Enhancement of Anaerobic Digestion of Cow Manure Through Inoculation Under Mesophilic Conditions

A wide range of techniques are currently used to improve the performance of the anaerobic digestion (AD) process, including co-digestion, inoculation, and addition of various accelerants. In the current study, ruminant intestinal waste inoculum effect on AD of cow manure was investigated. It was hypothesized that methanogens present in intestinal waste will enhance anaerobic digestion, which will result in increased biogas production. Five different cow manure/intestinal waste ratios (1:0, 9:1, 7:3, 1:1, 0:1) on wet weight basis were introduced in AD systems at mesophilic environment (37 °C) to find the best combination of cow manure and intestinal waste. Among tested cow manure/intestinal waste ratios, 9:1, 7:3, and 1:1 demonstrated a good synergistic effect and yielded higher cumulative biogas yields than cow manure and ruminant intestinal waste alone. The cow manure/intestinal waste (9:1) had the maximum cumulative biogas yield (239 ± 5.0 mL/g VS), chemical oxygen demand (COD) removal rate (40 ± 0.56%), volatile solid (VS) removal rate (27 ± 1%), and total solid (TS) removal rate (24 ± 0.52%). In the current study, nutrients’ status (nitrogen, N; phosphorus, P; and potassium, K) of digestate was determined while most of the previous studies are only on biogas production. A 9:1 ratio of cow manure to intestinal waste was found to be the best with a cumulative NPK of 4.53%. The inoculation technique using intestinal waste can be an effective means of increasing the efficiency of biodegradable waste.


Introduction
Pakistan being an agro-livestock country has more than 172 million livestock animals, of which 72 million are cows and buffalo, which can produce around 652 million kg of manure daily [1,2]. This massive amount of animal manure includes various pollutants, including nutrient, pathogenic organisms, testosterone, veterinarian antibiotic, and heavy metals, all of which can pollute soil, surface and groundwater [3]. In addition, many greenhouse gasses (GHG), such as N 2 O, CO 2 , and CH 4 , can be released into the atmosphere by animal manure [4]. Globally, the livestock sector contributes an estimated 18% of anthropogenic GHG emissions [5]. However, because of the negative effects on both the environment and human health, a refined method of safely treating animal manure while also protecting the environment is urgently required [6].
An increased interest in producing biogas from animal manure has been sparked by two factors: a desire to generate green energy and a desire to better manage livestock waste.
Mohsin Tariq and Ayaz Mehmood contributed equally to this work.
For example, Abbas et al. [7] produced green energy using animal manure, food waste, and cow intestinal waste of ruminant animals using AD technology, which is presently the most globally acknowledged technology for this purpose. The AD of animal manure is a bio-chemical process carried out in an anaerobic environment by a variety of microorganisms. One kilogram of cow manure can yield 40 L of biogas [8], and it is estimated that, probably, a medium size single cow and buffalo can produce 10 kg and 19-28 kg of manure per day, respectively [9,10]. Animal manure contains a large amount of organic matter that can be effectively converted into biogas by anaerobic microorganisms under oxygenfree conditions [11,12]. Biogas, as renewable clean energy, can replace traditional fossil fuels for generating heat and electricity; the digestate can be used for preparing organic fertilizer [13,14]. However, AD of dairy manure faces low biodegradation due to higher amount of lignocellulosic molecules (lignin, cellulose, and hemicelluloses), poor stability, and buffer capacity. To enhance the biodegradation of lignocellulosic molecules, economic technique is urgently required. Using microbe-rich inoculums is needed to address these issues in the AD process [7,15].
Use of microbe-rich biomaterials as inoculant could be a way to improve biogas production in AD systems. The microbes contained in an inoculum can enhance the enzyme activity which ultimately converts complex organic compounds to simple sugars (e.g., cellulase and xylanase are the main enzymes that convert lignocellulose to reducing sugars); furthermore, higher enzyme activities lead to higher substrate degradation and biogas production [16,17]. High biogas yield can be achieved by injecting inoculum to improve the biodegradation of organic matter [18] by several inoculums such as ruminant intestinal waste [7], digested sludge [19], animal manure [20], vinegar residue [21], sewage sludge [22], and dairy wastewater [23] that have been used to improve microbial growth and activity. The biogas yield increases linearly as the inoculum ratio in food waste increases [24,25]. Intestine waste and food waste mixing of 1:2 is found to be suitable in the enhancement of biogas production by 57% and 17% when compared with intestine waste and food waste alone [26]. Rumen fluid inoculums caused an increase in biogas production two to three times in comparison with cattle manure substrate without rumen fluid and proposed that rumen fluid in the range of 25-50% will give the best performance for biogas generation [27]. As a bonus, previous research has shown that bacteria in the digester led to an increase in CH 4 yield [28][29][30]. Based on these studies, this study investigates the impact of ruminant intestinal waste inoculation and its ratios on mono-digestion at mesophilic condition. To the best of the authors' knowledge, there has never been a study on mono-digestion in the presence of ruminant animal intestinal waste. Additionally, by avoiding the complexity of co-digestion, this study encourages greater green energy (biogas) via mono-digestion.
Anaerobic digestion was losing interest among masses due to some technical challenges, which include feedstock variability, pH, and C/N ratios [31]. The underlined scientific problem in this study is to check how the ruminant intestinal waste creates a favorable environment for anaerobic digestion by providing extra anaerobic microorganism, which can ultimately be checked by the biogas production. The primary goal of this study was to improve AD process of cow manure by inoculating it with ruminant intestinal waste in mesophilic conditions (37 °C). Basic parameters of the AD process such as chemical oxygen demand (COD) removal rate, pH, biogas yield, total solid (TS), and volatile solid (VS) removal rates were determined to evaluate the AD performance of cow manure. It was also examined that, whether digestates left over from AD can be used as a fertilizer by evaluating the fertility status (nitrogen, N; phosphorus, P; and potassium, K) of digestate.

