*3.1. Methane (CH4) Production*

The co-digestion mixtures 0–23% GVW.DM.FW.GW had a significantly higher percent CH<sup>4</sup> in the biogas compared to the mono-DM digestion (*p*-value < 0.0001; Table 3). However, there were no significant differences in the percent CH<sup>4</sup> among the co-digestion mixtures, with a non-significant trend in increasing percent CH<sup>4</sup> as the percent of GVW increased (Table 3). The cumulative CH<sup>4</sup> production over the 67 day AD period was normalized using two methods: (1) the total mass of the substrate added (mL CH4/g substrate), as this normalization provides an estimate of CH<sup>4</sup> production that can be readily used by farmers, and (2) the VS of the substrate (mL CH4/g VS added) for comparison with other studies [5].


\* The % CH<sup>4</sup> shown is the average value from Days 53–67 of the experiment. # The CH<sup>4</sup> production from the inoculum was subtracted from all treatments, resulting in zero values when the inoculum outperformed the treatment.

As expected, the co-digestion treatments (with and without GVW addition) produced 359–524% more CH<sup>4</sup> compared to mono-DM digestion, when normalized by the mass of substrate added (Table 3). Normalized CH<sup>4</sup> production in co-digestion without GVW (DM.FW.GW-only) was 11.6% lower than the 5% GVW.DM.FW.GW mixture, 14.5% lower than 9% GVW.DM.FW.GW mixture, and 36.3% lower than the 23% GVW.DM.FW.GW mixture (Table 3; Figure 1). The CH<sup>4</sup> production in the 23% GVW.DM.FW.GW mixture was the highest among all treatments, as expected. The total normalized volume of CH<sup>4</sup> increased linearly with the mass percent of GVW added (r<sup>2</sup> = 0.9866) (Figure 2). *Energies* **2019**, *12*, x FOR PEER REVIEW 5 of 12 GVW.DM.FW.GW (23% GVW) 71.1 ± 1.0 336 ± 12 76.3 ± 2.7 68.3 ± 16.6 15.5 ± 3.8 \* The % CH4 shown is the average value from Days 53–67 of the experiment. # The CH4 production from the inoculum was subtracted from all treatments, resulting in zero values when the inoculum outperformed the treatment.

**Figure 1.** Methane (CH4) production normalized by gram of substrate (mLCH4/g substrate) ((**A**), top) and by gram of volatile solids (mL CH4/g VS) ((**B**), bottom) in the batch digestion testing of gummy vitamin waste (GVW), grease waste (GW), food waste (FW), and dairy manure (DM) digested **Figure 1.** Methane (CH<sup>4</sup> ) production normalized by gram of substrate (mL CH<sup>4</sup> /g substrate) ((**A**), top) and by gram of volatile solids (mL CH<sup>4</sup> /g VS) ((**B**), bottom) in the batch digestion testing of gummy vitamin waste (GVW), grease waste (GW), food waste (FW), and dairy manure (DM) digested singularly and as a mixture (DM.FW.GW), with the percent inclusion of GVW shown for the co-digestion mixtures.

singularly and as a mixture (DM.FW.GW), with the percent inclusion of GVW shown for the co-

digestion mixtures. When the total CH4 produced was normalized by the quantity of organic material added (mL CH4/g VS), the 23% GVW.DM.FW.GW mixture was significantly lower than the DM.FW.GW mixture with 0% GVW (*p*-value = 0.0156) and 5% GVW.DM.FW.GW mixtures (*p*-value = 0.0122) (Table 3), with no significant differences between the other co-digestion treatment groups. Mono-GVW When the total CH<sup>4</sup> produced was normalized by the quantity of organic material added (mL CH4/g VS), the 23% GVW.DM.FW.GW mixture was significantly lower than the DM.FW.GW mixture with 0% GVW (*p*-value = 0.0156) and 5% GVW.DM.FW.GW mixtures (*p*-value = 0.0122) (Table 3), with no significant differences between the other co-digestion treatment groups. Mono-GVW digestion resulted in negligible CH<sup>4</sup> production (0 mL CH4/g VS) over 67 days of digestion due to subtraction of inoculum CH<sup>4</sup> production from each treatment, and higher CH<sup>4</sup> production values in the triplicate inoculum reactors

vessels (under pH 7) (Table 4).

