Potential for Methane Production from Anaerobic Co-digestion of Cheese Whey Samples with Sewage Sludge

Y.D. Yilmazel*, S. Alanya*, C. Park*, R.J. Eschborn**, M.I. Williams***, M. Duran*

*Villanova University, Department of Civil and Environmental Engineering, Villanova University, Villanova, PA 19085-1681, USA

(E-mail: yasemin.yilmazel@villanova.edu; sevda.alanya@villanova.edu; cheol.park@villanova.edu; metin.duran@villanova.edu) ** AECOM, 1700 Market Street, Philadelphia, PA 19103, USA (E-mail: ralph.eschborn@aecom.com ) *** Bergen Country Utilities Authority, Foot of Mehrhof Road, P.O. Box 9, Little Ferry, NJ 07643, USA (E-mail: mwilliams@bcua.org)

Abstract The feasibility of co-digestion of cheese whey (CW) and sewage sludge was evaluated in mesophilic (35oC), bench-scale (batch) digesters. Biochemical methane potential (BMP) tests were performed to test the performance of two CW samples obtained from two different waste streams: CW before centrifuge and after the reverse osmosis (RO) treatment. BMP results indicated that CW samples (Pre centrifuge or Post RO) do not have any inhibitory effects on the anaerobic digestion of sewage sludge. Both samples are anaerobically biodegradable. However, the ultimate 30-day biochemical methane potential of Pre centrifuge CW sample is 1.8-2.2 times higher than that of Post RO CW sample for the same CW loadings (5% and 10 % CW by volume). Therefore, it is more beneficial to co-digest Pre centrifuge CW with sewage sludge and the optimal Pre centrifuge CW loading is determined as 5% of the feed sludge because it gives the highest COD recovery (163%) and specific methane (CH4) production (644 L CH4/kg COD equivalent CW at 1 atm and 35oC). A logistic growth model agreed well with the results from the BMP tests suggesting that specific CH4 production under various CW and sludge loading at fullscale could be predicted using the kinetic model developed. Nutrient deficiency tests showed that BCUA digester biomass is not stimulated by supplemental addition of the macro- and micronutrients.

Keywords Anaerobic co-digestion; Biochemical Methane Potential (BMP); cheese whey; sewage sludge; kinetic modeling

INTRODUCTION Anaerobic digestion (AD) is a well established bioconversion technology used for the treatment of a wide range of organic wastes. As a result of AD process, organic matter is mainly converted into a mixture of methane (CH4), carbon dioxide (CO2) and new bacterial cells (Speece, 2008). The efficiency of the AD process is highly dependant on the waste characteristics such as organic strength, buffering capacity, solids and nutrient content, the reactor configuration and the operational parameters of the system. Anaerobic treatment may not seem to be an appropriate choice for a certain type of waste due to its characteristics; however, there are several key measures that can be taken to increase the digestibility. Co-digestion is an attractive alternative for the improvement of anaerobic degradation of wastes with different characteristics. Anaerobic co-digestion is the use of a co-substrate in the digestion medium, which mostly improves the biogas yields as a result of positive synergism established in the reactor and the supply of

