Biological process development with proteomic analysis for enhancing the production of medium-chain carboxylic acids by Megasphaera species

Abstract

Developing processes that produce biomass-based chemicals to cope with climate change and move towards a bio-based economy is essential. Recently, the process of converting substances such as ethanol, acetate and lactate into medium-chain carboxylic acids (MCCAs) using carbon elongation (CE) technology has attracted much attention. MCCAs have the advantage of a higher energy content and increased hydrophobicity compared to those of shorter carbon chains, making them easier to separate from cultures. When utilized in chemical processes, MCCAs can be converted into versatile materials, such as alkanes, ketones, alcohols, olefins, lactones, etc.

This study attempted to produce MCCAs by culturing isolated bacterial strains. For the first time, the extractive fermentation of caproate production by Megaphaera eldenii T81 using Jerusalem artichoke, an energy crop, and sugarcane juice were evaluated. Additionally, M. elsdenii T81 produced a large amount of butyrate, which was overcome by product extraction. A subsequent study was performed with a low butyrate-producing strain. The newly isolated bacterium, Megsaphaera hexanoica, has metabolic properties in which the product is determined by the type of electron acceptors added to the culture. Proteomic studies confirmed protein expression in the cells. The rapid response of the protein (acetyl-CoA transferase, ACT) that links the CoA functional group to the extracellular organic acid, along with the substrate preference for substances with long chains in the condensation reaction (acetoacetyl-CoA thiolase, Thl), are key factors of the high productivity and high selectivity of M. hexanoica. In addition, a study on the production of valerate using propionate, a less preferred substrate than acetate, was conducted to understand the metabolic properties of the isolated strain. Tracer experiments using a carbon isotope (13C) predicted that proteins of this species convert rapidly to carbon elongation using external organic acids. Due to these metabolic properties, M. hexanoica was cocultured with a butyric acid-producing strain. Providing optimal culture conditions for each bacterium to easily maintain the reactor state, a hollow fibre membrane was used. The culture of each strain was carried out in a separate container. When butyric acid produced by Clostridium tyrobutyricum was supplied directly to M. hexanoica without separation and purification, caproic acid production of 10 g L-1 was achieved. However, the disadvantage of using large amounts of sugar still has yet to be overcome. M. hexanoica was cocultured with the lactate-producing bacterium Lactobacillus amylovorus to mitigate the use of purified sugars. When using food waste materials, up to 10 g L-1 caproate, the maximum soluble concentration, was produced after six days. This study outlined a process that can achieve economically feasible caproic acid separation and purification with the use of economical electron donors and electron acceptors and exhibiting low butyric acid production. Among these suggested processes, utilizing Jerusalem artichokes reduced the operation costs by 6-23% compared to those of the process using sucrose. The operation cost decreased below current caproic prices when producing 2,000 tonnes of caproate per year. In addition, applying the yield of a coculture process using food waste can make a significant contribution to the “waste to energy”.

|기후변화에 대응하고 바이오 기반 경제로 나아가기 위해 바이오매스를 활용한 화학물질을 생산하는 공정을 개발하는 것은 필수적인 일이다. 최근 탄소 연장 (carbon elongation, CE) 기술을 이용하여 에탄올, 초산, 젖산과 같은 물질을 탄소 사슬이 조금 더 긴 유기산 (medium chain carboxylic acids, MCCAs)로 전환하는 공정이 주목받고 있다.

