Report No. 449 SUSTAINABLE ANAEROBIC CO-DIGESTION OF GREASE INTERCEPTOR WASTE By Tarek Aziz 1Department of Civil, Construction, and Environmental Engineering North Carolina State University Raleigh, NC [email protected] 919-515-1562 UNC-WRRI-449 The research on which this report is based was supported by funds provided by the North Carolina General Assembly and/or the US Geological Survey through the NC Water Resources Research Institute. Contents of this publication do not necessarily reflect the views and policies of WRRI, nor does mention of trade names or commercial products constitute their endorsement by the WRRI, the State of North Carolina, or the US Geological Survey. This report fulfills the requirements for a project completion report of the Water Resources Research Institute of The University of North Carolina. The authors are solely responsible for the content and completeness of the report. WRRI Project No. 13-01-W November 2014 Acknowledgments This work would also not have been complete without funding provided by the NC Water Resources Research Institute. The faculty and staff at NC WRRI have provided support and flexibility to enable us to exceed our initial objectives with this project. We believe the result of this study provide new insight into anaerobic co-digestion of grease interceptor waste, move municipalities closer to sustainable energy generation at wastewater treatment facilities, provide an infrastructure-safe disposal of GIW, and raise exciting new research questions that will ultimately lead to the sustained full-scale implementation of GIW co-digestion; benefitting state of North Carolina, the country, and the world. There are several people the PIs would like to acknowledge in the completion of this report. First, we’d like to recognize the hard work of our graduate students (Ling Wang and Elvin Hossein). Without them this work would not have been complete. They have lead the charge in not only carrying out the tasks put in front of them, but finding questions and curiosities that push the bounds of this research well beyond the initial research question. In addition, an undergraduate researcher, Liya Weldegebriel, provided vital support during our lab scale reactor operation. We would also like to recognize the support of our colleague Joel Ducoste for his contribution in discussions related to FOG. Several municipalities have played an important role in supporting this research: The City of Durham, The City of Raleigh (TJ Lynch), and the Town of Cary (Donald Smith). Their support has been both in the form of sample collection (City of Durham and the Town of Cary) as well as in the form of letters of support/information sharing/dialogue (all of the above). Support from local municipalities is vital in research of this kind and we are extremely grateful. We would like to recognize Hazen and Sawyer who has shown interest and support for this project from the very beginning. Last but not least we would like to acknowledge our families who provide support for all our endeavors. 1 1. Introduction   1.1 The Fat, Oil, and Grease Problem Fat, Oil, and Grease (FOG) in sewers can accumulate and congeal on pipe walls, forming hardened deposits through a chemical reaction or a physical aggregation process (He et al., 2011; NCDENR, 1999; US EPA, 2004a). Animal- and vegetable-based FOG present the greatest threat of obstruction in sewer lines, responsible for up to 47% of the reported blockages and 50% to 75% of sanitary sewer overflows as it tends to solidify, reduce conveyance capacity, and eventually block flow (Keener et al., 2008; Southerland, 2002; US EPA, 2003; US EPA, 2004a; US EPA, 2004b). In addition to waste animal fats and waste industrial FOG, common sources of FOG wastes are food service establishments (FSEs) such as restaurants and fast food outlets. At FSEs, grease abatement devices such as grease interceptors (GIs) and grease traps (GTs) are commonly installed to prevent FOG discharge from entering the collection systems. The entire contents of a GI or GT comprising FOG, food particles, and associated wastewater produced at FSEs are collectively referred to as grease interceptor waste (GIW) or grease trap waste (GTW). GIW/GTW is one of the most abundant lipid-based organic substrates in the U.S. with high methane potential (Austic, 2010; City of San José, 2007; Dayton, 2010; FOG Energy Corporation, 2012; Long et al., 2012; Wiltsee, 1998). A GT is a small passive or mechanized oil/water separator installed inside the FSEs with a