missing nutrients by the co-substrates (Mata-Alvarez, 2000). Co-digestion also brings several economical benefits due to sharing of equipment, easier handling of mixed wastes and the use of common access facilities. Moreover, the use of a co-substrate can also help establish the required moisture content in the digester. In addition, co-digestion becomes a viable option when the amount of one type of organic waste generated at a particular site at a certain time is not sufficient to make AD cost effective (Callaghan et al., 1999). This is particularly important for small industrial plants where low quantities of wastes are generated. Cheese whey (CW) is a by-product of the cheese production and it is rich in proteins and lactose (Spachos and Stamatis, 2011). It has a high organic matter content [up to 70,000 mg/L chemical oxygen demand (COD)] and very high biodegradability (approximately 99%) and relatively high alkalinity (about 2500 mg/L CaCO3) (Mawson, 1994; Erguder et al., 2001). The high organic content of CW makes the aerobic treatment challenging mainly due to the high cost of oxygen supplementation. Another option of handling CW is to use it for animal feeding; however, this is limited with the availability of nearby farms. A sustainable option for the handling of CW is anaerobic treatment, which does not require any oxygen supplementation and generates significant amounts of energy in the form of CH4. However, several researchers reported that the direct treatment of raw whey was problematic due to the tendency of rapid acidification of the waste (Kalyuzhnyi et al., 1997). Other problems associated with direct anaerobic treatment of CW include instability of the reactor, the difficulty to obtain granulation, and reduced sludge settleability due to the tendency to produce an excess of viscous exopolymeric materials of probable bacterial origin (Malaspina et al., 1995). In the literature co-digestion of whey and dairy manure was proved to be successful without any need of chemical addition (Gelenegis et al., 2007; Kavacik and Topaloglu, 2010). Another alternative co-digestion process is treating CW in sewage treatment plants which is advantageous because of the wide distribution of sewage treatment plants minimizes the transportation costs. Adding co-substrate in existing digesters also helps to utilize the availability of free capacities and increase the amount of biogas generation (Braun and Wellinger, 2005). This study investigated the feasibility of co-digestion of CW and sewage sludge using the standard Biochemical Methane Potential (BMP) test (Owen et al., 1979). The objective was to determine if CW generated by a local dairy plant could successfully be digested with sewage sludge, which in turn would lead to an increase in the overall CH4 yield from the full-scale anaerobic digesters operated by the Bergen County Utilities Authority (BCUA) in New Jersey, USA. The co-digestion study was performed using two different CW samples obtained from two different waste streams: CW before centrifuge and CW after the reverse osmosis (RO) treatment. In addition, a nutrient deficiency study was performed to assess any possible micro- and macro-nutrient supplementation needs of the anaerobic biomass in the BCUA digesters for optimal metabolism. The standard Anaerobic Toxicity Assay (ATA) was used to identify the potential beneficial effect of supplementing iron, nickel, cobalt, as well as that of a macronutrient, a trace metal cocktail, and their combination, known as the Vanderbilt Media. MATERIALS AND METHODS The objective of this study was to investigate feasibility of co-digestion of CW and sewage sludge in the anaerobic digesters operated by BCUA. The treatment plant in operation today uses conventional activated sludge process and flow through the plant in 2010 averaged 300,000 m3/day (80 million gallons per day). The BCUA operates five 24 metre diameter mesophilic anaerobic digesters of capacity about 18,750 m3 (4.85 million gallons). Dairy plant from which CW samples were collected is located near the treatment plant. The dairy processes include centrifugation as a

means of resource recovery, and reverse osmosis (RO) process for the treatment of wastewater to meet the requirements for discharge into sewer collection system. Waste Characteristics Sewage sludge was obtained from the influent of the anaerobic digesters of BCUA, NJ, while the CW samples were collected from the dairy operation. CW samples were obtained from two different locations of the dairy process, before centrifugation (referred to as Pre centrifuge CW) and after RO process (referred to as Post RO CW). All wastes were characterized and kept refrigerated at 4 oC until used. The waste characteristics are summarized in Table 1.
Table 1. Characteristics of the wastes used in the study

COD, mg/L TS, % VS, % Total ammonia, mg/L Alkalinity, mg/L as CaCO3 pH

Sewage sludge 45,500 3.62 2.78 -

Pre centrifuge CW 72,500 6.52 5.61 20 1400 5.75

Post RO CW 195,000 17.8 15.4 24 2600 5.87

CW: cheese whey; TS: total solids; VS: volatile solids COD: chemical oxygen demand

