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How to efficiently use agricultural waste to ferment biogas is a major challenge facing modern agriculture. Research institutions and universities both at home and abroad have conducted extensive research in this area. Liu Dejiang’s research shows that, compared to biogas production using a single fermentation substrate, mixing livestock manure or combining livestock manure with straw can effectively increase biogas output. When the proportion of cow manure in the fermentation substrate is increased, the CH4 content in the gas rises significantly. Meanwhile, pig manure tends to acidify during anaerobic fermentation, resulting in a decrease in biogas production or even a halt in gas generation, whereas sheep manure and cow manure are less prone to acidification and generally do not affect biogas production. Li Wenzhe’s research shows that when temperature and slurry concentration are high and the water residence time is long, the propionic acid content in the fermentation broth increases as well, which has a negative impact on the subsequent methanogenesis stage. The optimal carbon-nitrogen ratio for microbial production of biogas feedstock is 25:1, and a single fermentation feedstock fails to meet this requirement; therefore, it is significant to study the fermentation using different proportions of mixed feedstocks. This experiment investigated the effects of fermenting pig manure or sheep manure alone, as well as in different mixture ratios, at the same temperature on biogas production and CH4 content, aiming to provide a theoretical basis for improving biogas production and quality. 1 Materials and Methods 1.1 Materials The experimental materials were fresh cow manure and sheep manure obtained from the breeding farm located behind Shenyang Agricultural University ； The test inoculum was obtained from the sludge at the bottom of biogas digesters used by farmers in Dongling District, Shenyang. The carbon and nitrogen contents of the fermentation raw materials are shown in Table 1. 1.2 Methods 1.2.1 Fermentation test A constant-temperature anaerobic fermentation apparatus was designed by ourselves; this apparatus consists of three components: a fermentation flask, a gas collection bottle, and a water collection bottle. A 1500 mL plexiglass bottle was used as both the fermentation vessel and the gas collection vessel; holes were drilled in each of these bottles to serve as sampling ports and water outlet ports, respectively, with water stop clamps being used to seal the sampling ports. A 10mm×8mm silicone tube is used to connect the fermentation flask and the gas collection flask. A 100 mL measuring cylinder is used as the water collection bottle, and the gas is collected by allowing water to drain downward; all connections are sealed. The structural diagram is shown in Figure 1. This experiment used a fermentation concentration with a inoculum volume of 20% and total solids of 8%. The fermentation broth formulations are divided into the following 4 groups: Group I: Fermentation with pure pig manure ； Group II: Pure sheep manure fermentation ； Group III: Pig manure: Sheep manure (dry matter ratio) = 1:1 mixed fermentation ； Group IV: Pig manure : Sheep manure (dry matter ratio) = 1:2 fermentation. Two parallel experiments were conducted for the fermentation broth of each formulation. The four experimental groups I, II, III, and IV were placed in 1500 mL fermentation flasks and subjected to open-pretreatment for 9 days. Afterwards, they were sealed, equipped with air ducts and gas collection bottles, and the fermentation apparatus was placed in a constant-temperature incubator where anaerobic fermentation was carried out at a temperature of (28±1)°C for 30 days. 1.2.2 Analysis and determination methods: The daily gas production volume and cumulative gas production volume are measured using the downward drainage method. The pH value of the fermentation broth is determined with an intelligent pH meter (model PHS225), while the CH4 content in the gas is measured using a ZS22 biogas analyzer. 