**Abhinav Choudhury and Stephanie Lansing \***

Department of Environmental Science and Technology, University of Maryland, University of Maryland Energy Research Center, 1429 Animal Science/Ag Engineering Bldg., College Park, MD 20742, USA; abhinavc@terpmail.umd.edu

**\*** Correspondence: slansing@umd.edu; Tel.: +1-(301)-405-1197

Received: 1 November 2019; Accepted: 19 November 2019; Published: 23 November 2019

**Abstract:** Co-digestion of dairy manure with waste organic substrates has been shown to increase the methane (CH4) yield of farm-scale anaerobic digestion (AD). A gummy vitamin waste (GVW) product was evaluated as an AD co-digestion substrate using batch AD testing. The GVW product was added at four inclusion levels (0%, 5%, 9%, and 23% on a wet mass basis) to a co-digestion substrate mixture of dairy manure (DM), food-waste (FW), and grease-waste (GW) and compared to mono-digestion of the GVW, DM, FW, and GW substrates. All GVW co-digestion treatments significantly increased CH<sup>4</sup> yield by 126–151% (336–374 mL CH4/g volatile solids (VS)) compared to DM-only treatment (149 mL CH4/g VS). The GVW co-digestion treatments also significantly decreased the hydrogen sulfide (H2S) content in the biogas by 66–83% (35.1–71.9 mL H2S/kg VS) compared to DM-only (212 mL H2S/kg VS) due to the low sulfur (S) content in GVW waste. The study showed that GVW is a potentially valuable co-digestion substrate for dairy manure. The high density of VS and low moisture and S content of GVW resulted in higher CH<sup>4</sup> yields and lower H2S concentrations, which could be economically beneficial for dairy farmers.

**Keywords:** biochemical methane potential; biogas; anaerobic digestion

### **1. Introduction**

Anaerobic digestion (AD) of organic substrates with dairy manure, also known as co-digestion, can increase biogas production and result in higher return on investment for dairy farmers [1]. Biogas produced from AD is a combination of 50–75% methane (CH4) and 25–50% carbon dioxide (CO2), with trace levels (0.01–1%) of hydrogen sulfide (H2S) that can be used as a source of renewable energy for heat and power generation [2]. Limitations from mono-digestion of organic materials arise from substrate properties, such as unbalanced C:N ratios, recalcitrance in the feedstock, high concentrations of long chain fatty acids, and deficiency in trace minerals required for the growth of methanogens [1,3]. These limitations can lead to unfavorable economics for dairy farmers using AD to generate energy on-farm [1,4]. Furthermore, positive synergy from co-digestion of a mixture of substrates can lead to more CH<sup>4</sup> production than the addition of CH<sup>4</sup> produced from mono-digestion of each individual substrate. A review by Mata-Alvarez et al. (2014) reported that co-digestion of carbon (C)-rich organic matter with cattle and poultry manure resulted in up to 3.5 times more CH<sup>4</sup> production than the CH<sup>4</sup> potential of the individual substrates [3]. Lisboa and Lansing (2013) reported a maximum of 29.4 times more CH<sup>4</sup> yield when dairy manure was co-digested with chicken processing waste compared to mono-digestion of dairy manure [5]. Moody et al. (2011) determined the biomethane potential of a wide range of food waste substrates and concluded that co-digestion of manure and

organic waste has the potential to increase biogas production, and in turn, increase energy generation from AD [6]. However, often studies are only applied to individual substrates due to differences in organic waste composition and collection.