Inoculum The mixed anaerobic culture obtained from anaerobic sludge digesters of BCUA, NJ, was used as seed in this study. The composition of the seed culture was analyzed and stored at 4 oC prior to use. The characteristics of seed culture were; total solids (TS) 2.07%, volatile solids (VS) 1.42%, chemical oxygen demand (COD) 22,600 mg/L and alkalinity 4,600 mg/L as CaCO3. Analytical Methods COD tests were carried out according to the closed reflux colorimetric method as described in Standard Methods Section 5220D (Eaton et al., 1995). High range (0-1,500 mg/L) COD digestion vials (Hach Company, Loveland, CO) containing potassium dichromate were used and the COD content of the vials were determined by a spectrophotometer at 600 nm (Model DR 4000 U, Hach Company, Loveland, CO). Total ammonia (NH4+ and NH3 combined) was measured according to the Standard Methods Section 4500-NH3 D (Eaton et al., 1995). A glass ammonia selective electrode probe was used to quantify total ammonia after the pH of samples was raised above 11 using 10 N NaOH. Raising pH above 11 converted NH4+ into NH3 allowing measurement of total ammonia. The probe was calibrated with standard solutions of known concentrations of NH4+. pH of the samples was determined by a pH meter (Fisher Scientific Instrument, Accumet Model 50) equipped with a poly Accumet combination electrode with silver/silver chloride references. TS, VS and alkalinity measurements were conducted as described in Standard Methods, Section 2540B, 2540 E, and 2320 B, respectively (Eaton et al., 1995). Total biogas production in serum bottles was monitored by a liquid displacement device. The device used a 21 G 1 precision glide needle connected to a 100 mL burette which is connected to a 100 mL graduated cylinder filled with diluted acid solution. The amount of the gas produced in each bottle was measured by determining the volume of the liquid displaced by the gas vented into the device. Biogas composition was analyzed for CH4 and CO2 by a Hewlett Packard, Model 6890 gas chromatograph equipped with a thermal conductivity detector. An eight meter long HayeSep Q, 80/100 packed column was used to separate CH4 and CO2 (Alltech Associates Inc, Deerfield, IL). Helium was the carrier gas with a flow rate of 25 mL/min. Pure gases (MicromatTM

 14 Cylinder from Matheson Tri- Gas, Alltech Associates, Inc. Deerfield, IL) were used to develop calibration curves for CH4 and CO2. In addition, pure samples (10% methane) were run periodically as the quality check of the calibration. Samples were run for 2 minutes at 75 C. CH4 percentages were calculated by assuming other components are negligible. Experimental Set-up and Procedures BMP experiments. Anaerobic digestion of sewage sludge, and combination of CW samples (Pre centrifuge CW and Post RO CW) and sewage sludge at various ratios were studied in 165-mL capacity serum bottles with effective volume of 60 mL. The reactors were maintained at 352 oC in a temperature controlled room and they were shaken continuously at 135 rpm on a rotating shaker. In order to determine the effect of waste mixing ratio on the anaerobic biodegradability and CH4 generation, four different ratios were tested for each CW sample. The volume percentages used were 2%, 5%, 10% and 20% for the Pre Centrifuge CW sample and 1%, 2.5%, 5% and 10% for the Post RO CW sample. The concentrations, along with the corresponding CW to sewage sludge waste mixing ratios, are reported in Table 2. After the constituents were added, the serum bottles were purged with 30% CO2 and 70% N2 mixture for approximately 30 seconds to maintain anaerobic conditions and around neutral pH. Two control reactors were operated: (1) Seed Control that received only seed and diluted to 60 mL mark with sterilized tap water; and (2) Feed Control that received only sewage sludge and seed but no CW. All reactors were run in triplicate and the averaged values from triplicate are reported here.
Table 2. The ratios of CW and sewage sludge tested for co-digestion CW/SS CW/SS Sample (% by volume) (COD CW/ COD feed as %) 2 3.2 5 8 Pre centrifuge CW 10 16 20 32 1 2.5 Post RO CW 5 10 CW: cheese whey; SS: sewage sludge 4.3 10.71 21.4 43 CW (mg/L COD eq.) 145 363 726 1452 195 488 975 1950

