Electricity-Assisted Production of Caproic Acid from Grass
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Khor et al. Biotechnol Biofuels (2017) 10:180 DOI 10.1186/s13068-017-0863-4 Biotechnology for Biofuels RESEARCH Open Access Electricity‑assisted production of caproic acid from grass Way Cern Khor, Stephen Andersen, Han Vervaeren and Korneel Rabaey* Abstract Background: Medium chain carboxylic acids, such as caproic acid, are conventionally produced from food materi- als. Caproic acid can be produced through fermentation by the reverse β-oxidation of lactic acid, generated from low value lignocellulosic biomass. In situ extraction of caproic acid can be achieved by membrane electrolysis coupled to the fermentation process, allowing recovery by phase separation. Results: Grass was fermented to lactic acid in a leach-bed-type reactor, which was then further converted to caproic 1 acid in a secondary fermenter. The lactic acid concentration was 9.36 0.95 g L− over a 33-day semi-continuous ± 1 operation, and converted to caproic acid at pH 5.5–6.2, with a concentration of 4.09 0.54 g L− during stable pro- duction. The caproic acid product stream was extracted in its anionic form, concentrated± and converted to caproic acid by membrane electrolysis, resulting in a >70 wt% purity solution. In a parallel test exploring the upper limits of production rate through cell retention, we achieved the highest reported caproic acid production rate to date from 1 1 a lignocellulosic biomass (grass, via a coupled process), at 0.99 0.02 g L− h− . The fermenting microbiome (mainly consisting of Clostridium IV and Lactobacillus) was capable of producing± a maximum caproic acid concentration of 1 10.92 0.62 g L− at pH 5.5, at the border of maximum solubility of protonated caproic acid. ± Conclusions: Grass can be utilized as a substrate to produce caproic acid. The biological intermediary steps were enhanced by separating the steps to focus on the lactic acid intermediary. Notably, the pipeline was almost com- pletely powered through electrical inputs, and thus could potentially be driven from sustainable energy without need for chemical input. Keywords: Grass, Lactic acid, Caproic acid, Decane, Fermentation, Chain elongation, Electrolysis Background fermentation. Zhu et al. [3] recently demonstrated that a Caproic acid is a medium-chain carboxylic acid, its solution of lactic acid could enable caproic acid produc- demand has been growing due to its application as chem- tion through the microbial reverse β-oxidation pathway. ical commodity, feed additive and more recently as bio- Lactic acid is typically formed during the fermenta- based fuel precursor. Te medium-chain carboxylic acid tion/ensiling of grass. Grass is a widely available sub- market (caproic acid, caprylic acid, capric acid, and lau- strate presently underused and often overlooked as a ric acid) is predicted to reach USD 1.25 billion globally carbon source. In the US, grasslands represent an esti- 6 2 by 2020 [1]. Generally, these medium chain fatty acids mated 2.51 × 10 km of available biomass [4], which (MCFA) are derived from triglycerides of coconut and is either ensiled or lowly used. A grass-based fermenta- palm oil by fractional distillation, ozonolysis or catalytic tion can lead to the formation of lactic and acetic acid. reduction processes [2]. MCFA can also be microbially In an uncontrolled pH, mesophilic fermentation of grass, synthesized from alcohols and carboxylic acids through a concentration of 12.6 g L−1 lactic acid can be reached, usually along with 2.0 g L−1 of acetic acid [5]. Lactic acid *Correspondence: [email protected] and acetic acid are highly soluble in water, which makes Department of Biochemical and Microbial Technology, Centre the downstream extraction of lactic acid energy intensive. for Microbial Ecology and Technology (CMET), Ghent University, Coupure More hydrophobic products such as caproic acid can Links 653, 9000 Ghent, Belgium © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Khor et al. Biotechnol Biofuels (2017) 10:180 Page 2 of 11 −1 be produced through the reverse β-oxidation pathway stabilized at 9.36 ± 0.95 g L , and the acetic acid con- −1 with ethanol as the most common reducing substrate centration at 0.90 ± 0.14 g L (Fig. 2a). Te pH of the [6] and acetic acid as the electron acceptor. Studies have fermentation broth was between 4.5 and 5. Te lactic been carried out with pure cultures such as Clostridium acid concentration rapidly reached its fnal concentration kluyveri using ethanol [7, 8] and Megasphaera elsdenii within the frst day of fermentation, indicating that the using sugars and lactic acid [9]. Caproic acid produc- native microorganisms of grass were active as there was tion from