typical volume of around 190 L, while a GI is typically a larger (3,800 to 7,600 L) oil/water/food solids passive separator (Long et al., 2012). Adequate residence time allows the FOG to congeal and float to the top of the basin, the food solids to settle to the bottom, and the remaining wastewater to continuously flow down to the collection systems (Aziz et al., 2011; Chapin, 2008; Gallimore et al., 2011). As a result, GIW can be roughly divided into three layers: top layer of FOG, middle layer of associated wastewater, and bottom layer of settled food particles. The characteristics of this complex mixture may vary with time, the type of FSE, and configuration and maintenance frequency of GIs/GTs (Aziz et al., 2012; He et al., 2012; Lesikar et al., 2006). 1.2 Anaerobic Co-Digestion Biogas from anaerobic digestion can be used to offset thermal energy and electricity needs at wastewater treatment facilities (WWTFs) or exported as renewable energy to the grid. Despite the benefits of anaerobic digestion, of the 544 operational WWTFs in the United States with influent flow rates greater than 19 × 106 L/d employing anaerobic digestion, only about 19% use biogas to offset onsite energy demand and/or generate electricity, as identified by the 2004 Clean Watersheds Needs Survey (CWNS) (US EPA, 2007). A more recent update to the biogas utilization numbers in the U.S. stated that of the 1,351 WWTFs with influent flow rates greater than 3.8 × 106L/d operating anaerobic digesters, only 15% employ biogas utilization technologies (US EPA, 2011). Moreover, it was found that smaller WWTFs do not commonly use anaerobic digesters for solids management, let alone implement biogas utilization. In many cases, smaller facilities operating anaerobic digesters often find biogas utilization less economically or operationally appealing than flaring excess biogas (US EPA, 2007; US EPA, 2011).   2 One way to improve the economics of biogas utilization is to substantially increase biogas production. This can be achieved by co-digesting biosolids with substrates of high bioenergy potential such as FOG waste streams. FOG wastes are often high in biodegradable volatile solids (VS) ranging from 17% to 93% (w/w) and chemical oxygen demand (COD) up to 1,211 kg/m3 depending on their origins (Battimelli et al., 2010; Davidsson et al., 2008; Kabouris et al., 2009; Luostarinen et al., 2009). Similar FOG-based wastes include: animal fats and waste oils from food or processing plants, the edible oil industry, the dairy products industry, and slaughterhouses; mixed FOG wastes from receiving or dewatering facilities, and trapped grease wastes from WWTFs (Appels et al., 2011; Long et al., 2012; US EPA, 2004). Co-digestion of these wastes not only serves as a disposal method but enhanced bioenergy production can help WWTFs achieve energy independence. 1.3 The challenge: GIW overloading and inhibition Anaerobic digestion includes hydrolysis, acidogenesis, acetogenesis, and methanogenesis in either a mesophilic or a thermophilic environment. This complex metabolism involves many microbial groups and close syntrophic cooperation between acetogenic bacteria and methanogenic archaea (McCarty, 1964; Parkin and Owen, 1986; Speece, 1996). When introducing FOG-rich wastes that are high in biodegradable VS, COD, and lipid concentrations, the challenge is to avoid overloading and inhibition of methanogenesis. Efficient degradation of major intermediates such as long chain fatty acids (LCFAs), acetate, propionate, and butyrate is crucial to prevent drops in pH, imbalance of the major metabolic steps, and consequently, process failure. The inhibition and toxicity of LCFAs to microorganisms have been documented in many studies conducted with synthetic LCFAs (Angelidaki and Ahring, 1995; Hanaki et al., 1981; Hwu et al, 1998; Koster and Cramer, 1987; Palatsi et al., 2010; Rinzema et al., 1994). Inhibitory effects of LCFAs including substrate and product transport limitation, damage to cell membrane, increased lag phase of methane production, methanogenic activity loss, sludge flotation and washout, to name a few, were reviewed and summarized in Alves et al. (2009), Chen et al. (2008), and Long et al. (2012). To date most of the studies were conducted with single-source LCFAs, and the presence of commingled wastes such as GIW/GTW that comprises mixed FOGs, food residuals, wastewater, and possibly detergents and other substances derived from FSEs may further complicate and accentuate the inhibition. In addition to identifying the