digestion resulted in negligible CH4 production (0 mL CH4/g VS) over 67 days of digestion due to subtraction of inoculum CH4 production from each treatment, and higher CH4 production values in compared to the triplicate GVW-only AD reactors. Both treatments with negligible CH<sup>4</sup> production (mono-GVW and mono-FW) had low final pH levels in the digestion vessels (under pH 7) (Table *Energies*  4). **2019**, *12*, x FOR PEER REVIEW 6 of 12

**Figure 2.** Linear regression of normalized methane (CH4) production per gram of added substrate and percent gummy vitamin waste (GVW) within the co-digestion mixture. **Figure 2.** Linear regression of normalized methane (CH<sup>4</sup> ) production per gram of added substrate and percent gummy vitamin waste (GVW) within the co-digestion mixture.

**Table 4.** Average pH and volatile solids (VS) in all treatment mixtures pre-digestion (initial) and postdigestion (final). Initial VS data was calculated theoretically, and final VS data was determined experimentally. **Table 4.** Average pH and volatile solids (VS) in all treatment mixtures pre-digestion (initial) and post-digestion (final). Initial VS data was calculated theoretically, and final VS data was determined experimentally.


#### GVW.DM.FW.GW (23% GVW) 78.0 51.0 ± 2.6 34.6% 7.77 7.88 *3.2. Hydrogen Sulfide (H2S) Production*

levels, which led to low biogas production.

*3.2. Hydrogen Sulfide (H2S) Production*  The DM treatment produced biogas with a peak concentration of 2145 ppm H2S after 3 days of digestion (Figure 3). After this time, H2S levels decreased and no H2S was detected in the biogas by the 60th day of the experiment. The treatment with the next highest peak H2S concentration in the biogas was the 9% GVW.DM.FW.GW mixture (804 ppm H2S), which was 63% less than the DM treatment and 23% greater than the next highest treatment (DM.FW.GW-only mixture with 0% GVW) at 576 ppm H2S. The peak H2S concentrations for all treatments were observed within the first 2–3 days before peak CH4 production. The 23% GVW.DM.FW.GW treatment, DM and FW had detectable H2S concentrations in the biogas for the longest period (51 days). The mono-GVW treatment did not produce a measurable amount of CH4, but it had the shortest period of detectable levels of H2S (5 days). This is likely due to lowered microbiological activity within the digester due to the low pH The DM treatment produced biogas with a peak concentration of 2145 ppm H2S after 3 days of digestion (Figure 3). After this time, H2S levels decreased and no H2S was detected in the biogas by the 60th day of the experiment. The treatment with the next highest peak H2S concentration in the biogas was the 9% GVW.DM.FW.GW mixture (804 ppm H2S), which was 63% less than the DM treatment and 23% greater than the next highest treatment (DM.FW.GW-only mixture with 0% GVW) at 576 ppm H2S. The peak H2S concentrations for all treatments were observed within the first 2–3 days before peak CH<sup>4</sup> production. The 23% GVW.DM.FW.GW treatment, DM and FW had detectable H2S concentrations in the biogas for the longest period (51 days). The mono-GVW treatment did not produce a measurable amount of CH4, but it had the shortest period of detectable levels of H2S (5 days). This is likely due to lowered microbiological activity within the digester due to the low pH levels, which led to low biogas production.

