Complex organic particulate artificial sewage (COPAS) as surrogate wastewater in anaerobic assays

Ana L. Prieto *a, Craig S. Criddle b and Daniel H. Yeh c
aDepartamento de Ingeniería Civil, Universidad de Chile, Av. Blanco Encalada 2002, 3er piso, Santiago, Chile. E-mail:
bEnvironmental Engineering and Science Program, Department of Civil and Environmental Engineering, Stanford University, 473 Via Ortega, Room 151, Stanford, CA 94305, USA
cDepartment of Civil and Environmental Engineering, University of South Florida, 4202 East Fowler Ave. ENB 118, Tampa, FL 33620, USA

Received 1st May 2019 , Accepted 13th August 2019

First published on 23rd August 2019

Storage, preservation, and batch-to-batch variability of the influent composition are challenges to laboratory-scale research in the wastewater treatment field. Synthetic wastewaters are commonly used, but many fail to capture the complexity of actual wastewater, especially in terms of particulate organic matter. An alternative synthetic sewage, referred to as complex organic particulate artificial sewage (COPAS), is introduced in this study. COPAS is easily prepared and is based on a simple recipe that uses granular dried cat food as the source of particulate organic matter. On a weight basis, COPAS particles consist of proteins (40%), fats (17%), carbohydrates (43%), and trace nutrients, including vitamins and trace metals. Dissolution/hydrolysis batch tests of COPAS particles indicate that the dissolved organic carbon and nitrogen are released rapidly within the first two hours (approximately 10% C and 17% N). The remaining fraction of organic C and N remain in the particulate form for further dissolution and/or biodegradation. A khyd of 0.82 d−1 was calculated for COPAS based on its protein, fat, and carbohydrate contents. Further, anaerobic bioassays prove the biodegradability of COPAS with approximately 60% theoretical methane produced after 45 days of incubation.

Water impact

This investigation will benefit society by providing a simple and readily available substrate to use either in the lab or in industrial scale applications. Using complex organic particulate artificial sewage (COPAS) can decrease the start-up time of the wastewater treatment process by providing a suitable surrogate to actual wastewater, which can decrease wastewater treatment costs and energy use in centralized and decentralized applications. COPAS can be used to maintain viable sludge during temporary shutdowns of aerobic and anaerobic bioreactors. Reduction of chemical input and reduction of overall carbon footprint of the wastewater treatment process are potential outcomes from the application of COPAS in wastewater treatment.


Bench-scale wastewater treatment research is routinely conducted to provide insight into the physical, chemical, and biological processes that are operative in systems at a large scale or to test and develop new processes. Ideally, fresh sewage should be used for these studies. Important parameters such as CODt (200–1650 mg L−1), BOD5 (135–242 mg L−1), TN (18–180 mg N per L), TP (2–31 mg P per L), SS (40–440 mg L−1), and pH (6.9–8.3) can vary depending on the source. Table 1, for example, presents a summary of domestic wastewaters characterized for different treatment applications. Routine accessibility to sewage, its inherent variability, storage problems, health concerns, and batch-to-batch variability are problematic for laboratory-scale investigations. To overcome this problem, lab-scale research has often made use of synthetic wastewater.1,2 Ideally, synthetic sewage should mimic real domestic wastewater, but no standardized recipe is available. Instead, researchers have used a range of ingredients, including K2HPO4, MgSO4, urea, various food additives (e.g. starch, soy oil, beef extract, etc.), and animal feed (e.g. canned dog food). Table 2 summarizes the compositions, concentrations and specific applications of some synthetic sewage preparation methods used to date. Although some of these formulas aim to mimic typical domestic sewage, such as SYNTHO and SYNTHES,1–3 others are customized to the specific process under study.4,5 In other studies, the recipe includes expensive chemicals, and many involve the preparation of concentrated stock solutions that are refrigerated or acidified,6 which raises concerns about possible changes in composition during prolonged storage. Concentrations are then adjusted to achieve a desired chemical oxygen demand (COD) and nutrient concentration (especially nitrogen and phosphorous).7–11 An additional concern is the use of readily biodegradable and completely soluble carbon sources, such as glucose or acetate, as the sole source of COD. Furthermore, the absence of particulate matter fails to simulate the complexity of actual sewage. While slowly degradable constituents, such as starch, may be added to simulate particulates, such mono-component particles do not simulate the complex, heterogeneous particulates found in domestic sewage.
Table 1 Examples of domestic wastewater compositions used for treatment applications
Application Water quality parameters of raw water Source
Evaluation of protein, carbohydrate and lipid contents in domestic wastewater by using the Lowry, phenol and anthrone, and infrared lipid methods Percentages of total COD in the influent: proteins: 28 ± 4%; carbohydrates: 18 ± 6%; lipids: 31 ± 10%; other organics: 23%; VFA within other organics <1% 12
Comparison among CAS, Behrtest KLD4® and CAS-UCT for biological nutrient removal. Duffel WWTP (Belgium) CODt: 400 ± 200 mg L−1; TN: 25 ± 7 mg L−1; TP: 7 ± 3 mg L−1; pH: 6.9–7.5 13
Anaerobic digestion of domestic sewage and black water CODt: 634 mg L−1 and CODs: 217 mg L−1 14
Application of Moringa oleifera to a UASB reactor for process enhancement. Ossemeersen WTP (Ghent, Belgium) CODt: 320 ± 58 mg L−1; CODs: 140 ± 35 mg L−1; SS: 165 ± 41 mg L−1; VSS: 132 ± 22 mg L−1; TKN: 33 ± 12 mg L−1; NH4-N: 23 ± 9 mg L−1; TP: 10 ± 1 mg L−1; Alk: 412 ± 45 mg CaCO3 L−1; pH: 7.7 ± 0.2 15 and 16
Biodegradation of settleable organic matter based on interpretation of hydrolysis rate in an aerated batch reactor. Ataköy WTP (Istanbul, Turkey) CODt: 425 mg L−1; CODs: 120 mg L−1; SS: 240 mg L−1; VSS: 150 mg L−1 17
Application of UASB for treating domestic sewage at moderate temperatures based on COD removal efficiencies (Salta, Argentina) CODt: 224.2 ± 10.1 mg L−1 and CODs: 65.4 ± 5.5 mg L−1 18
Performance of a pilot-scale treatment wetland for low-cost domestic wastewater treatment (Santa Maria Nativitas, Mexico) CODt: 1569.2 ± 81.2 mg L−1; TN: 164.9 ± 14.3 mg L−1; NH4-N: 66.3 ± 4.5 mg L−1; NO3: 28.4 ± 7.3 mg L−1; DO: 1.9 ± 0.2 mg L−1; TSS: 406.1 ± 33.4 mg L−1; pH: 8.2 ± 0.1 19
Evaluation of wastewater characteristics and treatment of domestic sewage in tropical monsoon areas. Ruamrudee sewer pipe (Bangkok, Thailand) BOD5: 241.5 mg L−1; CODt: 320.6 mg L−1; TN: 42.4 mg L−1; NO3: 0.9 mg L−1 20
Evaluation of the performance of a DHS system for treating UASB effluent (Japan) BODt: 162 ± 37 mg L−1; BODs: 78 ± 19 mg L−1; CODt: 373 ± 83 mg L−1; CODs: 168 ± 38 mg L−1; SS: 134 ± 48 mg L−1; TN: 61 ± 11 mg L−1; NH4-N: 33 ± 6 mg L−1; pH: 7.3 21
Performance of submerged NF MBR for treating domestic wastewater (Tokyo Bay, Japan) TOC: 35.3–91.2 mg L−1; SS: 40–180 mg L−1; DO: 6.07–7.59 mg L−1; TN (dissolved): 7.2–31.9 mg N L−1; TP: 2.27–31.1 mg P L−1; pH: 7.27–7.85 22
Characterization of domestic wastewater and treatability approach. Beishiqiao Wastewater Purification Center (Xi'an, China) CODt: 257.8 mg L−1; BOD5: 134.7 mg L−1; SS: 162.3 mg L−1; TN: 38.8 mg L−1; NH3-N: 26.2 mg L−1; NO3-N: 0.48 mg L−1; TP: 8.16 mg L−1; pH: 7.6 23

