Enhancing Butanol Production under the Stress Environments of Co-Culturing <i>Clostridium acetobutylicum/Saccharomyces cerevisiae</i> Integrated with Exogenous Butyrate Addition

<div><p>In this study, an efficient acetone-butanol-ethanol (ABE) fermentation strategy integrating <i>Clostridium acetobutylicum</i>/<i>Saccharomyces cerevisiae</i> co-culturing system with exogenous butyrate addition, was proposed and experimentally conducted. In solventogenic phase, by adding 0.2 g-DCW/L-broth viable <i>S</i>. <i>cerevisiae</i> cells and 4.0 g/L-broth concentrated butyrate solution into <i>C</i>. <i>acetobutylicum</i> culture broth, final butanol concentration and butanol/acetone ratio in a 7 L anaerobic fermentor reached the highest levels of 15.74 g/L and 2.83 respectively, with the increments of 35% and 43% as compared with those of control. Theoretical and experimental analysis revealed that, the proposed strategy could, 1) extensively induce secretion of amino acids particularly lysine, which are favorable for both <i>C</i>. <i>acetobutylicum</i> survival and butanol synthesis under high butanol concentration environment; 2) enhance the utilization ability of <i>C</i>. <i>acetobutylicum</i> on glucose and over-produce intracellular NADH for butanol synthesis in <i>C</i>. <i>acetobutylicum</i> metabolism simultaneously; 3) direct most of extra consumed glucose into butanol synthesis route. The synergetic actions of effective amino acids assimilation, high rates of substrate consumption and NADH regeneration yielded highest butanol concentration and butanol ratio in <i>C</i>. <i>acetobutylicum</i> under this stress environment. The proposed method supplies an alternative way to improve ABE fermentation performance by traditional fermentation technology.</p></div

Economical evaluation of the proposed fermentation strategy using synthetic butyrate and butyrate fermentative supernatant.

<p>* Corn-starch consumption was calculated by 0.9×(glucose consumed amount) in ABE fermentation.</p><p>** Butyrate yield on glucose in butyrate fermentation was 0.38. The glucose consumed amount in butyrate fermentation was 10.5 g/L glucose (1/0.38×4 g-glucose/L).</p><p>Economical evaluation of the proposed fermentation strategy using synthetic butyrate and butyrate fermentative supernatant.</p

ABE fermentation performance with different operation strategies.

<p><b>(A)</b> ABE fermentation by <i>C</i>. <i>acetobutylicum</i> (control, case #a). <b>(B)</b> ABE fermentation by <i>C</i>. <i>acetobutylicum</i> with exogenous butyrate addition (case #b). <b>(C)</b> ABE fermentation by co-culturing <i>C</i>. <i>acetobutylicum</i>/<i>S</i>. <i>cerevisiae</i> (case #c). <b>(D)</b> ABE fermentation by co-culturing <i>C</i>. <i>acetobutylicum</i>/<i>S</i>. <i>cerevisiae</i> in coupling with exogenous butyrate addition (case #d). ●: butanol; ■: acetone; ▲: ethanol; ▽: butyrate; ○: glucose. <b>(E)</b>-<b>(F)</b> Change patterns of pH and gas production with different operation strategies. Thin line: case #a (control); dash dot line: case #b; broken line: case #c; bold line: case #d. Dashed arrow: the instant of initiating the consecutive butyrate additions (case #b); solid arrow: the instant of adding <i>S</i>. <i>cerevisiae</i> culture broth (case #c) and <i>S</i>. <i>cerevisiae</i> culture broth/concentrated butyrate solution (case #d).</p

Amino acids secretion patterns with different operation strategies.

<p><b>(A)</b> ABE fermentation by <i>C</i>. <i>acetobutylicum</i> (control, case #a). <b>(B)</b> ABE fermentation by <i>C</i>. <i>acetobutylicum</i> with exogenous butyrate addition (case #b). <b>(C)</b> ABE fermentation by co-culturing <i>C</i>. <i>acetobutylicum</i>/<i>S</i>. <i>cerevisiae</i> (case #c). <b>(D)</b> ABE fermentation by co-culturing <i>C</i>. <i>acetobutylicum</i>/<i>S</i>. <i>cerevisiae</i> in coupling with exogenous butyrate addition (case #d). Black: lysine; white: methionine; horizontal shadow: phenylalanine; slashed shadow: tyrosine.</p

Comprehensive performance comparison of ABE fermentation using different operation strategies.