ATA experiments. The effects of nutrient supplementation on BCUA anaerobic digester biomass were evaluated by slightly modified form of ATA, originally developed by Owen et al., (1979). Nutrient deficiency assay were performed in 165-mL capacity serum bottles with effective volume of 60 mL. Each serum bottle contained seed (biomass from BCUA digester), and 5,000 mg/L of calcium acetate as the primary substrate for aceticlastic methanogens, nutrient tested, and sterilized tap water for normalizing the total liquid volume. Serum bottles were supplemented with several concentration of iron, nickel, cobalt, a macro nutrient cocktail, a trace metal cocktail, and combination of macro nutrient and trace metal cocktails, called Vanderbilt Media, the compositions of which are given elsewhere (Park et al., 2010). During of the 30-day incubation period, biogas production and headspace gas composition in each reactor were periodically determined and recorded. After the digestion period was ended, all reactors (both BMP set and ATA set reactors) were subjected to COD determinations, in order to analyze the treatment efficiencies. RESULTS AND DISCUSSION The results of nutrient deficiency study showed that the cumulative CH4 generation in nutrient

supplemented serum bottles is almost the same as that in the acetate control serum bottles indicating that nutrient supplementation did not have any beneficial effect on CH4 production by aceticlastic methanogens, in the case of BCUA digester biomass. Co-digestion study The characterizations of CW samples are shown in Table 1. The data shows that both CW samples have high organic matter content. Pre centrifuge CW sample contains 72,500 mg/L of total COD and the RO process concentrates these organics to a 2.7 fold. Hence, in order to prevent overloading of the digesters the highest percentage of Post RO CW sample was kept at 10 %, whereas it was 20 % in the Pre Centrifuge sample (Table 2). Reactors were operated until no significant biogas production was detected. The cumulative CH4 generations from serum bottles that received sewage sludge and CW at various initial concentrations are presented in Figure 1. Each data point in Figure 1 is the average of cumulative CH4 production in triplicate serum bottles.
250 a) Cumulative CH Production (mL) 200

150

100

50

Seed control Feed control 2 % CW 5 % CW 10 % CW 20 % CW

0
250

Cumulative CH Production (mL)

b)
200

150

100

50

Seed control Feed control 1 % CW 2.5 % CW 5 % CW 10 % CW

10

15

20

25

30

35

Time (days) Figure 1. Cumulative methane production from co-digestion of sewage sludge and a) Pre centrifuge; b) Post RO CW samples at various concentrations

It is clear from Figure 1 that the serum bottles with increasing initial concentrations of CW resulted

in higher rates and amounts of gas production compared to the feed control. Same trend was observed for both CW samples. The fact that all of the active reactors supplemented with CW produced more CH4 than the feed control clearly indicates that CW has no inhibitory effect on digestion of sewage sludge, the feed. It also indicates anaerobic digestibility of CW. It is important in co-digestion feasibility studies to assess the optimal loading rate of the codigestion candidate that could be applied without any detrimental effect on the overall metabolism of digester biomass. The COD recovery at different loading rates is useful in that regard. Percent COD recovery is based on COD equivalent of CH4 and it is the ratio of CH4 generated (in COD equivalent) to the COD added. The COD equivalent of CH4 is 395 mL of CH4 per gram COD at 35 o C (incubation temperature), which is derived from the stoichiometry of CH4 oxidation (Speece, 2008). The estimate does not account for the fraction of substrate used for cell synthesis, which is typically less than 10% of the substrate digested. The COD recovery is based on net CH4 generated by digestion of CW. Net CH4 from digestion of CW is the difference between cumulative CH4 from active reactors and that from the seed control. Two control samples used in the study were seed and feed control. Seed control accounted for the CH4 that resulted from digestion of residual volatile solids in the digester sample (seed) collected from the BCUA plant. Feed control, on the other hand, was used to determine the amount of CH4 from digestion of sewage sludge. Figure 2 presents the percent COD recovered as a function of CW loading, expressed as initial CW concentration and initial CW to sewage sludge ratio in the feed.
250
a) Pre centrifuge CW Post RO CW b) Pre centrifuge CW Post RO CW

