Engineering Success

This study explored the efficiency and mechanism of rumen biomimetic microbiota for high-efficiency fermentation of corn straw to produce VFAs, and constructed the VFA production performance of rumen biomimetic microbiota at different substrate concentrations in an semi-continuous reactor.

We developed two versions of the system. Version I:Biomimicry Strategy primarily adopted a biomimetic approach, in which rumen biomimetic microbiota efficiently hydrolyzed and acidified corn straw. Version II:Beyond Biomimicry further advanced the process by transforming the traditional batch fermentation mode into a semi-continuous fermentation system, thereby improving the stability and efficiency of VFA production.

We conducted batch fermentation using serum bottles.Corn straw of 7.5 g, rumen liquid of 100 mL, and nutrient solution of 200 mL were mixed in 500 mL serum bottle to make a corn straw content of 2.5% (w/v). The nutrient solution including trace element solution, buffer solution, macroelement solution and reducing agent solution was prepared according to Menke and Steingass (1988) to buffer the system pH and provide the inorganic salts and trace elements for rumen microorganisms. The serum bottles were blown with nitrogen for 5 min to make anaerobic condition, and an airbag of 250 mL was connected to collect biogas. The fermentation systems were cultured at 39 ℃ in a constant temperature incubator (HZQ-F160, Shanghai Baidian Instrument Equipment Co., Ltd, China) at a stirring rate of 120 r/min for 72 h(see Fig 1). Three parallel experiments were performed, and 18 identical reactors were used for experiments. Sampling was conducted only one time in one reactor to prevent the influence from fermentation broth volume change. The fermentation broth samples were collected at 2, 6, 12, 24, 48, and 72 h, respectively, which were centrifuged at 10000 r/min for 10 min. The supernatant was used for VFA and SCOD determination, and centrifuged solid of three samples were mixed and put in a -80 ℃ refrigerator before metagenomic sequencing analysis. Corn straw residues were used for analyzing straw chemical composition.

Figure 1. Equipment Arrangement Diagram in Version I

We transformed the traditional batch fermentation mode into a semi-continuous fermentation process. We added feeding and discharging systems at the beginning and end of the fermentation tank(see Figure 2). Compared with the traditional method, this enables continuous fermentation, thereby increasing the output efficiency and facilitating future industrial scale-up.A long-term rumen fermentation experiment was carried out in a 1.2 L reactor with a 0.8 L working volume (see supplementary material). The feeding and discharging of the reactor were carried out by a peristaltic pump and the circulating heated water was used to maintain a constant temperature of 39℃ in the reactor. The 39℃ simulates the temperature in ruminant rumen and maintains the maximum activity of rumen microorganisms. A magnetic stirrer with continuous stirring at a speed of 140 r/min was used to simulate the peristaltic function. Hydraulic and solids retention time of this reactor was 8 d to prevent loss of rumen microorganisms and inadequate hydrolysis of substrate. Intermittent discharging and feeding were used to simulate the feeding behavior of ruminants. Corn straw load in this reactor increased sequentially from 2.5%, 5.0%, and 8.0%, respectively. This reactor was run for 60 d at each corn straw load for a total of 180 d. The long-term rumen fermentation experiment was started by mixing 20 g of corn straw with 267 mL of rumen liquid and 533 mL of artificial saliva. Every day, 100 mL of reactor mixture was removed and corn straw mixed with 100 mL of artificial saliva was added into this reactor. The pH, VS, VFAs, and SCOD concentration of the excluded mixture from the reactor were determined. The 5, 10, 30, 70, 90, 120, 140, 160, and 180 d mixture solids were subjected to subsequent amplicon sequencing. A higher 10% (w/v) corn straw load was tried in this reactor. However, the 10% (w/v) corn straw load hindered the operation of this reactor due to the high water absorption of the dried corn straw. Thus, subsequent experiments were not conducted with 10% (w/v) or higher corn straw loads.

Figure 2. Equipment Arrangement Diagram in Version II