Substrate and Inoculum
Cow manure, used as a substrate, was collected in plastic bags from a local dairy farm situated on the periphery of the University of Haripur, Khyber Pakhtunkhwa, Pakistan. Cow manure was then domesticated for 1 week in an airtight bag at room temperature. This practice allowed development of an optimum anaerobic environment in the AD system [32,33]. The inoculum used was obtained from a slaughterhouse in Haripur city. Because anaerobic microorganisms may be sensitive to air and sunlight, the inoculum was collected and kept in a plastic bottle that was enclosed in dark paper. The intestinal waste and cow manure's primary characteristics are mentioned in Table 1.

Batch Experimental Setup
In this study, a laboratory-scale experiment was carried out to evaluate the impact of cow intestinal waste inoculation on the performance of anaerobic digestion (AD) of cow manure under mesophilic conditions. A batch experimental setup consist of a 500-mL glass bottle with a working volume of 400 mL functioned as the anaerobic digester. A conical flask was used as the gas collection bottle. A plastic lab-bottle used for water collection bottle. Rubber bungs were used to seal the anaerobic digester and the gas collection bottle. Glass capillary tubes were used as outlet and inlet for gas from digester and gas-collected bottle, and rubber tubes were used for the delivery of gas and water. Five different ratios of cow manure/intestinal waste based on fresh weight (1:0, 9:1, 7:3, 1:1, 0:1) were introduced into different AD systems to find out the best performing ratio. The ratio 1:0 was treated as control check (CK). A certain amount of deionized water was used to balance the total volume of the digester and was placed in a thermostat water bath at 37 ± 1 °C throughout the digestion period. Before the calculation of biogas yield, each AD system was shaken manually. The water displacement method was used to measure the amount of biogas produced [34].

Analytical Methods
Before being fed into the digester, the substrate was chemically analyzed for pH, moisture content (MC), volatile solids (VS), and total nitrogen concentration (TN). The volume of biogas expressed in milliliters per gram (mL/g VS). Measurements were made using an elemental analyzer (Vario ELIII, Germany) to determine the amounts of N and C in intestine waste.

Statistical Analysis
All the analytical tests were conducted in triplicate. The values of performed parameters were expressed as the mean and standard deviation. The statistical analysis of parameters including cumulative biogas yield, initial pH, final pH, TS removal rate, VS removal rate, and COD removal rate was performed using a one-way analysis of variance in STATIX 8.1 software. Statistical significance of these parameters was compared using a 95% level of confidence Fisher test. Means were compared using the least significant difference (LSD) test at p ≤ 0.05 [38].