250

200 % COD recovered

200 % COD recovered

150

150

100

100

50

50

0 0 500 1000 Initial COD (mg\L) 1500 2000 0 5 10 15 20 25 CW / Sewage sludge (% by volume)

Figure 2. Relationship between % COD recovery and a) initial CW concentration expressed in mg COD equivalent per L of reactor volume; b) cheese whey to sewage sludge ratio as % by volume

Another way to determine an optimal load of CW is to look at the specific CH4 production under each CW loading. Specific CH4 is expressed as volume of CH4 produced per unit mass of CW fed, as COD equivalent of CW. The specific CH4 production for each CW sample and under each initial CW concentration is presented in Table 3.

Table 3. Specific CH4 production as a function of CW loading CW/SS Specific CH4 prd. Sample (L/kg COD eq.)a (% by vol.) 2 460 Pre centrifuge 5 644 10 CW 529 20 414 1 769 2.5 342 Post RO CW 5 291 10 299 a Reported at 1 atmosphere of pressure and 35oC temperature CW: cheese whey; SS: sewage sludge

Figure 3 as well as Table 3 indicates that Pre centrifuge CW has higher COD recovery, and thus higher specific CH4 production, at all loadings compared to Post RO CW sample. The COD recovery was between 74 and 195% for Post RO CW sample. The highest recovery, 195% at the lowest loading, is likely to be an outlier since the net CH4 generation is considerably small, only 9 mL over the 30-day incubation period, and as such even a small difference in recovered and expected CH4 translates into large percentage difference. The COD recovery for the Pre centrifuge CW samples on the other hand, varied between 105 and 163% suggesting that addition of Pre centrifuge CW has a synergistic effect leading to greater mass of sludge digestion. However, the solids and COD concentrations measured at the end of the incubation period were more or less the same in the control and Pre centrifuge CW added bottles indicating that there was not a greater degree of feed sludge digestion. The most likely explanation for greater than 100% COD recovery from Pre centrifuge CW sample is the fact that fats have higher energy content, on a mass per mass basis, than carbohydrates and proteins. Therefore, Pre centrifuge CW having greater fat content than the Post RO CW samples leads to over 100% CH4 recovery when it is anaerobically digested. Kinetic modeling of anaerobic cheese whey co-digestion at BCUA facility. It is difficult, and likely to be erronous, to use batch data gathered from BMP tests to predict the performance of an intermittently fed completely stirred tank reactor (CSTR), typical configuration for full-scale anaerobic digestors. Nevertheless, a model based on first order degradation kinetics, might provide at least some qualilative insight on CH4 yield as a function of time for full-scale co-digestion of CW. The modeling was carried out only for Pre centrifuge CW. The model developed is an logistic growth model that predicts CH4 yield as a function of time, roughly representing hydraulic retention time (HRT) of a full-scale digester, and it is expressed as follows: Y = Ymax(1-e-kt) where; Y: Cumulative CH4 yield (liter of CH4 at 35 oC per gram of COD added) Ymax: Maximum methane yield (L CH4/per gram COD added) k: First order reaction rate constant (1/day) t: Time (days) The cumulative CH4 production data from BMP tests under various CW loadings as percent of total feed volume (feed sludge plus CW) are shown in Figure 3. For each CW loading, the cumulative CH4 were normalized per active reactor volume. Then, the logistic growth model expressed by Equation 1 was applied to the normalized cumulative CH4 production data using Curve Expert software to determine the constants Ymax and k for each CW loading. It is important to note that Y (1)

in the model includes CH4 from degradation of feed sludge as well as of CW. These constants are presented in Table 4.
S = 0.00790396 r = 0.99719089
7 0.2

Y (L CH4/ g COD added)