본 학위 논문에서는 분리된 균주를 순수 배양하는 방식으로 MCCAs를 생산하였다. 처음으로, 에너지 작물인 돼지감자를 이용하여 Megaphaera eldenii T81이 카프로산을 생산하는 프로세스의 경제성을 평가하였다. 이때, M. elsdenii T81에서 많은 양의 butyrate가 생산되는 것을 product 추출로 극복하였는데, 이하의 연구는 butyrate 생산이 적은 균을 사용하고자 하였다. 새로 분리된 균인 Megsaphaera hexanoica는 배양액에 첨가한 유기산 종류에 따라 생산물이 결정되는 형태의 대사 특성을 가지고 있다. M. hexanoica의 대사학적 특성을 좀 더 정확히 파악하고자 일반적으로 아세트산보다 기질로 덜 선호되는 프로피온산을 이용해 발레르산 생산 연구를 먼저 진행하였다. 탄소 동위 원소를 이용한 추적 실험을 통해 이 종의 단백질이 외부 유기산을 활용하여 탄소를 연장하는데 빠르게 반응한다는 사실을 예측하였다. 추가적으로 단백체학 연구를 통해 실제로 세포 내부에서 예상한 단백질의 발현양과 발현 형태를 확인하였다. 외부 유기산에 CoA 작용기를 연결하는 단백질(acetyl-CoA transferase, ACT)의 빠른 반응과 축합 반응 단백질(acetoacetyl-CoA thiolase, Thl)의 더 긴 사슬을 가진 물질에 대한 기질 선호는 M. hexanoica의 주요 요소였다. 이러한 대사 특징에 기인하여 M. hexanoica와 부티르산 생산 균주를 공동 배양하였다. 각 균에 맞는 배양조건을 제공하여 반응기 상태 유지를 용이하게 유지할 수 있도록 중공사막을 이용한 반응기를 제작하여 분리된 공간에서 배양을 진행하였다. Clostridium tyrobutyricum을 이용한 부티르산을 분리 및 정제 과정 없이 바로 M. hexanoica에 제공하였을 때, 성공적으로 10 g L-1의 카프로산 생산이 이루어졌다. 그러나 아직도 너무 많은 당류가 사용되는 단점이 극복되지 않았다. M. hexanoica를 전분을 이용해 젖산을 생산하는 균인 Lactobacillus amylovorus와 공동 배양을 진행하여 당류 사용을 극복하고자 하였다. 이 때 음식물 폐기물을 이용하여 6일 동안 최대 수용 농도인 10 g L-1까지 카프로산이 생산되었다. 논문에서 제안된 공정 가운데, 돼지감자를 사용하는 공정이 자당을 사용하는 공정에 비해 6-23%가량 공정 운전 비용을 줄일 수 있으며, 연간 2,000 톤의 카프로산을 생산할 때 현재 카프로산의 가격 이하로 운전 비용을 줄일 수 있음을 확인하였다. 또한 음식물 쓰레기를 사용하는 공동 배양 공정의 수율을 적용하면 “폐기물에서 에너지로”에 크게 도약할 수 있다.; Developing processes that produce biomass-based chemicals to cope with climate change and move towards a bio-based economy is essential. Recently, the process of converting substances such as ethanol, acetate and lactate into medium-chain carboxylic acids (MCCAs) using carbon elongation (CE) technology has attracted much attention. MCCAs have the advantage of a higher energy content and increased hydrophobicity compared to those of shorter carbon chains, making them easier to separate from cultures. When utilized in chemical processes, MCCAs can be converted into versatile materials, such as alkanes, ketones, alcohols, olefins, lactones, etc.

This study attempted to produce MCCAs by culturing isolated bacterial strains. For the first time, the extractive fermentation of caproate production by Megaphaera eldenii T81 using Jerusalem artichoke, an energy crop, and sugarcane juice were evaluated. Additionally, M. elsdenii T81 produced a large amount of butyrate, which was overcome by product extraction. A subsequent study was performed with a low butyrate-producing strain. The newly isolated bacterium, Megsaphaera hexanoica, has metabolic properties in which the product is determined by the type of electron acceptors added to the culture. Proteomic studies confirmed protein expression in the cells. The rapid response of the protein (acetyl-CoA transferase, ACT) that links the CoA functional group to the extracellular organic acid, along with the substrate preference for substances with long chains in the condensation reaction (acetoacetyl-CoA thiolase, Thl), are key factors of the high productivity and high selectivity of M. hexanoica. In addition, a study on the production of valerate using propionate, a less preferred substrate than acetate, was conducted to understand the metabolic properties of the isolated strain. Tracer experiments using a carbon isotope (13C) predicted that proteins of this species convert rapidly to carbon elongation using external organic acids. Due to these metabolic properties, M. hexanoica was cocultured with a butyric acid-producing strain. Providing optimal culture conditions for each bacterium to easily maintain the reactor state, a hollow fibre membrane was used. The culture of each strain was carried out in a separate container. When butyric acid produced by Clostridium tyrobutyricum was supplied directly to M. hexanoica without separation and purification, caproic acid production of 10 g L-1 was achieved. However, the disadvantage of using large amounts of sugar still has yet to be overcome. M. hexanoica was cocultured with the lactate-producing bacterium Lactobacillus amylovorus to mitigate the use of purified sugars. When using food waste materials, up to 10 g L-1 caproate, the maximum soluble concentration, was produced after six days. This study outlined a process that can achieve economically feasible caproic acid separation and purification with the use of economical electron donors and electron acceptors and exhibiting low butyric acid production. Among these suggested processes, utilizing Jerusalem artichokes reduced the operation costs by 6-23% compared to those of the process using sucrose. The operation cost decreased below current caproic prices when producing 2,000 tonnes of caproate per year. In addition, applying the yield of a coculture process using food waste can make a significant contribution to the “waste to energy”.