potential inhibitors and inhibition mechanism, the limit for GIW/GTW addition where the microbial communities survived the inhibition was once identified in the range of 30 to 100% (w/w) of VS added (Davidsson et al., 2008). For anaerobic co-digestion of other FOG waste streams, a range between 64 to 71% (w/w) of VS added can be identified by combining the data of Davidsson et al. (2008), Kabouris et al. (2009), Luostarinen et al. (2009), and Wan et al. (2011). In general, co-processing FOG-rich materials with sewage sludge increased methane production by 9% to 317%, depending on the source of startup substrate and co-substrate, total organic loading rate (OLR), OLR of co-substrate, solid retention time (SRT), mixing intensity, and feeding strategy and frequency (Davidsson et al., 2008; Kabouris et al., 2009; Luostarinen et al., 2009; Wan et al., 2011; Wang et al., 2013). Successful applications of anaerobic co-digestion with FOG wastes have been reported worldwide in lab scale (Kabouris et al., 2009; Luostarinen et al., 2009; Wan et al., 2011; Wang et al., 2013), pilot   3 scale (Davidsson et al., 2008), and full scale (Bailey, 2007; Cesca et al., 2010; Downey et al., 2010; Gabel et al., 2009; Muller et al., 2010; Johnson et al., 2011; York et al., 2009). However, to date an in-depth evaluation of the threshold for GIW/GTW co-digestion is still lacking. Challenging the microbial community with GIW to enhance methane production increases the risk of co-substrate overdose and LCFA inhibition, but also creates an opportunity for microbial adaptation of methanogenic archaeal and LCFA-degrading bacterial communities. Exposures to inhibitors such as ammonia, sulfide, sodium, insoluble organic compounds, and LCFAs at various concentrations have been reported to lead to adaptation of microorganisms to various degrees, as summarized in Chen et al. (2008). Biomass adaptation and digester recovery from LCFA inhibition were observed in microbial communities treated with synthetic LCFAs (Alves et al., 2001; Cavaleiro et al., 2001; Cavaleiro et al., 2008; Cavaleiro et al., 2009; Nielsen and Ahring, 2006; Palatsi et al., 2009; Palatsi et al., 2010), and FOG-containing wastes (Nadais et al., 2006; Silvestre et al., 2010; Wang et al., 2013). The adaptation may be the result of population adaptation when the microbial population shifts towards the better adapted microorganisms or due to phenotypic adaptation (physiological acclimation) when internal changes of existing microorganisms occur (Chen et al., 2008; Palatsi et al., 2010; Zeeman et al., 1985). Although the exact mechanism is still not clear, it is believed that adaptation of microorganisms may decrease the inhibitory effect of toxicity shock and increase the biodegradability of undesired substrates (Chen et al., 2008; Silvestre et al., 2010; Stuckey et al., 1980; Wu et al., 1993). In our recent study we challenged an anaerobic digester with step feedings of GIW from 46%, 66%, to 84% (w/w as VS) in an attempt to induce microbial adaptation and evaluate the limit of anaerobic co-digestion of GIW (Wang et al., 2013). Unlike lab-scale experiments where GIW loading rate can be customized, full-scale WWTFs can encounter fluctuations in GIW loading because of availability, seasonal changes in characteristics and collection areas, and introduction of new sources. These factors may make a step feeding approach, which relies on a stable GIW loading that increases in discrete steps, difficult. Several researchers have used pulse feeds (short periods of overloading) of synthetic LCFAs and other waste-based materials to evaluate LCFA inhibition (Cavaleiro et al., 2001; Cavaleiro et al., 2008; Cavaleiro et al., 2009; Neves et al., 2009; Nielsen and Ahring, 2006; Palatsi et al., 2009; Palatsi et al., 2010). These studies demonstrate the possibility of using a pulse feeding strategy to induce tolerance and adaptation in methanogenic archaeal and syntrophic acidogenic populations. Pulse feeds of GIW may be a feasible feeding strategy instead of step feeding to better simulate the fluctuating strengths in feedstock and to evaluate the ability of digesters to accept GIW at different concentrations.       