<p>BtOH, ACE, EtOH and ABE refer to butanol, acetone, ethanol and total solvents; ratios of BtOH and B/A are butanol ratio over total ABE and butanol/acetone ratio; ABE prod. points to the total ABE productivity; GLC^SC refers to the glucose consumed amount by <i>S</i>. <i>cerevisiae</i>; Butyrate points to the final residual butyrate concentration. All yields (<i>Y</i>) are weight ratios (butanol-formed/glucose-consumed or ABE-formed/glucose-consumed, g/g); <i>Y'</i>_BtOH is the butanol yield on glucose consumed by <i>C</i>. <i>acetobutylicum</i>; <i>Y'</i>_ABE is the total ABE yield on glucose consumed by <i>C</i>. <i>acetobutylicum</i>. N/A: not available or measured.</p><p>¹: Control refers to ABE fermentation by <i>C</i>. <i>acetobutylicum</i>.</p><p>²: Butyrate addition refers to ABE fermentation by <i>C</i>. <i>acetobutylicum</i> with exogenous butyrate addition.</p><p>³: Co-culture refers to ABE fermentation by co-culturing <i>C</i>. <i>acetobutylicum</i>/<i>S</i>. <i>cerevisiae</i>.</p><p>⁴: Co-culture+Butyrate refers toABE fermentation by co-culturing <i>C</i>. <i>acetobutylicum</i>/<i>S</i>. <i>cerevisiae</i> in couple with exogenous butyrate addition.</p><p>⁵: Co-culture+Butyrate refers to ABE fermentation by co-culturing <i>C</i>. <i>acetobutylicum</i>/<i>S</i>. <i>cerevisiae</i> integrated with exogenous butyrate fermentative supernatant addition (4.0 g-butyrate/L-broth).</p><p>Comprehensive performance comparison of ABE fermentation using different operation strategies.</p

<i>S</i>. <i>cerevisiae</i> cells viability and theoretical glucose amount consumed by <i>S</i>. <i>cerevisiae</i> in the proposed co-culturing system.

<p>○ & ●: cell viability of <i>S</i>. <i>cerevisiae</i> in the co-culturing system without/with butyrate addition; □ & ■: theoretical glucose amounts consumed by <i>S</i>. <i>cerevisiae</i> in the co-culturing system without/with butyrate addition.</p

Simplified metabolic network map indicating <i>C</i>. <i>acetobutylicum</i> and <i>S</i>. <i>cerevisiae</i> metabolisms.

<p>Bold solid lines: enhanced metabolic fluxes; broken lines: weakened metabolic fluxes; dotted red lines: accumulation/secretion and assimilation/utilization directions of the amino acids favorable for butanol synthesis by <i>C</i>. <i>acetobutylicum</i> and <i>C</i>. <i>acetobutylicum</i> survival.</p

Investigation of interactions and dynamic behaviors of <i>S</i>. <i>cerevisiae</i> and <i>C</i>. <i>acetobutylicum</i> under each stress environments.

<p><b>(A)</b> Cells growth and ethanol synthesis in <i>S</i>. <i>cerevisiae</i> cultivation. Horizontal shadow: Initial cells concentration (OD₆₀₀); slashed shadow: increased cells concentrations (OD₆₀₀); white: ethanol concentration. <b>(B)</b> The concentrations of the four amino acids favorable for <i>C</i>. <i>acetobutylicum</i> survival and butanol synthesis in <i>S</i>. <i>cerevisiae</i> cultivation. Black: lysine; white: methionine; horizontal shadow: phenylalanine; slashed shadow: tyrosine. None and B&B referred to the cases of no any ingredients additions and simultaneously adding butanol and butyrate, respectively. <b>(C)</b> Impacts of exogenous ethanol addition on ABE fermentation by <i>C</i>. <i>acetobutylicum</i>. Black: butanol; white: acetone; slashed shadow: ethanol produced by <i>C</i>. <i>acetobutylicum</i>; horizontal shadow: added ethanol; ●: gas production amount. EtOH5 and EtOH10: 5 g-ethanol/L and 10 g-ethanol/L were added. <b>(A)</b>-<b>(B)</b><i>S</i>. <i>cerevisiae</i> seeds were incubated in a rotating shaker at 200 r/min and 30°C for 24 h and then transferred into the anaerobic bottles. The cultivation continued for 36 h. <b>(C)</b> The fermentations ended at 60 h, ethanol was added at 24 h.</p