Table 2 Examples of synthetic wastewaters used in the literature
Application Composition Water quality parameters Source
Development of a risk assessment tool for chemical fate prediction in aquatic environments Syntho (precursor): urea, ammonium chloride, uric acid, sodium acetate, dried yeast, lauric acid, diet fiber, LAS, AE, meat extract, peptone, potato starch, low fat milk powder, mineral salts and trace elements CODt: 390 mg L−1; TN: 34.6 mg L−1; TP: 7.9 mg L−1; pH: 7.25 1
Evolution of the sludge bed sedimentology for UASB SYNTHES: urea, NH4Cl, Na-acetate, peptone, MgHPO4·3H2O, KHPO4, FeSO4·7H2O, CaCl2, starch, milk powder, dried yeast, soy oil, trace metals CODt: 500 ± 50 mg L−1; CODs: 170 ± 40 mg L−1; TKN: 49 ± 8 mg L−1; NH4-N: 27 ± 7 mg L−1; PO4-P: 21 ± 2 mg L−1; SS: 200 ± 50 mg L−1; COD/N/P ratio: 30/3/1 2
Performance evaluation of BASR for wastewater treatment applications C2H5OH, K2HPO4, MgSO4, NH4Cl and trace elements SCOD: 298–694 mg L−1 5
COD/NH4-N: 6/1
COD/P: 78/1
Evaluation of the reliability of synthetic wastewater for breeding stable activated sludge in an SBR SYNTHO (modified): urea, NH4Cl, Na-acetate, peptone, MgHPO4·3H2O, KHPO4, FeSO4·7H2O, starch, milk powder, yeast, soy oil, trace metals COD: 439.47 mg L−1, N: 60.23 mg L−1 and P: 9.42 mg L−1 6
Performance of UASB and EGSB reactors for low strength wastewater treatment Ethanol or whey Whey CODt: 113–630 mg L−1; ethanol CODt: 146–722 mg L−1 7
Performance of an SMBR for the treatment of highly concentrated ammonia influent NH4HCO3, K2HPO4, MgSO4·7H2O, MnSO4·4H2O, FeCl3·6H2O and NaCl NH4-N: 180–1300 mg L−1 8
Comparison among CAS, Behrtest KLD4® and CAS-UCT for biological nutrient removal BSR3 and SYNTHO: urea, ammonium chloride, uric acid, dried yeast, lauric acid, sodium acetate, diet fiber, LAS, AE, meat extract, peptone, starch, low fat milk powder, mineral salts and trace elements BSR3 (Syntho precursor) CODt: 390 mg L−1, TN: 34.6 mg L−1 and TP: 7.9 mg L−1 13
SYNTHO CODt: 470 mg L−1, TN: 31.6 mg L−1 and TP: 8.3 mg L−1
Performance of an anaerobic–aerobic domestic sewage treatment system using UASB and SBR Meat extract, sucrose, starch, cellulose and vegetable oil CODt: 422 ± 68 mg L−1; CODs: 169 ± 45 mg L−1; BOD5: 257 ± 26 mg L−1; TSS: 246 ± 130 mg L−1; VSS: 158 ± 65 mg L−1; TKN: 57 ± 11 mg N L−1; NH4-N: 26 ± 7 mg L−1; Alk: 288 ± 85 mg CaCO3 L−1; pH: 7.0 ± 0.36 24
Evaluation of degradation kinetics and heat and mass transfer in an aerated static bed reactor Dry dog food and hard maple wood chips as bulking agent and carbon source Dog food, C: 44.6% and N: 5.3%; wood chips, C: 29.7% and N: 4.1% 25
Introduction of an improved option for synthetic sewage and potential applications for lab and pilot scale WWT SYNTHO CODt: 470 mg L−1, TN: 31.6 mg L−1 and TP: 8.3 mg L−1 26
Immobilization of sludge using PVA and performance evaluation Peptone, beef extract, NaCl, KCl, MgSO4·7H2O, Na2HPO4 and CaCl2·2H2O COD: 360 mg L−1, TKN: 48 mg L−1, BOD: 240 mg L−1 and TOC: 150 mg L−1 27
Evaluation of two-stage treatment configuration by comparison of CAS and MBR performance for reduced sludge production Skimmed milk powder and antifoam 100 g of dry skimmed milk powder contains: carbohydrates: 51.9 g; proteins: 35.5 g; lipids: 1 g; minerals: 7.8 g CODt: 360–1033 mg L−1; average C/N/P ratio: 100/8.3/4.0 28
Performance of ABR for treating complex (soluble and colloidal) dilute wastewaters Semi-skimmed milk (soluble feed), dry dog food and rice (colloidal feed >500 μm), and trace chemicals COD: 500 mg L−1 29
Evaluation of a novel biosensor for BOD measurement using HCF(III) as a mediator OECD synthetic sewage (also adapted by the EPA): peptone, meat extract, urea, NaCl, CaCl2·2H2O, MgSO4·7H2O and K2HPO4 BOD5 of solution: 14[thin space (1/6-em)]000 mg L−1 30–32
BOD values from 15 to 200 mg L−1 used to show sensor response
Evaluation of pH effect on anaerobic solubilization (hydrolysis/acidogenesis) of synthetic and domestic sludge Dry dog food Protein: 21%, fat: 8% and Fiber: 5% 33
VS: 90% of TS; VSS: 30[thin space (1/6-em)]000 mg L−1
Study of bacterial adaptation in aerobic MBR treatment of synthetic domestic wastewater Gelatin, starch, Tween-80, yeast extract, casamino acids, NH4Cl, NaHCO3, Na2HPO4, KH2PO4, MgCl2, CaCl2, and SL7 trace mineral solution 34
Performance of a microaerobic MBR and anaerobic granular sludge domestic wastewater treatment Sugar, potato starch, peptone, meat extract, urea, NH4Cl, KH2PO4, MgSO4·H2O, CaCl2·H2O, FeSO4·H2O and trace metals COD: 500 ± 46–214 ± 30 mg L−1 35
TN: 45.1 ± 2.2–18.9 ± 0.4 mg L−1
Performance of an AnMBr for dilute wastewater treatment Glucose, peptone, meat extract, urea, and NaHCO3 COD: 460 ± 20 mg L−1 36
Evaluation of alternating pumped sequencing batch biofilm reactor performance Glucose, yeast, extract, dried milk, NH4Cl, urea, Na2HPO4·12H2O, NaHCO3, MgSO4·7H2O, MnSO4·H2O, CaCl2·6H2O and KHCO3 CODt: 346 ± 32 mg L−1; CODs: 319 ± 25 mg L−1; TN: 33 ± 1.3 mg L−1; P: 18 ± 2.7 mg L−1 37
Evaluation of biofouling in attached and suspended growth media MBR Glucose, soy starch, NH4Cl, KH2PO4, CaCl2, MgSO4·7H2O, FeCl3 and NaHCO3 COD: 500 mg L−1 38
COD/N/P ratio: 100/10/2
Performance improvement of an aerobic MBR system by ozone gas backwashing as fouling control Glucose, poly-peptone, NH4Cl, CaCl2·2H2O, FeCl3·6H2O, MgSO4·7H2O, NaHCO3 and KH2PO4 BOD: 250 mg L−1, alkalinity: 270 mg CaCO3 L−1, NH4+-N: 22.3 mg N L−1, TN: 42.3 mg N L−1, TP: 6.8 mg P L−1 39
Evaluation of the degradation of non-ionic surfactants by activated sludge's bacterial community Peptone, yeast extract, urea, NaCl, CaCl2·2H2O, MgSO4·7H2O, K2HPO4, KH2PO4, and nonylphenol ethoxylates (NPEs) COD: 190 mg L−1 40
Determination of optimal carbon source in a SAAR/AR (sequencing anoxic/anaerobic and aerobic reactor) Acetate, propionate, glucose and methanol. Combinations of VFAs at different ratios. COD from 250 to 400, TN from 5.2 to 6.2 and TP from 9 to 41 41
Assessment of the fate of PPCPs in aerobic MBR treating domestic wastewater AcNa·3H2O, NH4Cl, Na2HPO4, KH2PO4, NaHCO3, and PPCPs 42