2 Results and Analysis 2.1 Dynamic changes in pH values of different fermentation groups during pretreatment The fermentation mixture prepared according to the formula was stirred evenly, and then subjected to aerobic fermentation at room temperature (20°C) in 1500 mL fermentation flasks for a period of 9 days. During the composting process, the pH value of the system was measured once a day; the changes in the pH value of the fermentation broth are shown in Figure 2. As can be seen from Figure 2, the pH value of Group I remained at a low level throughout the entire pretreatment process, remaining acidic. The pH value of Group II remained within the alkaline range and fluctuated until the end of the pretreatment ； In the mixed-material fermentation experiment group: Group III had a pH value of 7.50 at the start of the composting process; on the 3rd day, its pH value dropped to 6.95, indicating an acidic condition, after which it began to rise again and remained in an alkaline state. By the end of the pretreatment period, Group III’s pH value was 7.50 ； Throughout the entire pretreatment process, Group IV showed a pH trend similar to that of Group III, undergoing a pattern of first decreasing and then increasing before stabilizing. However, compared to Group III, Group IV experienced smaller fluctuations in pH, and its pH remained within the alkaline range throughout the pretreatment process. http://img5.wtoutiao.com/?url=http://mmbiz.qpic.cn/mmbiz/I3BFZqfWrdOyQcVicpIWn1RNbjHpbPt3uTKVJ37bGDJGMdhFsiblTs3FUZOY2Z3XGDJicaTiaGM01Yskwj81BRqpaA/640?wx_fmt=jpeg 2.2 Dynamic changes in pH value of different fermentation groups during anaerobic fermentation During the anaerobic fermentation experiments, the pH value of the fermentation broth was measured every 3 days. The changes in the pH value of the fermentation broth during the biogas fermentation experiment are shown in Figure 3. As shown in Figure 3, in Group I, the pH value dropped to 6.20 on the 9th day of fermentation; it was adjusted to 7.00 by adding alkali artificially. During the subsequent fermentation process, its pH value remained above 6.50, and at the end of fermentation, the pH value was 7.10 ； The pH value in Group II remained at a high level throughout the fermentation process; it dropped to 6.70 on the 15th day of fermentation, and by the end of fermentation it was 7.20. In the mixed raw material fermentation group: Group III experienced a rapid decrease in pH at the beginning of fermentation, dropping to 6.45 by day 12; thereafter it began to rise again and remained above 6.50. By the end of fermentation, the pH value of Group III was 7.30 ； Group IV exhibited the smallest range of pH changes; on the 6th day of fermentation the pH dropped to 6.90, after which it began to rise, reaching 7.31 on the 15th day, with a pH of 7.20 at the end of fermentation. http://img5.wtoutiao.com/?url=http://mmbiz.qpic.cn/mmbiz/I3BFZqfWrdOyQcVicpIWn1RNbjHpbPt3ubK0aNS4JzGgol1y8cq2fD3AjciaQlmokVHuf9JWnt0509byUYm4vGnQ/640?wx_fmt=jpeg 2.3 Changes in gas production depending on the ratio of different fermentation ingredients The changes in the daily gas production volume of the fermentation broth resulting from different ratios of fermentation ingredients are shown in Figure 4. As shown in Figure 4, Group I started producing gas the fastest; a small amount of gas was generated within 1 day of the start of anaerobic fermentation. Its daily gas production reached 100 mL on the 4th day, after which it declined rapidly. By the 8th day of fermentation, gas production in Group I came to a complete stop. After adding alkali to adjust the pH value of the system, gas production resumed, and it reached its peak of 246 mL/day on the 11th day ； Group II began to produce gas more slowly than Group I; gas production started on the 9th day of fermentation, reaching a peak of 325 mL/day on the 16th day, after which it gradually declined, yet its daily gas production level remained higher than that of Group I. In the mixed raw material fermentation group, Group III began to produce gas on day 6; its daily gas production reached a peak of 375 mL/d on day 10, after which it started to decline gradually. By day 24, this value had dropped to 175 mL/d, and by day 30 of fermentation, Group III’s daily gas production was only 47 mL/d ； Group IV began to produce gas on the 4th day of fermentation, and its daily gas production started to increase as fermentation progressed, reaching a peak of 350 mL/day on the 9th day of fermentation. http://img5.wtoutiao.com/?url=http://mmbiz.qpic.cn/mmbiz/I3BFZqfWrdOyQcVicpIWn1RNbjHpbPt3ujrmByvKlibcgnFstzdHrjgjvgxTVmuBJnz91XhlZo8PRGChyBl5zP6w/640?wx_fmt=jpeg The changes in CH4 content in the fermentation broth resulting from different ratios of fermentation substrates are shown in Figure 5. The CH4 content in Group I remained low, reaching 60% on the 11th day of fermentation, after which it began to decline rapidly; no CH4 was present in