Production of Lipase-producing Inoculum for Using in Fat Wastewater Treatment System

Abstract

The objectives of this research were to: 1) screen, isolate, and identify the potential lipase-producing microorganisms from fat wastewater treatment pond at a poultry processing plant, 2) investigate the optimum condition of microbial culture for lipase production, and 3) examine the efficiency of lipase-producing inoculum for fat hydrolysis in wastewater. First, the solid and liquid samples were collected from a wastewater treatment pond to primary screen lipase-producing microorganisms for culture in palm oil medium (PM) and Tween 80 agar. Then, titration and spectrophotometric methods were carried out for the quantitative screening of isolated lipase-producing strains. Their biosurfactant production was examined by the hemolytic method on sheep blood agar. The lipase-producing strains were identified by a molecular technique. Subsequently, Plackett-Burman Design ( PBD) and Box-Behnken Design ( BBD) were conducted to investigate the optimum condition of microbial culture for lipase production. Then, scaling up lipase production was performed in a 5-L bioreactor and examined the stability of lipase at different pH and temperatures. Finally, the efficiency of fat hydrolysis in wastewater through lipase-producing inoculum was evaluated in lab-scale and pilot-scale studies. In the lab-scale study, 1-5 % of A. baumannii RMUTT3S8-2 inoculum (liquid or powder form) was added to 10 L fat wastewater in 20 L tank and treated by a batch process. In the pilot-scale study, 5 % of A. baumannii RMUTT3S8-2 inoculum (powder in a gauze bag) was added to 150 L fat wastewater in 200 L tank and conducted by a continuous treatment process. Volatile fatty acid (VFA) obtained from fat hydrolysis was investigated by gas chromatography-mass spectrometry (GC-MS). The study found that thirty-one isolated strains produced lipase in PM and Tween 80 agar. The top five isolated strains of lipase production were quantitatively examined by spectrophotometric analysis. The result showed that isolate no. RMUTT3S8-2 provided the significantly highest lipase activity of 97.43 ± 4.29 U/mL, followed by RMUTT2S3-2, RMUTT2S4-2, RMUTT3S8-3, and RMUTT3S5-1, and the iosurfactant production was not found in isolate no. RMUTT3S8-2. Furthermore, the isolate no. RMUTT3S8-2 was olecularly identified as Acinetobacter baumannii and chosen to study lipase production. The optimum lipase-producing condition of A. baumannii RMUTT3S8-2 was peptone 24.49 g/L, yeast extract 33.82 g/L, and NaCl 6.21 g/L. Under optimum conditions, the lipase activity of A. baumannii RMUTT3S8-2 was 216. 23 ± 3. 69 U/ mL, which was higher than in unoptimized conditions by 2. 2 times. Moreover, lipase production in the bioreactor was 16 % higher than in the flask scale. Lipase stability was above 95 % in pH value ranging from 5.0 to 9.0 at 30 °C. The efficiency of fat hydrolysis using lipase-producing inoculum was examined. It was found that the highest fat hydrolysis was 41.94 ± 4.98 % at 12 days with 5 % of the powder inoculum under a batch experiment, and the highest fat hydrolysis was 79 % at 22 h conducted by a continuous process. When the VFAs were investigated by GC-MS, it was found that acetic acid was the main product of VFA in the batch process, while acetic and propionic acids were the main products of VFA found in a continuous process. In addition, VFAs were the highest acids in the continuous process of fat hydrolysis using lipase-producing inoculum in a gauze bag.