Standardization of synthetic sewage recipes is challenging, but the availability of an economical, consistent, and easy to obtain/preserve recipe would benefit laboratory research and facilitate comparison of results across laboratory studies. In the present study, another option for domestic synthetic sewage is examined. This recipe, hereafter referred to as complex organic particulate artificial sewage (COPAS), is based on the use of dried granular cat food. The use of a dry food source is advantageous because preservation steps (e.g., refrigeration and acidification) are not needed, and the amount of dry food used can be adjusted to match the TS and COD concentrations in domestic sewage. Using dry food avoids the use of complicated and concentrated chemical solutions, incorporates particulate material, and avoids loss of chemical integrity due to storage conditions. In particular, dried granular cat food is an inexpensive, commercially available product that does not require special preparation and can be tailored to the desired particle size by grinding the pellets. Compared to dog food, cat food kibbles are much easier to break and have more protein and fatty acids than dog food due to the dietary demands of the felines (Table 3).43 Cat food also includes minerals and trace elements that might be found in sewage. Although the commercially available recipes of cat food might vary from brand to brand, they must comply with the minimum dietary needs of felines as specified by the Association of American Feed Control Officials (AAFCO).43 To further assess the suitability of COPAS for lab-scale sewage treatment processes, we evaluated solution quality (COD, BOD5, TOC and TN) and its biodegradability under anaerobic conditions.

Table 3 AAFCO nutrient and mineral content requirements for dog and cat food. Minimum values for adult pet maintenance. Adapted from Dzanis, 1994 (ref. 43)
Feeding component Units, dry matter Dog food Cat food
Protein g kg−1 180 260
Fat g kg−1 50 90
Ca g kg−1 6 6
P g kg−1 5 5
K g kg−1 6 6
Na g kg−1 0.6 2
Chloride g kg−1 0.9 3
Mg g kg−1 0.4 0.4
Fe mg kg−1 80 80
Cu mg kg−1 7.3 5
Mn mg kg−1 5 7.5
Zn mg kg−1 120 75
I mg kg−1 1.5 0.35
Se mg kg−1 0.11 0.1

Materials and methods

COPAS characterization

The cat food used for this study was a commercial brand (Purina Friskies® Ocean Fish Flavour). To assess its nutritional composition, a sample of ground cat food (particle size: 0.472 to 1.7 mm) was sent to a registered animal feed analysis laboratory (Barrow-Agee Laboratories, LLC, Memphis, TN). The particle size was selected to represent the particulate fraction of sewage after preliminary treatment. Chemical characteristics of COPAS were calculated based on the dry weight of the COPAS sample. COD values were measured in triplicates using Hach HR COD digestion vials (Hach Company, Loveland, CO). The dissolved organic carbon (DOC) and dissolved nitrogen (DN) contents of liquid samples were measured in triplicates using a total organic carbon analyzer (Shimadzu TOC-V) coupled with a total nitrogen detector (Shimadzu TNM-1). A solid sample module (Shimadzu SSM-5000A) coupled to the TOC-V was used for measuring the particulate organic carbon (POC) in solid phase samples (Shimadzu, Columbia, MD). Biological oxygen demand (BOD) was measured in an external laboratory (Howard F. Curren Advanced Wastewater Treatment Plant (HFC AWTP), Environmental Laboratory, Tampa, FL) using Method 5210 for BOD5 and BOD20 as described in ALPHA 2008.44

Disintegration/dissolution tests

To evaluate the feasibility of COPAS as a readily available carbon and nitrogen source, a dissolution test was performed in a compact laboratory mixer unit (EC Engineering, Edmonton, Alberta, Canada) and the DOC and DN concentrations were monitored over time. For this test, two 1 liter batch reactors were filled with a COPAS solution of 500 mg L−1 (particle size between 0.425 to 1.7 mm). The reactors were mixed at controlled speeds of 20, 50 and 100 rpm. Triplicate samples were then filtered through a 0.45 μm SFCA syringe filter which had been pre-rinsed with Milli-Q water. The filtrate was acidified with 1 N HCl to a pH of 2 to 3 to avoid biological activity and to remove inorganic carbon. DOC and DN were measured using the Shimadzu TOC-V/TNM-1 analyzer.