Previous research on co-digestion of food waste and dairy manure has primarily focused on the CH<sup>4</sup> production potential of co-substrates [7–9], with limited data on the effects of co-digestion substrate selection on the production of H2S [10]. The production H2S in biogas occurs when sulfur-containing compounds, such as sulfates, sulfites, and thiosulfate, in AD substrates are reduced by sulfate-reducing bacteria (SRB) under anaerobic conditions [11]. High H2S concentrations in biogas (0.05–1% by vol.) can become a major problem when utilizing the biogas due to health concerns and corrosion of biogas equipment [12]. Combined heat and power (CHP) systems usually require H2S concentrations to not exceed 500 ppm to prevent reduced performance from corrosion, and H2S concentrations over 100 ppm can lead to severe adverse human health impacts [10]. Most dairy farms use CHP systems to generate energy for on-farm use and lower H2S concentrations can lead to improved energy generation efficiencies and reduced maintenance. Corro et al. (2013) observed a reduction in H2S concentrations when coffee waste was co-digested with dairy manure compared to digestion of dairy manure only, but there was no discussion of the cause for the observed H2S differences [13]. Research has shown that co-digestion of organic matter with higher C:N ratios in manure-based digesters can reduce ammonia inhibition and enhance methane production [3]. Co-digestion of carbon-rich organic matter with a low sulfur (S) content may also reduce the H2S concentration in the biogas when compared to the mono-digestion of dairy manure and prevent sulfide inhibition.

Industrial food waste comprises 5% of the total food waste generated globally [14]. Although the fraction of industrial food waste is significantly less than food waste from other sources, it has logistical and economic advantages due to its high-volume generation at specific points and homogenous nature. Valorization of these industrial food waste streams can help mitigate disposal costs in landfills, while providing a source of tipping fees for dairy farmers with AD systems. The waste produced from gummy vitamin industries is high in degradable C compared to dairy manure. Production of gummy vitamin waste (GVW) from a single manufacturing facility can be up to 10% of the total weight of the product produced [15]. For example, one multi-national gummy vitamin manufacturing company produces approximately 100 million gummy vitamins daily, with a daily production of 500 tons of gummy product (5 g per gummy vitamin), resulting in approximately 50 tons/day of GVW produced [16]. Most of this waste product is landfilled, with some composting and incineration being practiced in the EU [15,17]. The GVW material can contain up to 70% sugar and gelatin, with starch or pectin-based gels that create the unique structure that is characteristic of gummy candies [18]. Due to its high sugar content, GVW can be a valuable resource for AD, yet the dense jelly-like consistency may lead to issues, such as a slow degradation rate, increased hydraulic retention time, or possible pipe clogging within the AD system. It is also possible that GVW with a high C:S ratio could reduce the H2S concentration in the biogas when co-digested with dairy manure.

The main goal of the project was to evaluate a GVW product as a co-digestion substrate for AD. The specific objective was to evaluate the CH<sup>4</sup> and H2S production and VS degradation of a GVW substrate when co-digested with a dairy manure (DM), food waste (FW), and grease waste (GW) mixtures (DM.FW.GW). A co-digestion mixture was used for testing, as many on-farm digesters incorporate multiple waste streams and to highlight the benefits of testing co-substrates as both mixtures and single substrates. Co-digestion of the tested mixtures was expected to produce a significantly higher amount of CH<sup>4</sup> and lower H2S compared to the mono-digestion of DM.

#### **2. Materials and Methods**

#### *2.1. Sample Collection*

Anaerobic digester effluent (inoculum source) and the GVW product were collected from a Northeastern US farm. The farm co-digested dairy manure from heifers with gummy vitamin waste, food waste, and grease waste at a 64% DM, 9% GVW, 16% FW, and 11% GW ratio, by mass. The AD effluent sample was utilized as an inoculum source, as it had been pre-acclimated to the GVW material used at the farm. The GW and FW were collected from a local supermarket. Un-separated dairy manure from the USDA Beltsville Agricultural Research Center (BARC) in Beltsville, MD, USA, was utilized as the DM substrate. Field samples were collected and brought back to lab on ice. The mean total solids (TS) and volatile solids (VS) data for the substrates used in the experiment are shown in Table 1.

**Table 1.** Total and volatile solids content of the individual substrates (gummy vitamin waste, food waste, grease waste, dairy manure) and digester effluent (inoculum) used for the experiment.