4 1.4 Research Objectives The objectives of this study were to: O1 To determine the limits of anaerobic co-digestion operation with varied composition of GIW (i.e. the ratio of FOG/Food Solids) as the co-digestion feedstock. O2 Explore bioreactor operation and microbial communities that are functionally resilient, robust and resistant to variations in FOG loading O3 Evaluate the steady-state stability of co-digested biosolids during experimentation. Objective 1 (O1) was investigated through a series of bio-methane potential (BMP) tests to explore methane yield from FOG and food solids (Sections 2.6 and 3.4). O3 was explored in parallel with O2 during operation of lab-scale anaerobic digesters. O2, the most complex of the objectives, required the most time and was investigated through a combination of experimental reactor operation and next-generation sequencing (Sections 2.1-2.5, 3.1-3.3, 4.1-4.5). Two sets of lab-scale anaerobic digestion experiments were performed in this study. In experiment I, we continued our previous study (Wang et al., 2013) to further evaluate: (i) if step feeding can create more robust microbial communities against GIW inhibition by inducing microbial adaptation; (ii) if both GIW-adapted and non-GIW-adapted communities can recover from a onetime loading shock and if so (iii) if they can achieve the same level of methane production or even tolerate a higher GIW loading rate. Additionally, in experiment II we sought to evaluate: (i) if challenging the anaerobic co-digester with periodic pulse feeds can result in more robust microbial communities, (ii) if pulse feeding can enhance methane production, and (iii) the use of ecological parameters such as resistance (magnitude of change in accumulated concentration of major intermediates) and resilience (time taken by the accumulated intermediates to return to its referential state) (Botton et al., 2006; Werner et al., 2011) as measures of digester robustness. As data was collected over the course of experimentation and analysis, various aspects of the project were modified. The most significant modification was the shift to a next-generation sequencing procedure (Section 2.5) for the analysis of microbial population shifts resulting from GIW loading. This approached was deemed more rigorous and useful for the objectives of this study. Another substantial change to the initially proposed research was the use of the biochemical methane potential test for evaluating GIW variation. This change was based on reviewer comments and the research team believed this approach to be better than the one initially proposed.       5 2. Methods   2.1. Experimental setup The anaerobic co-digestion experiments were conducted using two identical anaerobic reactor systems operating in parallel as shown in Figure 1, each configuration consists of a feeding and decanting system, a reactor system, and a biogas collection system. Figure 1 – Reactor set-up. The reactor chamber was a Plexiglas tube with an inner diameter (ID) of 15 cm and a total volume of 8 L (working volume of 6 L).The cover was clamped to the reactor flange with 12 bolts and a lubricated O-ring between the reactor cover and the grooved flange kept the reactor airtight. The cover had ports serving as a feeding port, a biogas/foam outlet, and a liquid recycle line. Four staggered nylon tube fitting adapters were fitted along the side of the chamber with the bottom opening serving as a decant port. Openings that were not in use were sealed using screw tubing clamps. Two other adapters were inserted along the side of the chamber to connect with a Masterflex peristaltic pump (Cole-Parmer Instrument Company, Vernon Hills, IL) that generated a circular mixing pattern. The peristaltic pumps were controlled by a ChronTrol timer (Chrontrol Corporation, San Diego, CA). The initial mixing intensity for the start-up period (phase 1, Figure 2) was 7.5 W for 2 min/hr, providing a 2.1×10-5 m3/s jet flow rate. Mixing intensity was subsequently adjusted by increasing the mixing duration. Constant mixing during feeding was provided by the mixer. Periodic feeding and decanting were performed through the feeding and decant ports using a Masterflex peristaltic pump (Cole-Parmer Instrument Company, Vernon Hills, IL). Biogas and foam produced from the reactor were led to a recycle bottle and excessive foam was directed back to the reactor through the recycle line. The biogas passed through a hydrogen   6 sulfide (H S) scrubber bottle with steel wool then through a 3-way stopcock where biogas 2 sampling took place, and finally to a wet tip gas meter (Wet Tip Gas Meter.com, Nashville, TN) for biogas production measurement. FOG 10% v/v Food particles 40% v/v Water 50% v/v TWAS 110 100 90 v/v) 80 % ( 70 nt e 60 nt o k c 50 c o st 40 d e e 30 F 20 10 0 1 2 3 4 5 Phase   Figure 2 – Reactor loading schedule. 