Biodegradation assays

Anaerobic biodegradability was assessed in serum bottles seeded with anaerobic digester sludge from a local wastewater treatment plant (HFC AWTP, Tampa, FL.). The mean total solids (TSs), volatile fraction, total volatile fatty acids (VFAs), alkalinity, and pH for the seed sludge are 23 ± 1 g L−1, 72.8 ± 0.5%, 334 ± 101 mg L−1, 5648 ± 295 mg L−1, and 7.47 ± 0.05; respectively. For the serum bottles (clear 118 ml glass bottles, 60 ml liquid sample) (Fisher Scientific, Pittsburg, PA), the sludge was previously screened using a 1 mm wire mesh to remove grit, hair, and other things that might interfere in the biodegradability assay. The serum bottles were maintained at 37 °C and fed with COPAS at different solid concentrations (in triplicates) within a typical range for domestic wastewater (i.e., 500, 1000 and 2000 mg L−1). COD, VFAs, biogas production and POC were continuously measured in each bottle. The sludge samples were centrifuged at 3000 rpm for 20 minutes and the supernatant was used for dissolved COD and VFA measurements. Dissolved COD was measured using Hach HR COD digestion vials (Hach Company, Loveland, CO). Biogas volume was determined using a water displacement burette. Gas composition was analyzed for CO2 and CH4 using a gas chromatography (GC) unit (Agilent 7820A) equipped with a thermal conductivity detector (TCD) and a 30 m J&W 113-3133 GS-CarbonPLOT, 0.32 mm diameter, column (Agilent Technologies, Lexington, MA). The inlet, oven and detector temperatures were set at 185 °C, 50 °C, and 160 °C, respectively. Helium was used as a carrier gas at 1.3 mL min−1. VFAs (i.e. acetic, propionic, butyric and valeric acids) were monitored using a GC unit equipped with a flame ionization detector (FID) and a 30 m Restek 11025 Stabilwax DAm 0.53 mm ID column (Restek Corp., Bellefonte, PA). The inlet and detector temperatures were both set at 250 °C. Helium was used as a carrier gas at 4.5 mL min−1 and the following program was used for the oven temperature: 90 °C for 0.5 min, 2 °C min−1 to 100 °C, 6 °C min−1 to 120 °C, and 30 °C min−1 to 230 °C for 15 min. The total run time was 27.5 min.

Results and discussion

COPAS characterization

A single batch of COPAS can maintain its chemical composition during extended storage (room temperature of 25 °C to 28 °C). Lab analysis of two different lots of cat food purchased 1 year apart suggest that key characteristics of the stored feed (moisture, fat, protein, fiber, carbohydrates, ash, nitrogen and phosphorus) varied less than five percent (<5%) over time (data not shown). On a dry weight basis, the organic portion of the COPAS granules consists mainly of proteins (40%), carbohydrates (43%) and fats (17%). Other constituents such as metals are found in trace concentrations and are accounted in the ash fraction of COPAS particles. The measured values of carbon, nitrogen, and phosphorus were 52.54% C, 6.35% N and 1.57% P of the organic fraction. Based on the measured C, N, and P values and a COD/wt of 1.26, an empirical formula for COPAS was derived as C9.5H10.3O5.05N1P0.11. A common empirical formula for domestic wastewater organic matter is C10H18O3N, suggesting that sewage organic matter has 60% C, C[thin space (1/6-em)]:[thin space (1/6-em)]N of 8.6[thin space (1/6-em)]:[thin space (1/6-em)]1, COD/wt of 1.96, and COD/TOC of 3.3.45 Other authors have identified the empirical formula for sewage particulate organics as C3.5H7O2N0.196, corresponding to 50% C, C[thin space (1/6-em)]:[thin space (1/6-em)]N of 15.3[thin space (1/6-em)]:[thin space (1/6-em)]1, COD/wt of 1.57, and COD/TOC of 3.1.46 COPAS, on the other hand, has 52.5% C, C[thin space (1/6-em)]:[thin space (1/6-em)]N of 8.2[thin space (1/6-em)]:[thin space (1/6-em)]1, COD/wt ratio of 1.26 and COD/TOC of 2.4. Although the C[thin space (1/6-em)]:[thin space (1/6-em)]N and COD/wt ratios seem lower than those previously reported, COPAS fat, protein, and carbohydrate contents are comparable to typical values reported for sewage in the literature. According to Raunkjaer et al. (1994),12 sewage contains 40–50% carbohydrate, 40–60% protein, and ∼10% fat. COPAS organics contain 43% carbohydrate, 40% protein, and 17% fat (Table 4).
Table 4 COPAS composition
Characteristics Total weight Dry wt solids Organic solids
a BA lab results. b HFC lab results. c This study.
% of total weight 100% 97.35% 91.55%
Moisture (%) 2.65 n/a n/a
Ash (%) 8.23 8.45 n/a
Carbohydrates (%)a 38.27 39.31 42.94
Protein (%)a 35.35 36.31 39.67
Fat (%)a 15.50 15.92 17.39
Fiber (%) 2.40 2.47 2.69
Phosphorous (%) 1.40 1.44 1.57
TKN (%)b 4.53 4.65 5.08
Nitrogen (%)a 5.66 5.81 6.35
Nitrates (%) 0.00192 0.00197 0.00215
Nitrite (%) 0.00121 0.00124 0.00136
Urea (%) 0.120 0.123 0.135
COD/wt (g g−1)c 1.15 1.3
COD/OC (g g−1)c 2.5