the gas produced after day 25 of fermentation. Group II had a CH4 content of 21% at the start of gas production on day 9 of fermentation, reached a peak of 79% on day 15, then decreased gradually; by the end of fermentation, its CH4 content was 30%. In Group III, the CH4 concentration reached 35% on the 5th day of fermentation once gas production began; it then continued to rise, reaching a peak of 82% on the 9th day of fermentation. It remained at this high level until the 14th day of fermentation, after which it started to decline gradually. By the end of fermentation, the CH4 concentration was still at 30% ； Group IV reached a peak CH4 concentration of 70% on the 8th day of fermentation, after which it began to decline gradually; by the end of fermentation, its CH4 concentration was only 10%. In the mixed feed fermentation group, Group III had a higher peak CH4 concentration and a longer duration compared to Group IV. The cumulative gas production and dry matter gas yield of the fermentation broths with different ratios of fermentation raw materials are shown in Table 2. As shown in Table 2, after 30 days of anaerobic fermentation at a constant temperature of 28°C, the order of cumulative gas production from highest to lowest among different fermentation substrates was: Group III > Group IV > Group II > Group I. Group I had the lowest cumulative gas production (2749 mL), which was only half of that of Group III (with a cumulative gas production of 6258 mL). The cumulative gas production of mixed raw materials was higher than that of single raw materials, and the cumulative gas production of Group III was higher than that of Group IV. In terms of cumulative gas production alone, Group III is significantly superior to Group IV, with a difference of 922 mL. The dry matter gas production rate, from highest to lowest, is: Group III > Group IV > Group II > Group I. http://img5.wtoutiao.com/?url=http://mmbiz.qpic.cn/mmbiz/I3BFZqfWrdOyQcVicpIWn1RNbjHpbPt3uDNYwUV7CwaJcpV0y9VDc7ZJzRkfdO5yuHt5jBJ2P7XqESH5LarsdibA/640?wx_fmt=jpeg 3 Discussions Conducting an aerobic pretreatment of the mixture of fermentation raw materials and inoculants for 9 days prior to anaerobic fermentation helps to break down the complex organic compounds in these materials and produce acid; pig manure generates acid the fastest, while sheep manure does so the slowest. The pH values of the fermentation broths made from the 4 different raw materials all showed a pattern of first decreasing and then increasing. This was because acid-producing bacteria broke down the complex organic substances in the raw materials to produce volatile fatty acids (VFA) and organic acids, which caused the pH value to drop. Subsequently, as the acids were broken down through the metabolism of gas-producing bacteria, the pH value began to rise and stabilized. Pig manure tends to acidify during anaerobic fermentation, which leads to a decrease or even cessation in gas production; therefore, it is necessary to artificially adjust the pH of the system in order to maintain gas production ； The sheep manure and mixed material fermentation group did not exhibit acidification. Due to the high content of crude fat and crude protein in pig manure, it is decomposed by microorganisms to produce acid during the initial stage of anaerobic fermentation, causing the pH value of the system to drop rapidly and suppressing the activity of methanogenic bacteria. The gas production initiation in the sheep manure fermentation group was relatively delayed, as microorganisms decompose cellulose and hemicellulose at a slow rate, preventing methanogens from obtaining sufficient metabolic substrates. In the mixed feed fermentation group, the acid produced by the acidification of pig manure can damage the chemical structure of the lignocellulosic feedstock; this facilitates the hydrolysis of lignocellulose, increases its biodegradability, and accelerates the decomposition of high-cellulose feedstocks to produce acid. As a result, the gas production and CH4 content increase, leading to better fermentation results. This study shows that, under the same dry matter concentration and temperature conditions, the cumulative gas production and dry matter gas production rate rank from highest to lowest as: Group III > Group IV > Group II > Group I. A ratio of pig manure to sheep manure (on a dry matter basis) of 1:1 is the optimal mixing ratio for fermentation materials. (Li Muzi, Sun Junde, School of Land and Environment, Shenyang Agricultural University)