2 0.2 8 0.1 3 0.1 9 0.0 4 0.0 0 0.0

0.0

5.5

11.0

16.5

22.0

27.5

33.0

Time (days)
Figure 3. Model results for 5% (vol./vol.) Pre centrifuge cheese whey in feed (s:standard error; r: correlation coefficient)

In each case, data fit well to the model as indicated by r2 values 0.98 or above. Figure 3 shows an example best fit for CW loading of 5% of total feed volume. The kinetic model results were compared with full-scale operation data. However, the full scale data could only be obtained for sewage sludge digesters which are currently in operation. The retention time of full scale digesters are about 35 days and the calculated yield ranges between 0.22-0.31 L CH4/kg COD added with an average Y value of 0.25 L CH4/kg COD added depending on the feed rate and characteristics. The model result gives 0.20 L CH4/kg COD added for the same retention time. The difference between the model and full scale data ranges between 9 and 35 %, and considering the fact it is a full-scale data used for calibrating a model based on bench-scale batch data, this discrepancy appears reasonable.
Table 4. Kinetic constants, under various cheese whey load, for exponential growth model CW/SS Ymax (L CH4/g COD added) (% by vol.) 0 ( sewage sludge only) 0.1995 2 0.2122 5 0.2354 10 0.2524 20 0.2557 CW: cheese whey; SS: sewage sludge k (1/day) 0.978 0.921 0.823 1.13 0.982

CONCLUSIONS This study evaluated the potential benefits of co-digestion of sewage sludge and CW on biogas yield of BCUA digesters. The evaluation was performed using CW samples obtained before centrifugation (Pre centrifuge CW) and from the effluent of RO process (Post RO CW). In addition, possible nutrient deficiency in the BCUA digesters was evaluated by supplemental addition of iron, nickel, cobalt and other macro- and micro-nutrients in the Vanderbilt media. Nutrient deficiency tests showed that BCUA digester biomass is not stimulated by supplemental addition of the aforementioned nutrients. In other words, aceticlastic methanogens in the BCUA digesters have all

the required nutrients and thus no benefit would be realized by nutrient supplementation at the fullscale operation. The results of the co-digestion of CW and sewage sludge indicated that CW samples (Pre centrifuge or Post RO) do not have any inhibitory effects on the anaerobic digestion of sewage sludge. Both samples are anaerobically biodegradable. However, the ultimate 30-day Biochemical methane potential of Pre centrifuge CW sample is higher than that of Post RO CW sample (Table 3). For 5 % CW loading (by volume) the specific CH4 yield is calculated as 644 L /kg COD equivalent in the case of Pre centrifuge CW sample whereas the specific CH4 yield for the same loading rate is 291 L /kg COD equivalent. Thus, it is advantageous to co-digest CW before centrifuge treatment. In addition, elimination of centrifuge and RO process is beneficial in terms of operating cost of dairy plant as running centrifuge and RO processes are energy intensive and costly. The optimal Pre centrifuge CW loading seems to be 5% of the feed sludge as that loading led to the highest COD recovery (163%) and specific CH4 production (644 L/kg COD equivalent CW at 1 atm and 35 oC). Even higher Pre centrifuge CW loads resulted in more than 100% COD recovery. If the optimal Pre centrifuge CW loading of 5% of the feed is applied at the full-scale, daily expected CH4 production would be slightly over than 3,500 m3. Although greater than 100% COD recovery from Pre centrifuge CW suggest a synergistic effect and thereby improved destruction of VS in the feed sludge, it is more likely that this is an artifact of the fact that Pre centrifuge CW contain fats. According to stoichiometric relationships, 395 mL of CH4 at 35oC is equivalent to 1 g of COD removed from wastewater if the COD is removed via methane production only. Higher than 100 % COD recovery rates reported here may be due to the high fat content of Pre Centrifuge CW sample. We speculate that standard COD test (the closed reflux colorimetric method, Section 5220D) might underestimate COD content of wastewaters with high fat contents as some fats may not be fully oxidized due to their immiscible nature and/or some fats might be in the gas phase at high temperature COD test is conducted at, 150oC.

References
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