An important component of COPAS is the particulate fraction missing in other synthetic recipes. Table 5 presents a comparison between COPAS characteristics and those of raw sewage. Depending on the solid concentrations of the COPAS solution, available nutrients and organic matter can be tuned to desired concentrations. For instance, a solution of 1000 mg L−1 COPAS solids provides sufficient COD and TKN to simulate raw WWTP influent as standardized in the literature (Table 5). Nevertheless, COPAS has a larger fraction of hardly degradable organic matter compared to municipal wastewater, which can be more representative of domestic sewage or black water (i.e., wastewater from toilet flushing and kitchen waste). According to Henze (2002),47 the BOD5/COD for domestic wastewater and black water ranges from 0.33 to 0.47. In terms of nitrogen content, COPAS has a high COD/TN ratio compared to other wastewaters. The values of COD/TN in domestic sewage range from 6 to 16 as reported by Henze et al. (2002).47 For the TS concentration of COPAS presented in Table 5, the COD/TN values range from 23 to 32. These COD/TN ratios are similar to those reported by Jönsson et al. (2005) for feces (COD/TN of 24.9),48 and makes COPAS more suitable to anaerobic degradability studies. For laboratory experiments that require a different concentration of nitrogen per sample, other compounds such as ammonium chloride or urea can be easily added to match the desired requirements.

Table 5 Comparison of COPAS to actual sewage
Sample TS (mg L−1) TSS (mg L−1) COD (mg L−1) BOD5 (mg L−1) BOD20 (mg L−1) BOD5/COD TKN (mg L−1)
a As reported by the HFC Environmental lab. b Corresponds to BOD7 value. c Corresponds to TN value.
COPASa 500 215 625 191 540 0.31 19.8
1000 520 1250 382 906 0.31 53.9
2000 1184 2500 680 3144 0.27 92.1
HFC AWTP primary influent 146 402 212 0.53 33.8
WWTP influent49 720–1200 220–350 500–1000 220–400 0.40–0.44 40–85
Household WW (raw)50 1028 232 849 420 0.5 57
Domestic wastewater47 210–740 100–350 0.47 17.5–92.5c
Black water47 900–1500 300–600 0.33–0.40 100–300c
Feces48 2.75 × 105 2.28 × 105 3.41 × 105 2.06 × 105b 0.60 1.37 × 104c
Feces and toilet paper48 4.80 × 105 4.34 × 105 5.80 × 105 3.08 × 105b 0.53 1.36 × 104c

Disintegration/dissolution tests

The addition of COPAS to water at a concentration of 500 mg L−1 dropped the pH from 7.10 ± 0.10 to 6.52 ± 0.05 at t = 30 min. These pH values do not differ much from those of actual domestic wastewater. Fig. 1 illustrates the dissolution profiles of organic carbon (DOC) and nitrogen (DN) when COPAS is mixed with water. Both carbon and nitrogen followed similar dissolution patterns: rapid dissolution in the first hour (stage I), followed by slow dissolution over the following 23 h (stage II). Based on the total organic C and N found in COPAS, the measured dissolved concentrations after 1 day of experiment were approximately 25 mg C per L and 5 mg N per L, corresponding to 10% and 17% of the carbon and nitrogen present in the dry solids, respectively. The remaining 90% of TOC and 83% of TN was still available in particulate form upon further dissolution and/or biodegradation. The first stage of dissolution most likely corresponds to the breakage or disintegration of COPAS particles into smaller primary particles due to the dissolution of the gelling agent (e.g., gums, gelatin, carrageenan, or other starches and thickeners) used to aggregate the cat food. This could simulate the soluble organic matter fraction entering a WWT reactor. The latter stage of dissolution is due to organic carbon leaching from the primary particles plus additional DOC from the gelling agent (Fig. 2). The dissolution rate of COPAS was not affected by different mixing speeds over the evaluated range (e.g., 20, 50 and 100 rpm), suggesting diffusion limitation.
image file: c9ew00365g-f1.tif
Fig. 1 Summary of COPAS dissolution curves for different mixing conditions. The dissolution stages correspond to I) breakage of secondary particles (i.e. gelling agent dissolving in water) and II) gradual disintegration of primary particles.

image file: c9ew00365g-f2.tif
Fig. 2 Schematic of COPAS particles and different components. At t > 0, the gelling agent dissolves and primary particles are released (i.e. particle disintegration starts). At t > 1 h, the primary particles start dissolving into the water. Hydrolysis occurs in the presence of biological enzymatic activity.

Anaerobic biodegradability assays

During biodegradability assays, the distribution of the available COPAS for degradation was evaluated by continuously measuring methane production and the fraction of substrate dissolved in the liquid. Measurements were corrected for the background activity of the anaerobic digester seed. The COD corresponding to CH4 generation was calculated based on the theoretical COD equivalence of 350 ml CH4 per gram of COD degraded at STP (0 °C, 1 atm). By correcting this value for an incubation temperature of 37 °C, the COD equivalents corresponding to methane generation can be expressed as:
CODmethane = VCH4 ÷ 397.4 ml CH4 per g COD(1)

Results for the specific methane production after 45 days of incubation indicate that 59 ± 11% of COPAS organics were converted to methane for the studied concentrations. The methane content of the produced biogas was 57 ± 8% v/v. An important fraction of biodegradable COPAS remained in the particulate form. Taking into account the heterogeneous composition of COPAS, this hardly biodegradable fraction can be attributed to bones and other recalcitrant components in animal feed; most of the time disregarded in other sewage synthetic recipes.

Substrate availability

With respect to bioreactor design, the availability of secondary soluble COD released from particulates in domestic sewage is typically difficult to simulate at a lab scale with synthetic feeds. For COPAS in particular, slow dissolution of particles occurs in the second stage of the batch dissolution tests (Fig. 1). If this second stage of the particulate fraction dissolution is modeled as a first-order process, the dissolution kinetics constant of the particulate substrate can be derived from the following expression:
image file: c9ew00365g-t1.tif(2)
where image file: c9ew00365g-t2.tif is the initial biodegradable fraction of COPAS, kdiss is the dissolution/hydrolysis rate (h−1) and Sp is the concentration of particulates in the water phase (mg particulate COD per L). The biodegradable particulate COD in the initial sample is:
image file: c9ew00365g-t3.tif(3)
where γ is the COD/wt ratio in COPAS (1.3), β is the biodegradable fraction of COPAS (57 ± 8%) and Xin is the concentration of COPAS in the influent.

