DataSheet1_Butanol as a major product during ethanol and acetate chain elongation.docx

Chain elongation is a relevant bioprocess in support of a circular economy as it can use a variety of organic feedstocks for production of valuable short and medium chain carboxylates, such as butyrate (C4), caproate (C6), and caprylate (C8). Alcohols, including the biofuel, butanol (C4), can also be generated in chain elongation but the bioreactor conditions that favor butanol production are mainly unknown. In this study we investigated production of butanol (and its precursor butyrate) during ethanol and acetate chain elongation. We used semi-batch bioreactors (0.16 L serum bottles) fed with a range of ethanol concentrations (100–800 mM C), a constant concentration of acetate (50 mM C), and an initial total gas pressure of ∼112 kPa. We showed that the butanol concentration was positively correlated with the ethanol concentration provided (up to 400 mM C ethanol) and to chain elongation activity, which produced H2 and further increased the total gas pressure. In bioreactors fed with 400 mM C ethanol and 50 mM C acetate, a concentration of 114.96 ± 9.26 mM C butanol (∼2.13 g L−1) was achieved after five semi-batch cycles at a total pressure of ∼170 kPa and H2 partial pressure of ∼67 kPa. Bioreactors with 400 mM C ethanol and 50 mM C acetate also yielded a butanol to butyrate molar ratio of 1:1. At the beginning of cycle 8, the total gas pressure was intentionally decreased to ∼112 kPa to test the dependency of butanol production on total pressure and H2 partial pressure. The reduction in total pressure decreased the molar ratio of butanol to butyrate to 1:2 and jolted H2 production out of an apparent stall. Clostridium kluyveri (previously shown to produce butyrate and butanol) and Alistipes (previously linked with butyrate production) were abundant amplicon sequence variants in the bioreactors during the experimental phases, suggesting the microbiome was resilient against changes in bioreactor conditions. The results from this study clearly demonstrate the potential of ethanol and acetate-based chain elongation to yield butanol as a major product. This study also supports the dependency of butanol production on limiting acetate and on high total gas and H2 partial pressures.

链延长（Chain elongation）是支撑循环经济的关键生物过程，可利用多种有机原料生产高价值短链及中链羧酸盐，如丁酸（butyrate, C4）、己酸（caproate, C6）与辛酸（caprylate, C8）。链延长过程中还可生成包括生物燃料丁醇（butanol, C4）在内的醇类，但目前学界对利于丁醇合成的生物反应器调控条件仍尚不明确。本研究针对乙醇与乙酸链延长过程中的丁醇（及其前体丁酸）生成情况展开探究。实验采用半间歇式生物反应器（semi-batch bioreactors，0.16 L血清瓶），设置梯度乙醇浓度（100~800 mM C）、恒定乙酸浓度（50 mM C），初始总气压约为112 kPa。研究结果表明，丁醇浓度与添加的乙醇浓度（最高至400 mM C乙醇）及链延长活性呈显著正相关；链延长过程会产生氢气（H₂），并进一步提升反应器内总气压。在添加400 mM C乙醇与50 mM C乙酸的反应器中，经过5次半间歇循环后，丁醇浓度可达114.96±9.26 mM C（约2.13 g·L⁻¹），此时总气压约170 kPa，氢气分压约67 kPa。该组反应器同时实现了丁醇与丁酸的摩尔比为1:1的产物比例。在第8轮循环启动时，实验人员刻意将总气压降至约112 kPa，以探究丁醇生成对总气压及氢气分压的依赖关系。总气压降低后，丁醇与丁酸的摩尔比降至1:2，同时打破了氢气生成的停滞状态。实验阶段内，反应器中存在大量扩增子序列变异体（amplicon sequence variants），包括此前被证实可产丁酸与丁醇的克氏梭菌（Clostridium kluyveri）以及与丁酸生成相关的拟杆菌属（Alistipes），这表明反应器内微生物组对生物反应器条件变化具有较强的抗逆性。本研究结果明确证实，基于乙醇与乙酸的链延长工艺具备将丁醇作为主要产物的应用潜力；同时验证了丁醇生成受乙酸浓度限制，且依赖于高总气压与高氢气分压的结论。