The quantity of H2S produced showed an increasing trend with increases in the percent of GVW inclusion (0–23%) when normalized by kilograms of substrate addition (5.3–15.5 mL H2S/kg substrate; Table 3, Figure 4). The H2S production in the DM treatment (17.4 mL H2S/kg substrate) was

**Figure 3.** Hydrogen sulfide (H2S) concentration (ppm) in the biogas over time in the batch digestion testing of gummy vitamin waste (GVW), grease waste (GW), food waste (FW), and dairy manure (DM) digested singularly and as a mixture, with the GVW inclusion shown for each co-digestion mixture tested. **Figure 3.** Hydrogen sulfide (H2S) concentration (ppm) in the biogas over time in the batch digestion testing of gummy vitamin waste (GVW), grease waste (GW), food waste (FW), and dairy manure (DM) digested singularly and as a mixture, with the GVW inclusion shown for each co-digestion mixture tested.

The quantity of H2S produced showed an increasing trend with increases in the percent of GVW inclusion (0–23%) when normalized by kilograms of substrate addition (5.3–15.5 mL H2S/kg substrate; Table 3, Figure 4). The H2S production in the DM treatment (17.4 mL H2S/kg substrate) was significantly higher than the treatments co-digested with GVW (*p*-value = 0.0046). However, in the DM.FW.GW treatment (0% GVW), the normalized H2S production was the lowest among the co-digested treatments (5.3 mL H2S/kg substrate), and significantly lower than 23% GVW.DM.FW.GW (*p*-value = 0.0106) and DM (*p*-value = 0.0023) treatments. However, there were no significant differences for normalized H2S production between the 5–23% GVW inclusion (*p*-value = 0.633) treatments. **Figure 3.** Hydrogen sulfide (H2S) concentration (ppm) in the biogas over time in the batch digestion testing of gummy vitamin waste (GVW), grease waste (GW), food waste (FW), and dairy manure (DM) digested singularly and as a mixture, with the GVW inclusion shown for each co-digestion mixture tested.

**Figure 4.** Normalized hydrogen sulfide (H2S) production per kilogram of added substrate and percent gummy vitamin waste (GVW) within the co-digestion mixture. **Figure 4.** Normalized hydrogen sulfide (H2S) production per kilogram of added substrate and percent gummy vitamin waste (GVW) within the co-digestion mixture.

0.0003). However, there were no significant differences within the 5–23% GVW.DM.FW.GW

treatments (*p*-value = 1.000).

When the total H2S was normalized by the amount of VS added, the DM treatment (212 mL

When the total H2S was normalized by the amount of VS added, the DM treatment (212 mL H2S/kg VS) produced a significantly larger amount of H2S compared to all co-digestion treatments (*p*-value < 0.0001) (Table 3). The addition of GVW (68–72 mL H2S/kg VS) showed a significant increase in H2S production compared to the DM.FW.GW (0% GVW) treatment (35 mL H2S/kg VS; *p*-value = 0.0003). However, there were no significant differences within the 5–23% GVW.DM.FW.GW treatments (*p*-value = 1.000).

#### *3.3. E*ff*ect of Retention Time and Solids Degradation*

The percentage of CH<sup>4</sup> in the biogas of the DM treatments rose above 25% on the 11th day of digestion, while the treatments containing additional substrates (FW, GW, and GVW) had a longer lag phase and started producing higher quantities of CH<sup>4</sup> after 20 days of digestion (Figure 1), which is a relatively long lag-time for BMP analyses. The DM treatment produced 43% of its total cumulative CH<sup>4</sup> within the first 20 days, while all other treatments had less than 10% of the total cumulative CH<sup>4</sup> production during this time (Table 5). By the 41st day of the experiment, 89% of the total cumulative CH<sup>4</sup> from the mono-DM treatment had been produced, but the percent of total cumulative CH<sup>4</sup> from the GVW.DM.FW.GW and DM.FW.GW treatments by Day 41 varied from 57–80% of the cumulative CH<sup>4</sup> after 67 days of digestion. The effect of the longer retention times on GVW degradation was seen, as the CH<sup>4</sup> production rate for co-digestion was highest when no GVW was added (DM.FW.GW), with a maximum CH<sup>4</sup> production rate of 16.8 mL CH4/VS.day). The maximum CH<sup>4</sup> production rate decreased with increasing GVW inclusion (10.6–11.6 mL CH4/VS.day). The maximum CH<sup>4</sup> production rate was the lowest for DM (6.0 mL CH4/VS.day) for the treatments with CH<sup>4</sup> generation.