Data fitting of the DOC dissolution profile as COD (COD/OC = 2.5) to first order kinetics results in a kdiss of 0.12 × 10−2 h−1 or 0.029 d−1. The actual hydrolysis kinetics in a biological reactor is faster due to the presence of microorganisms and enzymatic activity compared to that in their absence. Other authors have recognized hydrolysis as the limiting step in the biodegradation of complex particulate organics under anaerobic conditions.51–53 Gujer and Zehnder (1983) identified the hydrolysis rate constants of complex organic biopolymers such as lipids (0.08–1.7 d−1), proteins (0.02–0.03 d−1), cellulose (0.04–0.13 d−1), and hemicellulose (0.54 d−1).51 Miron et al. (2000) identified the khyd for lipids and carbohydrates in primary sludge to be 0.842 d−1 and 0.153 d−1; respectively.54 García-Heras (2003) reported the hydrolysis kinetic constants for biopolymers in complex wastes to be 0.5 to 2 d−1 for carbohydrates, 0.1 to 0.7 d−1 for lipids and 0.25 to 0.8 d−1 for proteins.55 In a more general case, Ristow et al. (2006) defined a khyd of 0.992 d−1 for the hydrolysis of primary sludge under methanogenic conditions.53 Although the kdiss = 0.029 d−1 found in this study under abiotic conditions does not fall far from the khyd values of proteins reported by Güjer and Zehnder, an approximation to the hydrolysis kinetics can be estimated using García-Heras' values of khyd for proteins (0.53 d−1), lipids (0.4 d−1) and carbohydrates (1.25 d−1),55 prorated to the contents of proteins (40%), lipids (17%) and carbohydrates (43%) in COPAS. The estimated khyd of COPAS is 0.82 d−1.

Using the above kinetics in a bioreactor model (e.g. steady state continuous flow stirred tank reactor), a mass balance of the particulate substrate can be expressed as:

image file: c9ew00365g-t4.tif(4)
image file: c9ew00365g-t5.tif(5)
where Sp is the particulate COD exiting the reactor, image file: c9ew00365g-t6.tif is the particulate COD entering the reactor, V is the reactor volume, and θ is the hydraulic retention time that is equal to the solids' residence time (θx) in a continuous flow stirred tank reactor. On the other hand, mass balance of soluble COD in the bioreactor can be expressed as:
Sin − CODeff + Sp hydrolizedSuptake = 0(6)

The available COD is provided by the incoming soluble COD to the reactor and the soluble COD released by dissolution/hydrolysis of particulate COD. For a well-mixed reactor, the effective soluble COD can be expressed as:

image file: c9ew00365g-t7.tif(7)

By substituting eqn (5) in eqn (7):

image file: c9ew00365g-t8.tif(8)
where image file: c9ew00365g-t9.tif is the effective soluble COD, S° is the soluble COD entering the reactor, V is the reactor volume, and θ is the hydraulic retention time that is equal to the solids' residence time (θx) in a continuous flow stirred tank reactor. Fig. 3 illustrates the image file: c9ew00365g-t10.tif or COD available for biodegradation at different θx values. A comparison between image file: c9ew00365g-t11.tif as a function of kdiss and image file: c9ew00365g-t12.tif as a function of khyd shows that the modeled values of image file: c9ew00365g-t13.tif using kdiss underrepresent the amount of substrate available for biodegradation in a CSTR over time. The modeled data of image file: c9ew00365g-t14.tif using khyd shows a more conservative approach to the COD profile available for biodegradation if biological activity was present in the reactor.

image file: c9ew00365g-f3.tif
Fig. 3 Effective substrate availability (Seff) of COPAS at 500, 1000 and 2000 mg L−1 TS, when using khyd (continuous line) vs. kdiss (dashed line).

Fig. 4 presents a comparison between the modeled image file: c9ew00365g-t15.tif (as a function of khyd) available for anaerobic microorganisms (eqn (8)) and the measured dissolved COD and methane COD equivalent data (CODd+g). Little COD remained in the dissolved fraction after 5 days of digestion and almost no dissolved COD was detected after 15 days. Most of the available COD for biodegradation was utilized for methane production. Although additional tests would be required to assess the kinetics of the hydrolysis of COPAS, the first order kinetic data found in the literature for complex substrates showed a good approximation of the soluble COD pathways in the present study.

image file: c9ew00365g-f4.tif
Fig. 4 Comparison between the modeled Seff (continuous line) and measured COD dissolved in the liquid phase and in the gas phase (CODd+g) (dashed line). COD profiles for 500, 1000 and 2000 mg L−1 COPAS solids.


COPAS provides an alternative surrogate to raw domestic wastewater in lab-scale studies. This study showed that COPAS can mimic sewage composition of complex organic matter (fats (17%), proteins (40%), and carbohydrates (43%)) from animal and plant origins. A solution of COPAS can be tuned to fulfill the COD and TKN requirements of laboratory studies that use raw sewage. By comparing the COD and TKN concentrations of COPAS to those of other sources of wastewater (e.g. primary influent), its characteristics are found to be more closely related to those of primary sludge, which can be better suited for anaerobic studies. Anaerobic biodegradability assays at 500, 1000 and 2000 mg COPAS per L showed an average biodegradability of 59 ± 11%, while 57 ± 8% of COPAS was converted to CH4 after 45 days of incubation. Mechanical grinding of COPAS can provide synthetic wastewater with a particle size tailored to a range of interests. In summary, COPAS makes a good alternative to raw sewage in lab-scale studies targeting the direct treatment of screened sewage such as anaerobic digestors and/or AnMBRs.56,57

Conflicts of interest

There are no conflicts to declare.


