Abstract: Abstract: Disordered heap and unreasonable disposition of livestock manure in Erhai Lake Basin was the main pollution resource to Erhai. There exist problems in middle temperature biogas engineering, such as low gas production, unstable and easy to crusting. In order to control the farming wastes' pollution and select the suitable middle temperature biogas engineering operation parameters for the anaerobic fermentation process in Erhai Lake basin, taking the dairy manure as sampling, this study investigated the effects of temperature, substrate concentration and inorganic stimulating factors on the middle temperature anaerobic fermentation efficiency. What's more, the amount of trace elements was optimized with response surface method in the paper. The experimental conditions were 600 mL total volume of fermentation broth, 8% total solids content with a 30 days fermentation period. The single factor experiment of fermentation temperature selected three levels of 30, 35 and 45℃ for comparison. The results showed that the proportions of gas production in the first 20 days were respectively 93.7%, 93.5% and 95.3% accounted for the total gas production. The total gas production were respectively 0.187, 0.239 and 0.158 L/g at the end of the fermentation period. Thus, under the condition of fermentation temperature of 35℃, the gas production was greater than that at 30 and 45℃, it was conductive to the production of volatile fat acids (VFA), the reuse of methane bacteria, and the 4 kinds of microbial populations (aerobic acid-producing bacteria, anaerobic acid-producing bacteria, anaerobic cellulose decomposing microorganisms, methanogenic bacteria) were more than that at 30 and 45℃ in each stage of the fermentation. Therefore, fermentation temperature at 35℃ had a highest biogas production rate and a most proportion rate of functional microorganisms. The single factor experiment of substrate concentration selected 4 levels of 0, 10%, 20%, 30% and 40% for comparison. The total gas production were respectively 10 060, 10 622, 10 064, 9 358 and 8 558 mL at the end of the fermentation period. Therefore, the inoculation amount of 10% had the maximum total gas production, and the higher inoculation amount with a faster inoculation fermentation start-up speed. Meanwhile, the addition of inorganic stimulating factor can promote the whole fermentation process. It was investigated by the central composite experimental and response surface regression analysis. The results showed a fine effect between real values and predicted values of central composite experimental used quadratic polynomial fitting regression method. Response surface figures of Ni2+-Co2+, Fe2+-Ni2+ and Fe2+-Co2+ effects on accumulated biogas production were shown according to the central composite experimental results. And Ni2+-Co2+ and Fe2+-Ni 2+ did not have interaction，while Fe2+-Co2+ have interaction. Verification experiments were conducted, the results showed that the error between 3 parallel experimental values and predicted values was only 3.6%. Thus, the optimum concentrations was 5.00 mg/L Fe2+, 21.40 μg/L Ni2+, 28.82 μg/L Co2+. Under these conditions, it had a best gas production effect. Compared with the anaerobic fermentation system without addition of Fe2+, Ni2+ and Co2+, the biogas production was increased by 30%. The results will provide a basis for the normal operation of the rural farming manure anaerobic fermentation project in Erhai Lake basin.