Biogas Yield
Daily and cumulative biogas yields with varying manure to intestinal waste ratios (1:0, 9:1, 7:3, 1:1, 0:1) over 35-day digestion period is given in Fig. 1a-b. The highest daily biogas yield from cow manure/intestinal waste of 7:3 was 15.89 ± 1.31 mL/g VS, 9:1 (14.47 ± 1.25 mL/g VS), 1:1 (13.5 ± 1.13 mL/g VS), 0:1 (10.87 ± 1.02 mL/g VS), and 1:0 (9.8 ± 1.13 mL/g VS). It was observed from Fig. 1a that the biogas yields from all the AD systems were very low during the first 5 days of digestion period. It may be due to the lag phase of anaerobic microbes and slow breakdown of lignocellulose present in the cow manure. After 5 days, the biogas yields increased when anaerobic microbes became fully active, breaking the lignocellulosic molecules and produced more biogas yield. It was clearly noted that the biogas yield from the digester containing cow manure/ intestinal waste of 0:1 increased from the fourth day due to more active anaerobic microbes in intestinal waste. During the digestion period of 35 days, two representative peaks of daily biogas production were observed in all AD systems. The first peak appeared on the 7th-9th day, representing the degradation of carbohydrates, while the second peak occurred on the 13th-18th day describing the decomposition of crude proteins, as shown in Fig. 1a. These results are similar to earlier studies conducted by Huang et al. and Han et al. [11,13]. These results discovered that carbohydrates are easily degradable as compare to proteins [39]. It was clearly observed that the heights of representative peaks of daily biogas production from cow manure/intestinal waste ratio of 1:0 (contained only intestinal waste) and 0:1 (cow manure only) were low that may be attributed to already degraded organic matter and deficiency of anaerobes, respectively.
The cumulative biogas yields with the different cow manure to inoculum ratios are shown in Fig. 1b. The cumulative biogas yields (239.31 ± 5.0, 217 ± 3.5, 199 ± 4.5, 178 ± 2.7, and 141 ± 4.9 mL/g VS) were observed from the AD systems amended with cow manure/intestinal waste ratios of 9:1, 7:3, 1:1, 1:0, and 0:1, respectively. The cumulative biogas yield varied significantly among all the treatments (p < 0.004) and is given in Table 2. The treatment containing cow manure/intestinal waste of 9:1 produced higher biogas (239.31 ± 5.0 mL/g VS) followed by cow manure/ intestinal waste of 7:3 (217 ± 3.5 mL/g VS), cow manure/ intestinal waste of 1:1 (199 ± 4.5 mL/g VS), cow manure/ intestinal waste of 1:0 (178 ± 2.7 mL/g VS), and the statistically lowest biogas yield was recorded in cow manure/intestinal waste of 0:1 (141 ± 4.9 mL/g VS). Anaerobic digestion system with 0:1 of cow manure/intestinal waste ratio produced the lowest biogas yield (141 ± 4.9 mL/g VS) because of already degraded organic matter in the AD system. The highest biogas yield from cow manure/intestinal waste of 9:1 may be attributed to enhance the microbe biodiversity in the digester and improved process stability [40,41]. Furthermore, the AD system with a 1:0 cow manure/intestinal waste ratio has a high amount of organic matter in the digester containing only cow manure and no intestinal waste, indicating low methanogenic activities due to the deficiency of microorganisms [7] and thus producing less biogas. The above findings reckon that intestinal waste may contain an abundance of the anaerobic microorganisms required for the AD process. Consequently, intestinal waste can improve the performance of AD process.
Consequently, intestinal waste contains more and active anaerobes, which can increase the biogas yield by improving biodegradation of organic matter. Moreover, the AD of various organic wastes with intestinal waste can provide a better way of solid waste management by producing bioenergy and organic fertilizer. However, the exact working mechanism of intestinal waste in the AD system is not clear, which needs to be further investigated. Table 2 shows the calculated values of pH, COD, TS, and VS removal rates before and after the AD process. The  recommended optimal pH range for the AD process is 6.8 to 7.2 [32]. However, the AD process can tolerate a pH of 8.0 [42]. The pH of an AD system has a considerable effect on microbial growth and production and affects biogas production [43]. Figure 2a demonstrates that the pH values of all AD systems (6.09 ± 0.01 to 7.2 ± 0.04) were within the desirable range during the digestion period of 35 days. The pH values decreased (5.76 ± 0.05-6.81 ± 0.03) up to 5th day due to the production of fatty acids in the AD system [44]. After the 5th day, methanogen started to use the fatty acid, which increased the pH values gradually, except for the AD system contained cow manure/intestinal waste of 0:1 (Fig. 2a). The unusual pH observed from the cow manure/intestinal waste of 0:1 can be linked with insufficient nutrients, such as the unavailability of the substrate to methanogens for degradation in the AD system. Similar results about pH were also described by some previous studies [44,45]. The pH of the AD system containing cow manure/intestinal waste of 9:1 varied between 6.11 ± 0.02 and 6.85 ± 0.06 which is an optimum pH range for AD process. This optimum pH maintained the process stability by providing a favorable environment to anaerobic bacteria and showed a stable process, resulting in a higher biogas yield. The pH value of the AD system containing cow manure/intestinal waste of 0:1 varied between 5.47 ± 0.03 and 6.66 ± 0.01 and most of the time during the experiment it was below 6.5 (considered as not good pH for methanogenic bacteria) which shows prolonged acidification period and weaken the activity of the anaerobic microbes; thus, it produced low biogas. The pH values are consistent with the findings of Macias-Corral et al. (2008). The results suggest that AD of intestinal waste alone cannot maintain a stable fermentation environment and cannot promote biogas production. The chemical oxygen demand (COD) rate of degradation is a critical indicator for characterizing the breakdown of a substrate. Commonly, an increased COD degradation rate indicates complete digestion in AD systems and a less negative impact on the environment. Chemical oxygen demand removal rate was varied significantly with change in ratio of cow manure to intestinal waste (p value < 0.001) and is given in Table 2. The cow manure/intestinal waste of 9:1 showed the highest COD removal rate of 40 ± 0.56% followed by cow manure/intestinal waste of 7:3 (37.4 ± 0.64%), 1:1 (35.6 ± 0.65%), and 1:0 (33.88 ± 0.59%). The lowest COD removal rate of 24.6 ± 0.43% was observed from the cow manure/intestinal waste 0:1. The low COD removal rate of 0:1 (cow manure/intestinal waste) was due to the limited availability of organic matter for biodegradation in the anaerobic digester. The digester with cow manure/intestinal waste ratio of 9:1 has the highest COD removal rate, which indicates that the substrate degradation in the digestion system is greater than in other cow manure/intestinal waste. The COD removal rates increase in direct proportion to the level of biogas production and showed a positive correlation (r = 0.90), as shown in Fig. 3a. In general, the above results indicate that biomass-derived carbon-based composite accelerants are helpful to improve the AD performance, which could create a comfortable digestion environment that in turn promotes the biogas yield and organic matter degradation.