  1. G. Boeije, Chemical fate prediction for use in georeferenced environmental exposure assessment, Ph.D. Thesis, University of Ghent, Belgium, 1998 Search PubMed.
  2. S. Aiyuk and W. Verstraete, Sedimentological evolution in an UASB treating SYNTHES, a new representative synthetic sewage, at low loading rates, Bioresour. Technol., 2004, 93, 269–278 CrossRef CAS PubMed.
  3. DIN-38412-T24, DIN-38412-T24, German standard methods for the analysis of water, wastewater and sludge; bio-assays (Group L): determination of biodegradability by use of special methods of analysis (L 24), GmbH, Hennef, Beuth Verlag, 1981.
  4. M. Iaquinta, M. Stoller and C. Merli, Development of synthetic wastewater from the tomato industry for membrane processing purposes, Desalination, 2006, 200, 739–741 CrossRef CAS.
  5. H. Lin, S. L. Ong, W. J. Ng and E. Khan, Performance of a biofilm airlift suspension reactor for synthetic wastewater treatment, J. Environ. Eng., 2004, 130(1), 26–36 CrossRef CAS.
  6. I. Nopens, C. Capalozza and P. A. Vanrolleghem, Stability analysis of a synthetic municipal wastewater. Technical report, Department of applied mathematics, biometrics and process control, University of Ghent, Belgium, 2001.
  7. M. T. Kato, J. A. Field and G. Lettinga, The anaerobic treatment of low strength wastewaters in UASB and EGSB reactors, Water Sci. Technol., 1997, 36(6–7), 375–382 CrossRef CAS.
  8. M. Gao, M. Yang, H. Li, Y. Wang and F. Pan, Nitrification and sludge characteristics in a submerged membrane bioreactor on synthetic inorganic wastewater, Desalination, 2004, 170, 177–185 CrossRef CAS.
  9. R. Kurian, G. Nakhla and A. Bassi, Biodegradation kinetics of high strength oily pet food wastewater in a membrane-coupled bioreactor (MBR), Chemosphere, 2006, 65, 1204–1211 CrossRef CAS PubMed.
  10. A. N. Kofina and P. G. Koutsoukos, Spontaneous Precipitation of Struvite from Synthetic Wastewater Solutions, Cryst. Growth Des., 2005, 5(2), 489–496 CrossRef CAS.
  11. A. L. Prieto, L. H. Sigtermans, B. R. Mutlu, A. Aksan, W. A. Arnold and P. J. Novak, Performance of a composite bioactive membrane for H2 production and capture from high strength wastewater, Environ. Sci.: Water Res. Technol., 2016, 2, 848–857 RSC.
  12. K. Raunkjaer, T. Hvitved-Jacobsen and P. Nielsen, Measurement of pools of protein, carbohydrate and lipid in domestic wastewater, Water Res., 1994, 28(2), 251–262 CrossRef CAS.
  13. A. Rottiers, G. Boeije, R. Corstanje and K. Decraene, Adaptation of the CAS test system and synthetic sewage for biological nutrient removal part II: design and validation of test units, Chemosphere, 1999, 38(4), 711–727 CrossRef CAS PubMed.
  14. T. Elmitwalli, J. Soellner, D. Keizer, H. Bruning, G. Zeeman and G. Lettinga, Biodegradability and change of physical characteristics of particles during anaerobic digestion of domestic sewage, Water Res., 2001, 35(5), 1311–1317 CrossRef CAS PubMed.
  15. Y. Kalogo, A. M'Bassiguié Séka and W. Verstraete, Enhancing the start-up of a UASB reactor treating domestic wastewater by adding a water extract of Moringa oleifera seeds, Appl. Microbiol. Biotechnol., 2001, 55, 644–651 CrossRef CAS PubMed.
  16. S. Aiyuk, J. Amoako, L. Raskin, A. Van Haandel and W. Verstraete, Removal of carbon and nutrients from domestic wastewater using a low investment, integrated treatment concept, Water Res., 2004, 38, 3031–3042 CrossRef CAS PubMed.
  17. D. Orhon, D. Okutman and G. Insel, Characterisation and biodegradation of settleable organic matter for domestic wastewater, Water SA, 2002, 28(3), 299–305 CrossRef CAS.
  18. L. Seghezzo, R. G. Guerra, S. M. González, A. P. Trupiano, M. E. Figueroa, C. M. Cuevas, G. Zeeman and G. Lettinga, Removal efficiency and methanogenic activity profiles in a pilot-scale UASB reactor treating settled sewage at moderate temperatures, Water Sci. Technol., 2002, 45(10), 243–248 CrossRef CAS PubMed.
  19. M. A. Belmont, E. Cantellano, S. Thompson, M. Williamson, A. Sanchez and C. D. Metcalfe, Treatment of domestic wastewater in a pilot-scale natural treatment system in central Mexico, Ecol. Eng., 2004, 23, 299–311 CrossRef.
  20. R. R. Giri, J. Takeuchi and H. Ozaki, Biodegradation of domestic wastewater under the simulated conditions of Thailand, Water Environ. J., 2006, 20, 169–176 CAS.
  21. M. Tandukar, I. Machdar, S. Uemura, A. Ohashi and H. Harada, Potential of a combination of UASB and DHS reactor as a novel sewage treatment system for developing countries: long-term evaluation, J. Environ. Eng., 2006, 132(2), 166–172 CrossRef CAS.
  22. J. H. Choi, K. Fukushi and K. Yamamoto, A submerged nanofiltration membrane bioreactor for domestic wastewater treatment: the performance of cellulose acetate nanofiltration membranes for long-term operation, Sep. Purif. Technol., 2007, 52, 470–477 CrossRef CAS.
  23. X. Wang, P. Jin, H. Zhao and L. Meng, Classification of contaminants and treatability evaluation of domestic wastewater, Front. Environ. Sci. Eng. China, 2007, 1(1), 57–62 CrossRef.
  24. J. T. Sousa and E. Foresti, Domestic sewage treatment in an upflow anaerobic sludge blanket – sequencing batch reactor system, Water Sci. Technol., 1996, 33(3), 73–84 CrossRef.
  25. J. S. VanderGheynst, J. M. Gossett and L. P. Walker, High-solids aerobic decomposition: pilot-scale reactor development and experimentation, Process Biochem., 1997, 32(5), 361–375 CrossRef CAS.
  26. G. Boeije, R. Corstanje, A. Rottlers and D. Schowane, Adaptation of the CAS test system and synthetic sewage for biological nutrient removal part I: development of a new synthetic sewage, Chemosphere, 1998, 38(4), 699–709 CrossRef.
  27. K. C. Chen, S. C. Lee, S. C. Chin and J. Y. Houng, Simultaneous carbon-nitrogen removal in wastewater using phosphorylated PVA-immobilized microorganisms, Enzyme Microb. Technol., 1998, 23, 311–320 CrossRef CAS.
  28. W. Ghyoot and W. Verstraete, Reduced sludge production in a two-stage membrane-assisted bioreactor, Water Res., 1999, 34(1), 205–215 CrossRef.