**Table 5.** Normalized methane production (mL CH<sup>4</sup> /g VS) after 20, 46, and 67 days, with the percentage of the cumulative CH<sup>4</sup> (Day 67) by Days 20 and 46 shown in parentheses.


The C:N ratios of the GVW (196:1) was high due to the high C (255 g C/kg GVW) and low N content (1.3 g N/kg GVW), which was much higher than the dairy manure (7.7:1) and inoculum (8.0:1) utilized. The TS and VS concentrations of the GVW showed that the VS comprised 99.7% of the total solids content (46.4% of the wet GVW). While a high percentage of the GVW was degradable, there was only a 34–35.2% degradation of VS during digestion (Table 4). While there was no CH<sup>4</sup> production from the mono-FW and mono-GW treatments, there was a decrease of >30% of the initial VS content, which can be attributed to the initial breakdown of the organic matter, resulting in CO2-enriched biogas production. Biogas volume for these treatments was over 200 mL during the first two days, with less than 0.5% CH<sup>4</sup> and over 35% CO<sup>2</sup> for mono-FW and over 50% CO<sup>2</sup> for mono-GVW treatments.

#### **4. Discussion**

Increasing the amount of GVW during digestion did increase CH<sup>4</sup> production, as expected. The GVW appeared to completely hydrolyze during digestion, with no visible trace of solid GVW in the post-BMP samples after 67 days of digestion. The GVW accounted for 5–23% of the total mass of substrate added, corresponding to 15–50% of the VS inclusion. The GVW product could be beneficial for farmers interested in co-digestion waste substrates that increase CH<sup>4</sup> production, but the longer retention time of the GVW compared to DM digestion should be taken into consideration.

The negligible CH<sup>4</sup> production and low pH values in the mono-GVW, FW, and GW treatments compared to the higher CH<sup>4</sup> production (336–374 mL CH4/g VS) and pH range (7.88–7.95) in treatments that co-digested GVW, FW, GW, and DM showed that the buffering capacity of the added co-substrates is important to mitigate accumulation of volatile fatty acids (VFA) and lowered pH [3,21]. Carbon-rich substrates can have a poor buffering capability, leading to an increased rate of VFA production and methanogenesis inhibition [3]. The mono-GW treatment had an initial pH of 7.79 but did not produce significant amounts of CH4, possibly due to the slow degradation rate of lipids in the grease waste. Previous studies have also shown that digestion of lipids without co-digestion required the use of lime as a pH stabilizer [22]. The use of a buffer for pH control in the experiment was avoided since the study was originally conducted to emulate the source farm conditions. The AD system on farm did not use any pH stabilizers, as the manure provided sufficient buffering capacity for the digestion process. Generally, the high alkalinity of manure increases digester resistance to acidification for high-fat and sugar content wastes and adds a nitrogen source for micro-organisms [23]. Another important parameter that likely resulted in negligible CH<sup>4</sup> production in the mono-GVW treatment was the high C:N ratio of GVW (196:1). High C:N ratios have been shown to result in low pH values during the digestion process and high VFA production [24]. As DM had a C:N ratio of 7.7:1 in this study, which is typical for DM, the resulting mixture in the co-digestion treatments likely increased the C:N ratio within the ideal range of 20–30 for AD, resulting in large increases in CH<sup>4</sup> yield for the co-digestion mixtures compared to the mono-digestion treatments [25].

