Syntrophy of Crypthecodinium cohnii and immobilized Zymomonas mobilis for docosahexaenoic acid production from sucrose-containing substrates

Highlights

• Immobilized Z. mobilis converted sucrose to ethanol that was consumed by C. cohnii.

• Levansucrase (sacB) and ndh-negative double mutant Zm6-ndh-sacB was constructed.

• Immobilized Zm6-ndh-sacB converted sucrose to ethanol aerobically with a high yield.

• Fed-batch with Zm6-ndh-sacB fermentation medium and cocultivations were carried out.

• Syntrophy with immobilized Zm6-ndh-sacB yielded C. cohnii biomass with up to 3% DHA.

Abstract

Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated ω−3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6-ndh, with high ethanol yield both under anaerobic and aerobic conditions, was taken as the genetic background for inactivation of levansucrase (sacB). SacB mutation eliminated the levan-forming activity on sucrose. The double mutant Zm6-ndh-sacB cells were immobilized in Ca alginate, and applied for syntrophic conversion of sucrose to DHA of C. cohnii, either taking the ethanol-containing fermentation medium from the immobilized Z. mobilis for feeding to the C. cohnii fed-batch culture, or directly coculturing the immobilized Zm6-ndh-sacB with C. cohnii on sucrose. Both modes of cultivation produced C. cohnii CCMP 316 biomass with DHA content around 2–3 % of cell dry weight, corresponding to previously reported results for this strain on glucose.

Introduction

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid (PUFA) that belongs to the ω-3 group. DHA is one of the most important long-chain polyunsaturated fatty acids (LC-PUFAs), having numerous health benefits (Adarme-Vega et al., 2014). Marine fish oil has been the most common source of DHA in the last decades but it is not able to meet the increasing demand of DHA due to the depletion of wild fish stocks and pollution of marine environment (Sijtsma and De Swaaf, 2004; Ji et al., 2015). Besides, the fish and other animals lack certain fatty acid desaturases that are required for the de novo synthesis of LC-PUFAs, hence the fatty acid profiles of their oils depend on their dietary inputs but not on the metabolic capabilities of the organism itself. Plants are commercially important source of oils and fats, yet they also do not synthesize LC-PUFAs (Ruiz-Lopez et al., 2015). Efforts to explore alternative sources of DHA during the last decades have largely focussed on DHA-producing microalgae and protists (Santoz-Sánchez et al., 2016). Microorganisms synthesize all their cell lipid fatty acids de novo, and unlike plants and animals, they can produce also significant amounts of LC-PUFAs. Among the microalgae, Crypthecodinium cohnii, a marine heterotrophic dinoflagellate, is regarded as one of the most perspective industrial producers of DHA, because its cells under aerobic growth conditions accumulate DHA at high concentrations (Mendes et al., 2009).

Glucose, ethanol and acetate are the best established carbon substrates for cultivation of C. cohnii (Sijtsma et al., 2010). No, or marginal growth on sucrose, fructose, maltose, rhamnose, arabinose, lactose and galacturonic acid has been reported (Mendes et al., 2009). In general, for bioprocess applications cheap industrial by-products, like e.g. lignocellulose hydrolysate, cheese whey, or molasses are the preferable feedstocks. Molasses is a by-product of sugar industry, consisting of up to 50 % (w/w) total sugars (predominantly sucrose, but also glucose and fructose), minerals, vitamins and other components (Patil and Patil, 2017). It might be the renewable substrate of choice for production of DHA (Gong et al., 2015), yet C. cohnii does not consume sucrose and fructose. To convert renewable, sucrose-containing substrates into DHA, a cofermentation approach may be a feasible alternative. Here we investigate a syntrophic cofermentation, involving generation of ethanol from sucrose (with a further perspective to use molasses as the substrate) by bacterium Zymomonas mobilis, and the following ethanol conversion to biomass and DHA by C. cohnii.

The alpha-proteobacterium Zymomonas mobilis is an unusual facultatively anaerobic bacterium, isolated from sugar-rich tropical plant saps (Swings and De Ley, 1977), able to grow on glucose, fructose and sucrose-containing substrates, like sugary plant saps or molasses (Behera et al., 2012), and synthesize ethanol as its dominant fermentation product with high rate and yield under anaerobic conditions (Rogers et al., 1982; Sprenger, 1996). This bacterium tolerates high sugar (up to 40 %) and ethanol (up to 16 %) concentrations (Swings and De Ley, 1977; Sprenger, 1996). Furthermore, Z. mobilis is a typical example of the ‘uncoupled growth’ phenomenon (Belaïch and Senez, 1965): its catabolic rate is loosely related to the demands of its anabolism, and thus, fermentation proceeds rapidly both in growing and non-growing cells (Strazdina et al., 2018; Fuchino et al., 2020). Such properties make Z. mobilis an ideal candidate for sugar conversion to ethanol by immobilized cell preparations, e.g., using cells entrapped in calcium alginate (Fu et al., 2009; Behera et al., 2012), or poly vinyl alcohol (PVA) (Wirawan et al., 2012, 2020). Immobilized cell preparations offer several advantages over growing cultures or non-growing planktonic cell suspensions. Catabolism in immobilized preparations may proceed even under conditions where culture growth is largely hampered. In general, higher ethanol productivity can be reached with immobilized Z. mobilis (Kannan et al., 1998). In cocultivations immobilized cells can be selectively removed from the medium when needed, and used repeatedly afterwards. While Z. mobilis is known to inhibit other microbial species during cocultivations, immobilization in alginate is reported to mitigate its inhibitory effects (Fu et al., 2009).

However, to run aerobic cocultivations of immobilized Z. mobilis with C. cohnii on sucrose, two specific limitations needed to be overcome. First, under aerobic conditions Z. mobilis metabolism shifts from ethanol production to acetaldehyde accumulation, because part of reducing equivalents required for ethanol synthesis from acetaldehyde get oxidised by the respiratory chain (Kalnenieks et al., 2019). Second, one of the three sucrose-hydrolyzing enzymes in Z. mobilis is levansucrase (sacB), which builds the fructose polymer levan, thus preventing the fructose moiety of sucrose from being converted to ethanol (Bekers et al., 2002). Also, it has been demonstrated that levan accumulation destabilizes the alginate beads (Kannan et al., 1998). We here aimed to resolve these problems by construction and use of a double knock-out mutant strain. Since the aerobic decrease of ethanol yield is due to respiration, the respiratory NADH dehydrogenase (ndh) mutant with low rate of oxygen consumption (Kalnenieks et al., 2008) and high aerobic ethanol yield (Hayashi et al., 2012; Kalnenieks et al., 2019) was taken as the genetic background. The task of the present study was to construct a levansucrase-deficient Z. mobilis double mutant strain ndh sacB, and to employ immobilized cells of this strain for syntrophic bioprocess with C. cohnii on sucrose-containing media.