  29. A. Langenhoff, N. Intrachandra and D. C. Stuckey, Treatment of dilute soluble and colloidal wastewater using an anaerobic baffled reactor: influence of hydraulic retention time, Water Res., 2000, 34(4), 1307–1317 CrossRef CAS.
  30. Organization for Economic Corporation and Development, OECD Guidelines for Testing Chemicals, Section 2: Effects on Biotic Systems. Test No. 209: Activated Sludge, Respiration Inhibition Test, 1991.
  31. US Environmental Protection Agency, Ecological Effects Test Guidelines OPPTS 850.6800, Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals, EPA 712-C-96-168, Washington, DC, 1996.
  32. N. Yoshida, K. Yano, T. Morita, S. J. McNiven, H. Nakamura and I. Karube, A mediator-type biosensor as a new approach to biochemical oxygen demand estimation, Analyst, 2000, 125, 2280–2284 RSC.
  33. C. Y. Gomec, M. Kim, Y. Ahn and R. E. Speece, The role of pH in mesophilic anaerobic sludge solubilization, J. Environ. Sci. Health, Part A: Toxic/Hazard. Subst. Environ. Eng., 2002, 37(10), 1871–1878 CrossRef.
  34. C. G. Klatt and T. M. LaPara, Aerobic Biological Treatment of Synthetic Municipal Wastewater in Membrane-Coupled Bioreactors, Biotechnol. Bioeng., 2003, 82(3), 313–320 CrossRef CAS PubMed.
  35. L. Chu, X. Zhang, F. Yang and X. Li, Treatment of domestic wastewater by using a microaerobic membrane bioreactor, Desalination, 2006, 189, 181–192 CrossRef CAS.
  36. A. Y. Hu and D. C. Stuckey, Treatment of dilute wastewaters using a novel submerged anaerobic membrane bioreactor, J. Environ. Eng., 2006, 132(2), 190–198 CrossRef CAS.
  37. M. Rodgers, X. Zhan and E. O'Reilly, Small-scale domestic wastewater treatment using an alternating pumped sequencing batch biofilm reactor system, Bioprocess Biosyst. Eng., 2006, 28, 323–330 CrossRef CAS PubMed.
  38. K. Sombatsompop, C. Visvanathan and R. Ben Aimb, Evaluation of biofouling phenomenon in suspended and attached growth membrane bioreactor systems, Desalination, 2006, 201, 138–149 CrossRef CAS.
  39. J. O. Kim, J. T. Jung, I. T. Yeom and G. H. Aoh, Effect of fouling reduction by ozone backwashing in a microfiltration system with advanced new membrane material, Desalination, 2007, 202, 361–368 CrossRef CAS.
  40. M. Lozada, L. Basile and L. Erijman, Impact of non-ionic surfactant on the long-term development of lab-scale-activated sludge bacterial communities, Res. Microbiol., 2007, 158, 712–717 CrossRef CAS PubMed.
  41. Z. Ahmed, B. R. Lim, J. Cho, K. G. Song, K. P. Kim and K. H. Ahn, Biological nitrogen and phosphorus removal and changes in microbial community structure in a membrane bioreactor: Effect of different carbon sources, Water Res., 2008, 42, 198–210 CrossRef CAS PubMed.
  42. R. Reif, S. Suárez, F. Omil and J. M. Lema, Fate of pharmaceuticals and cosmetic ingredients during the operation of a MBR treating sewage, Desalination, 2008, 221, 511–517 CrossRef CAS.
  43. D. Dzanis, The association of American feed control officials dog and cat food nutrient profiles: substantiation of nutritional adequacy of complete and balanced pet foods in the United States, J. Nutr., 1994, 124, 2535s–2539s CrossRef CAS PubMed.
  44. APHA, AWWA and WEF, Standard methods for the examination of water and wastewater, 21st edn, 2005 Search PubMed.
  45. B. E. Rittmann and P. L. McCarty, Environmental Biotechnology: Principles and Applications, McGraw-Hill Professional, 2001 Search PubMed.
  46. S. W. Sötemann, P. van Rensburg, N. E. Ristow, M. C. Wentzel, R. E. Loewenthal and G. A. Ekama, Integrated chemical/physical and biological processes modelling part 2: anaerobic digestion of sewage sludges, Procs. Water Institute of Southern Africa Biennial Conference (WISA 2004), Cape Town, 2004 May. ISBN 1-920-01728-3.
  47. M. Henze, P. Harremoes, J. la Cour Jansen and E. Arvin, Wastewater Treatment: Biological and Chemical Processes, 2nd edn, Springer, Berlin, Germany, 2002 Search PubMed.
  48. H. Jönsson, A. Baky, U. Jeppsson, D. Hellström and E. Kärrman, Composition of urine, faeces, greywater and biowaste for utilisation in the URWARE model, Urban Water Report, Chalmers University of Technology, Gothenburg, Sweden, 2005 Search PubMed.
  49. G. Tchobanoglous, F. Burton and H. D. Stensel, Wastewater Engineering Treatment and Reuse, Metcalf & Eddy, McGraw Hill, 2003, p. 1819 Search PubMed.
  50. WERF, Influent Constituent Characteristics of the Modern Waste Stream from Single Sources Executive Summary, 2008.
  51. W. Gujer and A. J. B. Zehnder, Conversion processes in anaerobic digestion, Water Sci. Technol., 1983, 15(8/9), 127–167 CrossRef CAS.
  52. J. T. O'Rourke, Kinetics of Anaerobic Waste Treatment at Reduced Temperatures, PhD thesis, Department of Civil Engineering, Stanford University, 1968 Search PubMed.
  53. N. E. Ristow, S. W. Sötemann, M. C. Wentzel, R. E. Loewenthal and G. A. Ekama, The effects of hydraulic retention time and feed COD concentration on the rate of hydrolysis of primary sewage sludge under methanogenic conditions, Water Sci. Technol., 2006, 54(5), 91–100 CrossRef CAS PubMed.
  54. Y. Miron, G. Zeeman, J. van Lier and G. Lettinga, The role of sludge retention time in the hydrolysis and acidification of lipids, carbohydrates and proteins during digestion of primary sludge in CSTR systems, Water Res., 2000, 34(5), 1705–1713 CrossRef CAS.
  55. J. L. García-Heras, in Reactor sizing, process kinetics and modelling of anaerobic digestion of complex wastes, ed. J. Mata-Alvarez, Biomethanization of the Organic Fraction of Municipal Solid Wastes Padstow, TJ International Ltd. IWA Publishing, Cornwall, UK, 2003, pp. 21–62 Search PubMed.
  56. D. W. Gao, T. Zhang, C. Y. Tang, W. M. Wu, C. Wong, Y. Lee, D. H. Yeh and C. Criddle, Membrane fouling in an anaerobic membrane bioreactor: Differences in relative abundance of bacterial species in the membrane foulant layer and in suspension, J. Membr. Sci., 2010, 364(1–2), 331–338 CrossRef CAS.
  57. A. L. Prieto, H. Futselaar, P. Lens, R. Bair and D. H. Yeh, Development and start up of a gas-lift anaerobic membrane bioreactor (GL-AnMBR) for conversion of sewage to energy, water and nutrients, J. Membr. Sci., 2013, 441, 158–167 CrossRef CAS.

This journal is © The Royal Society of Chemistry 2019