2.2. Substrates and inoculum Sludge from the anaerobic digester at the South Durham Water Reclamation Facility in North Carolina was used as inoculum, and thickened waste activated sludge (TWAS) obtained from the North Cary Water Reclamation Facility in North Carolina was used as feedstock. GIW provided by a FSE in Cary, North Carolina was used as co-substrate. FOG, food particles, and wastewater portions within GIW were separated upon collection at the FSE with the help of professional grease waste haulers. The feedstock, co-substrate, and inoculum were stored at 4°C within 3 hours of collection. Food particles collected were further mixed using a blender station to obtain homogeneous mixtures. TWAS and GIW were mixed thoroughly into desired portions an hour before feeding in the temperature-controlled room (37°C) in a feedstock container (Figure 1) using a mixer. 2.3. Experimental design The anaerobic digesters were operated in a temperature-controlled room to maintain mesophilic conditions (37°C) and fed every other day in a draw-and-fill semi-continuous mode with a solids   7 retention time (SRT) of 20 days. Each feed time, 600 mL effluent was removed through the decant port while 600 mL feedstock was pumped into the reactor through the feeding line. Experiment I was conducted to explore the limit of anaerobic co-digestion of sewage sludge with GIW by stepwise increase of GIW loading rate. During phase 1 (start-up), both reactors (control and treated) were fed only TWAS. A mixing intensity experiment was implemented in phase 2 for digester B. After the optimal mixing intensity was obtained, the mixing duration of digester A (control) was adjusted to this desired value. In phases 3 to 5, GIW addition in the treated digester was increased from 10 to 40% (v/v), or 46 to 84% (w/w as VS), to evaluate the optimal GIW fraction for maximum biogas production without digester inhibition, followed by perturbation and recovery tests at 66 and 75% (w/w as VS). In experiment II, six research phases were designed to evaluate the feasibility of applying repeated pulses of GIW to anaerobic digesters to develop more robust communities and produce higher methane production. During the start-up periods, both reactors were fed only TWAS at an OLR of 1.33 g L-1 day-1 in phase 1, and a mixture of TWAS (70% (w/w) as VS) and GIW VS (30% (w/w) as VS) at an OLR of 1.64 g L-1 day-1 in phase 2. In phase 3, four pulse-fed cycles VS were applied to the treated digester at an OLR of 2.24 g L-1 day-1 which corresponds to a GIW VS input of 60% (w/w as VS) in addition to regular feeding at 1.64 g L-1 day-1 (30% (w/w) as VS). VS During phases 4 to 6, to evaluate whether more robust microbial populations can be enhanced by pulse treatment, both digesters were challenged at higher GIW loads of 70 and 90% (w/w) as VS at 2.95 and 4.48 g L-1 day-1, respectively. VS 2.4. Analytical methods Biogas production was measured with a wet tip gas meter (Wet Tip Gas Meter.com, Nashville, TN) and normalized to STP based on the daily local climatological data (NCDC, 2012; NCDC, 2013). Methane content in biogas was analyzed by a gas chromatograph (GC, SRI 8610C) equipped with a thermal conductivity detector. Total solids (TS), VS, alkalinity, and pH were measured according to Standard Methods (APHA, 2005). Concentrations of individual volatile fatty acids (VFA; e.g., acetic, propionic, butyric, and valeric acids) were determined by acidification, centrifugation, filtration, and direct injection into a GC (GC-2014 Shimadzu) equipped with a flame ionization detector according to the VFA gas chromatographic method (5560 D) in Standard Methods (APHA, 2005). Total VFA (TVA; VFAs up to six carbon atoms) concentration was measured by centrifugation, acidification, distillation, and titration according to the TVA distillation method (5560 C) in Standard Methods (APHA, 2005). 2.5. Illumina sequencing of genomic DNA obtained from experiments I and II Molecular analysis using next generation sequencing (Illumina) assessed microbial population dynamics inside the mother digesters (i.e. the reactors described in Figure 1 used for Experiment I and II). Genomic DNA (gDNA) samples were extracted from effluent sludge using aluminum sulfate DNA extraction method. Forward and reverse primer pair sequences, modified 341F (CCTAYGGGRBGCASCAG) and modified 806R (GGACTACNNGGGTATCTAAT), respectively, were used to amplify a DNA fragment of ~460 bp length flanking the V3 and V4 regions of the 16S ribosomal RNA (rRNA) gene of bacteria and archaea in the gDNA samples   8