#### *2.2. Experimental Design*

The GVW product was added to individual batch digesters at four inclusion levels (0%, 5%, 9%, and 23% on a wet mass basis) to a co-digestion substrate mixture of dairy manure (DM), food-waste (FW), and grease-waste (GW) and compared to mono-digestion of the GVW, DM, FW, and GW substrates, with an inoculum control. The 9% GVW treatment (64% DM, 16% FW, 11% GW by mass) represented the mixture that was used at the farm during the time of AD effluent collection. An inoculum-to-substrate ratio (ISR) of 1:1 (VS basis) was used for the experiment. Table 2 shows the experimental design and the descriptions of the treatment levels for the experiment, with each treatment conducted using triplicate AD reactors. All mass data are expressed on a wet mass basis.

**Table 2.** Experimental design using a 1:1 inoculum-to-substrate ratio, with the calculated initial total solids (TS) and volatile solids (VS) of the treatment mixtures. The percent of gummy vitamin waste (GVW) inclusion was based on mass. All treatments were conducted in triplicate.


### *2.3. Biochemical Methane Potential (BMP) Test Procedures*

The batch laboratory testing followed the biochemical methane potential (BMP) protocol, which is a laboratory batch study used to characterize CH<sup>4</sup> production potential [6]. Substrate and inoculum were added into 300 mL serum bottles, purged with N<sup>2</sup> gas to establish anaerobic conditions, capped, and incubated at 35 ◦C in an environmental chamber. Biogas, CH4, and H2S concentrations were monitored at regular intervals for 67 days, at which point the daily biogas production was less than 1% of the cumulative biogas production for the treatments, indicating biogas production had largely ceased. The mass of substrate and inoculum in each bottle ranged from 31.4 to 58.8 g (Table 2) to keep the ISR at 1:1 for all treatments.

The quantity of biogas produced was measured using a graduated, gas-tight, wet-tipped 50 mL glass syringe inserted through the septa of the digestion reactors and equilibrated to atmospheric pressure. Biogas samples were collected in 0.5 mL syringes and tested on a gas chromatograph (Agilent 7890) using a thermal conductivity detector (TCD) at a detector temperature of 250 ◦C for CH<sup>4</sup>

and H2S concentrations. The average CH<sup>4</sup> and H2S production in the triplicates from the inoculum control was subtracted from the other treatments to present the total CH<sup>4</sup> production from the waste substrates only.

#### *2.4. Analytical Methods*

The treatment mixtures were analyzed for pH before and after digestion using an Accumet AB15 pH meter. Triplicate samples were tested for TS and VS, according to Standard Methods for the Examination of Water and Wastewater (APHA-AWWA-WEF, 2005) within 24 h of collection. For TS analysis, triplicate 10.0 mL samples were pipetted into pre-weighed porcelain crucibles. The samples were then dried at 105 ◦C until a constant mass was obtained for the TS concentration. The crucibles were then placed in a furnace at 550 ◦C until a constant weight was obtained to determine VS concentration. The gummy waste, dairy manure, and inoculum (digester effluent) were tested for total metals (iron, zinc) and sulfur using ICP-MS (inductively coupled plasma mass spectrometry), and total nitrogen using A3769 Methods for Manure Analysis at Agrolabs Inc., Harrington, DE, USA, [19]. The C:N ratio was calculated using the conversion factor from Adams et al. (1951) stating that 55% of the VS content is carbon [20]. The calculated C value and the measured N value were used to derive the C:N ratio.

#### *2.5. Statistical Analysis*

Collected data were reviewed in accordance with QA/QC procedures and analyzed for significant differences in biogas quantity, CH4, H2S, TS, VS, and pH using ANOVA and Tukey-Kramer post-hoc multiple mean comparison tests of the reviewed data using SAS ® statistical software package. Tests of significance were conducted with an alpha value set at 0.05. Data are reported as averages with standard errors (SE).

#### **3. Results**