All treatments produced large amounts of biogas during the first two days of digestion (ranging from 39 mL for DM to 379 mL for 23% GVW.DM.FW.GW), mostly composed of CO2. The biogas volume dropped sharply for all treatments (<10 mL per day) after Day 2, and the mono-DM treatment recovered the earliest (Day 11) and started producing > 50 mL biogas per day. The reduction in VS in the treatments with negligible CH<sup>4</sup> production for FW, GVW, and GW (Table 4) can be attributed to this initial burst of CO<sup>2</sup> enriched biogas production due to the initial breakdown of complex organic molecules. Bujoczek et al. (2000) showed that high organic loading rates may initially lead to large amounts of biogas, composed mainly of CO2, after which biogas production slows down [26]. In their study, the biogas production recovered after 30 days of digestion with CH<sup>4</sup> as the main component, similar to the results seen in this experiment. The authors also reported that the highest TS content for feasibility of digestion was 10%, while the shortest lag phase was obtained for 2.7% TS. The TS content in our experiment varied from 7.1% for DM to 11.6% for FW and showed similar CH<sup>4</sup> production trends to their study. The longer lag phase associated with a high TS content could be due to either high VFA concentrations or high ammonia concentrations or a combination of the two factors [26]. The CH<sup>4</sup> production in this study recovered after the lag phase, indicating acclimatization of the methanogenic bacteria to the initial inhibitory conditions, but the quantity of CH<sup>4</sup> generated from the DM treatment (149 ± 11 mL CH4/g VS) was 38–44% lower than the results obtained by Moody et al. (2011) for dairy manure (239–264 mL CH4/g VS) [6]. Witarsa and Lansing (2015) showed that the normalized CH<sup>4</sup> production on a VS basis is often lower for unseparated dairy manure due to the recalcitrant nature of the manure solids, leading to lower VS conversion efficiency [27].

It was expected that CH<sup>4</sup> production normalized by VS in the GVW co-digested treatments would be similar, but a decreasing trend with increasing percent GVW was observed. Normalization by VS illustrates the efficiency of organic material conversion to CH4. As GVW is a dense substrate in terms of grams of VS per gram of substrate, the increase in GVW inclusion decreased the efficiency and rate of converting the VS to CH4. The longer lag phase and the larger CH<sup>4</sup> production rates in the GVW treatments compared to DM.FW.GW and DM-only, from Days 41 to 67, suggests that long retention times would be needed to receive the full increase in expected CH<sup>4</sup> production. This effect was also seen by Kaparaju et al. (2002) when black candy, chocolate, and confectionary by-products were digested with dairy manure for 160 days in order to obtain a complete cumulative CH<sup>4</sup> value, with similar normalized CH<sup>4</sup> production for the confectionary waste (320–390 mL CH4/g VS) compared to the GVW.DM.FW.GW treatments (336–374 mL CH4/g VS) [28].

In all treatments, the VS degradation was low compared to studies conducted by Lisboa and Lansing (2013) and Li et al. (2013), where the VS degradation rates ranged from 48–93% [5,29]. Only 19.3% of the initial VS content of the mono-DM treatment was degraded at the end of the experiment, illustrating recalcitrance in the manure feed. The VS degradation was consistent with co-digestion studies of forage radish and dairy manure by Belle et al. (2015b), which used the same manure source as this study with a 21.3% reduction in VS concentration in the mono-DM treatment [30]. The VS degradation of our study (30.9–35.2%) was also comparable to the aforementioned study (30.8–39.7%), with 50–80% co-digestion substrate with dairy manure.

