Consistent 1,3-propanediol production from glycerol in mixed culture fermentation over a wide range of pH

Moscoviz et al. Biotechnol Biofuels (2016) 9:32 DOI 10.1186/s13068-016-0447-8 Biotechnology for Biofuels Open Access RESEARCH Consistent 1,3‑propanediol production from glycerol in mixed culture fermentation over a wide range of pH Roman Moscoviz, Eric Trably* and Nicolas Bernet Abstract Background: Glycerol is currently an over-produced chemical that can be used as substrate for the production of high value products such as 1,3-propanediol (1,3-PDO) in fermentation processes. The aim of this study was to investigate the effect of initial pH on a batch mixed culture fermentation of glycerol, considering both the bacterial community composition and the fermentation patterns. Results: For pH values between 5 and 9, 1,3-PDO production yields ranged from 0.52 ± 0.01 to 0.64 ± 0.00 mol1,3-PDO mol−1 glycerol, with the highest values obtained at pH 7 and 8. An Enterobacteriaceae member closely related to Citrobacter freundii was strongly enriched at all pH values. Within the less dominant bacterial species, two different microbial community structures were found, one at acid pH values and another at neutral to basic pH values. Conclusions: 1,3-PDO production was improved at pH values over 7. It was anti-correlated with lactate and ethanol production but positively correlated with acetate production. No direct correlation between 1,3-PDO production and a specific family of bacteria was found, suggesting functional redundancies in the microbial community. However, 1,3-PDO production yield remained high over the range of pH studied and was comparable to the best obtained in the same conditions in the literature. Keywords: 1,3-PDO, Metabolic patterns, Microbial consortia, Dark fermentation, Biodiesel Background In order to reduce their fossil fuel dependency, several countries have favored the production of biofuels such as bioethanol or biodiesel. The European Union voted in 2009 a resolution to raise the share of EU energy consumption produced from renewable resources to 20 %, while reaching a 10 % share of renewable energy in the transport sector. Biodiesel is currently produced from transesterification of animal or vegetal oils. However, approximately 100 kg of glycerol are co-produced per ton of biodiesel produced [1]. This has led to an increase in world glycerol production over the last decade. This production reached about 3 million tons in 2011 and 4.7 million tons are expected to be produced in 2020 [2]. Therefore, it is a major issue to find a recycling solution *Correspondence: INRA, UR0050, Laboratoire de Biotechnologie de L’Environnement (LBE), Avenue des étangs, 11100 Narbonne, France for this glycerol to make the biodiesel production more sustainable. Glycerol can be used as an inexpensive carbon substrate for fermentation to produce many economically interesting chemicals including 1,3-propanediol (1,3-PDO). 1,3-PDO is used for the production of solvents, cleaners, adhesives, resins, and cosmetics. It can also be used as a monomer for the production of polytrimethylene terephthalate (PTT) further used in textile industry [3]. Many micro-organisms from the Enterobacteriaceae and Clostridiaceae families are known as natural producers of 1,3-PDO from glycerol. So far, most studies about 1,3PDO production from glycerol fermentation have focused on the use of pure cultures such as Clostridium butyricum [4] or Klebsiella pneumoniae [5]. High yields, productivities, and final 1,3-PDO concentrations have been achieved with pure cultures which require sterile conditions and the use of yeast or meat extract in the culture medium. © 2016 Moscoviz et al. 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. Moscoviz et al. Biotechnol Biofuels (2016) 9:32 To reduce the production costs, only few articles have reported the use of mixed cultures to convert crude glycerol from biodiesel production into 1,3-PDO under nonsterile conditions. Dietz et al. [6] successfully used mixed cultures from municipal wastewater treatment plant and reached yields between 0.56 and 0.76 mol1,3-PDO mol−1 glycerol with a minimal culture medium containing crude glycerol. These production yields were slightly higher than the theoretical maximum yield of 0.72 mol1,3-PDO mol−1 glycerol [6] because of the impurities contained in crude glycerol that could be used as additional substrates. Selembo et al. [7] and Liu et al. [8] achieved 1,3-PDO production yields close to the theoretical maximum (resp. 0.69 and 0.65 mol1,3-PDO mol−1 glycerol) when using mixed culture on glycerol fermentation. Previous reported results using mixed cultures were obtained in different experimental conditions and, in particular, with pH values ranging from 5.5 to 8 and with different sources of glycerol [6–10], making difficult to outline the effects of pH. As reported by Samul et al. [11], the effects of crude glycerol impurities on the fermentation patterns can substantially vary, depending on their composition and the source of micro-organisms. The aim of this work was to investigate the effect of initial pH on batch production of 1,3-PDO under non-sterile conditions using a mixed culture as inoculum. Hence a minimal culture medium containing only pure glycerol with no additives such as yeast extract was used in order to reduce the sources of variability other than pH. Methods Inoculum The microbial inoculum used in this work was a mixed culture issued from a long-term continuous dark fermentation lab-scale reactor operated at pH 6.5 under microaerobic conditions for the production of H2 from glycerol [12]. It was stored at 4 °C for 1 month before use. Fermentation medium The composition of the fermentation medium (per liter of water) was modified from Dietz et al.’s as follows: 1.66 g glycerol, 1 g NH4Cl, and 0.5 g NaCl for pH-buffered experiments or 23.50 g glycerol, 2.5 g NH4Cl and 1.0 g NaCl for pH-regulated experiments (Sigma-Aldrich, ≥99 %). In all experiments, 20 mL of a trace element solution (1.5 g/L nitrilotriacetic acid; 3.0 g/L MgSO4·7H2O; 0.50 g/L MnSO4·H2O; 1.0 g/L NaCl; 0.10 g/L FeSO4·7H2O; 0.18 g/L CoSO4·7H2O; 0.10 g/L CaCl2·2H2O; 0.18 g/L ZnSO4·7H2O; 0.01 g/L CuSO4·5H2O; 0.02 g/L KAl(SO4)2·12H2O; 0.01 g/L H3BO3; 0.01 g/L Na2MoO4·2H2O; 0.03 g/L NiCl2·6H2O; 0.30 mg/L Na2SeO3·5H2O; 0.40 mg/L Na2WO4·2H2O) and 150 mM phosphate buffer were added. Page 2 of 11 pH‑buffered fermentation set‑up Batch experiments were performed in triplicates in glass bottles containing 200 mL of solution a (...truncated)