lignocellulosic material [10] and a route using no lag time for lactic acid production. In this study, lactic lactic acid was frst considered in 1956 [11], with atten- acid and acetic acid were the main products during fer- tion returning to this process in recent years, through the mentation due to low pH and short retention time. reverse β-oxidation pathway pure culture fermentations Te conversion of organic compounds to lactic acid was [12], and mixed culture community fermentations [3, low relative to our previous study [5] (0.065 g g−1 in this 13–17]. Zhu et al. proposed the stoichiometry of caproic study compared to 0.136 g g−1 grass volatile solid), while − −1 −1 acid formation via lactic acid as 3 CH 3CHOHCOO + 2 the rate of lactic acid production was 0.197 g L h in + − H → CH3(CH2)4COO + H2O + 2 H2 + 3 CO2, with this study. Te conversion rate and efciency of biomass −1 Gibbs free energy of −123.1 kJ mol [3]. to lactic acid were relatively low in this study, which is Te hydrophobicity and low water solubility of caproic likely due to the limited pretreatment and reactor design acid allow extraction of caproic acid from the fermenta- involving a higher ratio of solution volume relative to mass tion broth by phase separation. If the concentration of of grass. Pretreatment (e.g. grinding, steam-explosion, caproic acid in its protonated form exceeds the solubil- alkaline) can be performed to improve the biomass bio- ity limit (11.0 g L−1 at 20 °C), caproic acid will form an degradability, while taking into consideration the cost-ben- immiscible layer and phase separate from the fermenta- eft-sustainability nexus. For instance, mild pretreatment tion broth along with other hydrophobic chemical spe- methods such as lime pretreatment can be applied with- cies. Recently, Xu et al. [18] managed to extract n-caproic out introducing excessive cost, to improve conversion ef- acid from a bioreactor broth using an in-line membrane ciency and production rate, and minimize carbon loss [19]. electrolysis system, while minimizing caustic and acidic In earlier studies, it was shown that pretreatment, such as dosing with electrochemistry. extrusion, can improve the degradability of the grass [19]. Here, we studied the feasibility of a complete pipeline At the time of this manuscript, the equipment was not for caproic acid generation from grass via microbial fer- available; however, similar results to Khor et al. [5] can be mentations and electricity-driven processes alleviating expected, where a conversion increase of 109% was noted. the use of chemicals, and completely avoiding energy While pretreatment can enhance biomass biodegradabil- intensive dewatering and distillation steps on the car- ity, it is also necessary to consider the biological impact of boxylic acid intermediates. Lactic acid fermentation the pretreatment. Phenolic compounds and furfural can broth from grass elongates to caproic acid by reverse be released from lignin if the pretreatment is severe (e.g. β-oxidation. Te caproic acid extracts via membrane high temperature, low pH), which can then interfere with electrolysis delivering the acid concentration of caproic fermentative processes and inhibit the growth of micro- acid. As fnal proof of concept, we also converted caproic organisms. Hence, a substrate purifcation step (e.g. metal acid to decane via Kolbe electrolysis. For each process catalysts, adsorption or chemical oxidation) to remove rate, efciencies and intermediary concentrations were inhibitory compounds prior to biological processes could determined. Te overall process is presented in Fig. 1. be benefcial to improve product yield. In our study, the protein fraction of grass is assumed to be well retained Results and discussion and not degraded after fermentation due to the short Semi‑continuous fermentation of lactic acid from grass retention time and low pH of the process, and no ammo- Semi-continuous fermentation of lactic acid using grass as nia gas was detected. In a 2nd generation biorefnery pro- substrate was performed for 33 days, with a solids and liq- cess, complete conversion of lignocellulosic biomass into uid retention time of 2 days. Te lactic acid concentration a single targeted compound is highly unlikely due to the Fig. 1 Overall process of conversion of grass into lactic acid, caproic acid and decane Khor et al. Biotechnol Biofuels (2017) 10:180 Page 3 of 11 Fig. 2 Carboxylate profle and bacterial community of fermentation and elongation system: a carboxylate profle of fermentation system, b bacte- rial community for fermentation system, c carboxylate profle of elongation system, d bacterial community for elongation system. The concentric circles represent the bacterial taxonomy from phylum (centre) to genus (outermost).