pH, TS, VS, and COD Degradation Rates
As essential parameters reflecting the quantities of degraded biomass wastes in AD systems, the VS and TS reductions were also calculated, as shown in Table 2. From  Fig. 2c, it was clearly noted that, the highest TS removal rate of 24 ± 0.52% was found in cow manure/intestinal waste of 9:1 ratio, which gave highest biogas yield. Total solid removal rate significantly varied with cow manure to intestinal waste ratios (p value < 0.003). Total solid removal rate was highest in 9:1 (CM/IW) and was statistically similar with 7:3 followed by 1:0 and 1:1, while 0:1 was lowest in TS removal rate. The lowest TS removal rate (13% ± 0.47%) of cow manure/intestinal waste of 0:1 resulted in lowest biogas yield. In the case of volatile solid (VS) removal rate, the highest (27 ± 1%) and lowest (19 ± 0.76%) VS removal rates were also observed from cow manure/intestinal waste of 9:1 and 0:1, respectively (Fig. 2d). Volatile solid removal rate demonstrate the system's increased biodegradation of substrates, which ultimately increase biogas production [45]. The cow manure/intestinal waste of 9:1 ratio was significantly higher in VS removal rate than to 7:3, 1:0, and 1:1 while the lowest VS removal rate was recorded in 0:1. Furthermore, the trend of TS and VS removal rates from the anaerobic digester was consistent with cumulative biogas yields and showed a positive correlation (r = 0.90 and r = 0.82, respectively) (Fig. 3b-c), confirming the reason for the significant difference in cumulative biogas yields.
In general, the above results indicate that intestinal waste of ruminants, as inoculum is helpful to improve the AD performance, which could create a comfortable digestion environment that in turn promotes the biogas yield and organic matter degradation. Moreover, these findings will provide a novel avenue for developing an economical

Fertility Status of the Digestate
As important nutrients for plants, potassium (K), phosphorus (P), and nitrogen (N) are largely remained in the digestate [46]. The application of the digestate as a component of biofertilizer is considered to be advantageous and could improve the structure of soil [13]. Generally, when the digestate was used for agricultural production, the high content of nutrients in the digestate could enhance the fertility of the soil. To further confirm the potential fertilizer utilization of the digestate with inoculation of intestinal waste of ruminants, the TK, TP, and TN were measured in the present work.
In this study, the highest total nutrient content of 4.53% was found in cow manure/intestinal waste of 9:1 followed by 4.32% for cow manure/intestinal waste of 7:3. The CK (reference group) had total nutrient content of 3.42% which may show that increased inoculum concentrations contained more organism which may subsequently improve the mineralization of digestate resulted in more availability of N, P, and K (Fig. 4).

Conclusion
In this study, an economically and eco-friendly feasible inoculation method was established to improve the AD of cow manure. Various substrate/inoculum (cow manure/ intestinal-tract waste) ratios were tested for their performance concerning biogas yield, removal rates of COD, TS, VS, and digestate's nutrient content (NPK). The results showed that 9:1 ratio of cow manure/intestinal-tract waste revealed significant performance and produced the highest maximum cumulative biogas yield, COD, TS, and VS removal rates. Moreover, the digestate of this ratio also contained 4.53% major nutrients (NPK) which can be used as organic fertilizer to improve the soil fertility. This study can provide an opportunity to improve the biocircular economy by producing bioenergy (biogas) and economical and lowcost organic fertilizer from organic solid waste. Co-digestion and accelerant-introduction techniques in combination with inoculation with intestinal fluid will be applied in the future to increase biogas yield and improve digestate's NPK contents.