5a (4 times), S. enterica (8 times), and other Gram-negative strains including antibiotic-resistant E. coli BL21 (8 times) and K. pneumoniae (16 times). Moreover, both fluorescent imaging and photoinactivation measurements also demonstrated that the dimeric PpIX-YI13WF conjugate could selectively recognize bacterial strains over mammalian cells and generate less photo damage to mammalian cells. We believed that the enhanced fluorescence and bacterial inactivation were probably attributed to the higher binding affinity between dimeric photosensitizer peptide conjugate and LPS components on the surface of bacterial strains, which were the results of efficient multivalent interactions

Chemical Characterization of Low Molecular Weight Soluble Microbial Products in an Anaerobic Membrane Bioreactor

Effluents from wastewater treatment systems contain a variety of organic compounds, including end products from the degradation of influent substrates, nonbiodegradable feed compounds, and soluble microbial products (SMPs) produced by microbial metabolism. It is important to identify the major components of these SMPs to understand what is in wastewater effluents. In this study, physical pretreatments to extract and concentrate low molecular weight SMPs (MW< 580 Da) from effluents were optimized. Liquid–liquid extraction (LLE) of a 200 mL effluent sample showed the best performance using a mixture of <i>n</i>-hexane, chloroform, and dichloromethane (70 mL) for extraction. For solid phase extraction (SPE), two OasisHLB cartridges were connected in-line to optimize recovery, and the eluted samples from each cartridge were analyzed separately to avoid overlapping peaks. Four solvents varying from polar to nonpolar (methanol, acetone, dichloromethane, and <i>n</i>-hexane) were selected to maximize the number of compound peaks eluted. A combination of SPE (OasisHLB) followed by LLE was shown to maximize compound identification and quantification. However, the compounds identified accounted for only 2.1 mg of chemical oxygen demand (COD)/L (16% of total SMP as COD) because many SMPs have considerably higher MWs. Finally, the method was validated by analyzing a variety of different reactor effluents and feeds