In a review conducted by Xie et al. (2018), it was reported that addition of a carbon-rich substrate to sewage sludge digestion may lower the H2S concentration due to a dilution effect [31]. This dilution effect can be attributed to a proportionally higher biogas yield compared to the additional H2S produced from the co-digested substrates. The S concentration for GVW (212 ppm S) was lower than the inoculum source (368 ppm S), and unseparated dairy manure slurries with a TS content of 7% (average 400 ppm S) [32]. The low sulfur concentrations combined with the high VS content (46.3%) of GVW, in comparison to DM (8.2 % VS), provide more evidence to the dilution effect observed in the study, as previously hypothesized. Since more biogas was produced in the GVW treatments compared to the DM treatments, the relative percent of the biogas attributed to manure in the mixed substrate treatments was lowered, and thus, the relative contribution of H2S from the manure substrate also decreased. Furthermore, the contribution of H2S from GVW was comparatively lower due to its low sulfur content, leading to the overall decrease in H2S concentrations in the biogas. However, it should be noted that the GVW addition as a co-digestion substrate increased total normalized H2S production when compared to co-digestion with 0% GVW addition (DM.FW.GW). A co-digestion substrate with negligible S content could have led to further decreases in H2S concentrations and total yield. Some gummy vitamins are fortified with Fe, but the concentrations seen in this study (4.3 ppm Fe) was lower than the Fe concentrations in food waste (4800 ppm) and unlikely to have affected H2S production in our study [33].

The sulfurous compounds in the feedstock were primarily utilized during the initial phase of digestion as most of the H2S was produced within the first 20 days, after which the CH<sup>4</sup> percentage started rising for all treatments. Similar results were also observed by Belle et al. (2015b) when co-digesting different mass fractions of forage radish with dairy manure in BMP experiments [30]. Forage radish has a high sulfur content and increasing the forage radish percentage led to an expected increase in H2S production initially, but all the treatments had lowered and similar H2S production by the end of the study. Belle et al. (2015a) also conducted a pilot-scale study on the same substrates and showed an increased rate of H2S production during the first two weeks of digestion, after which, the concentration decreased by >75% of the maximum H2S concentration for the remainder of the digestion period (33 days total) [10]. These observations can be attributed to increased SRB activity during the initial digestion phase, as SRBs can outcompete methanogens when the availability of biodegradable sulfur is higher.

#### **5. Conclusions**

Results from the BMP study suggested that gummy waste is a potentially valuable co-digestion substrate with dairy manure. The mixture of substrates containing gummy waste, food waste, grease waste, and dairy manure enhanced CH<sup>4</sup> yields compared to digestion of dairy manure alone. The high density of VS and low moisture content of the gummy waste results in high CH<sup>4</sup> yields per gram of the substrate, but due to the slower degradation rate of the GVW, higher retention times may be needed to yield these higher CH<sup>4</sup> potentials. Co-digestion of GVW with dairy manure lowered the H2S yield and maximum H2S concentration compared to mono-digestion of dairy manure due to its low sulfur content. The research highlighted the significance of testing co-digestion mixtures in conjunction with single substrates for both CH<sup>4</sup> and H2S to provide beneficial information for researchers and AD practitioners. Co-digestion of industrial byproducts and food waste mixtures in farm-scale biogas digesters could provide economic incentives for farmers through tipping fees and increased biogas production while redirecting valuable waste products from the landfills.

**Author Contributions:** Conceptualization, A.C. and S.L.; Data curation, A.C.; Formal analysis, A.C.; Funding acquisition, S.L.; Investigation, A.C.; Methodology, A.C. and S.L.; Project administration, S.L.; Resources, S.L.; Software, A.C.; Supervision, S.L.; Validation, A.C.; Visualization, S.L.; Writing—Original draft, A.C.; Writing—Review & editing, A.C. and S.L.

**Funding:** This material is based upon work supported by the National Institute of Food and Agriculture at the U.S. Department of Agriculture through a Northeast Sustainable Agriculture Research and Education (SARE) grant (# LNE15-341).

**Acknowledgments:** The authors would also like to thank the participating farmer for his enthusiasm and assistance with the project.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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*Article*
