1 Introduction
Compared with the Embden-Meyerhof-Parnas (EMP) pathway, which is commonly used by other microorganisms, the ethanologenic bacterium Zymomonas mobilis employs the Entner-Doudoroff (ED) pathway for glycolysis with less ATP produced for lower biomass accumulation, as ATP is dissipated predominantly through biosynthesis, particularly cell growth of cells (Xia et al., 2019). From the viewpoint of mass balance, more sugar can be directed to ethanol production with improved yield, which is most important for producing ethanol as a biofuel with a major cost from sugar consumption (Gombert et al., 2015). On the other hand, the bacterial cells are smaller than the brewing yeast Saccharomyces cerevisiae due to a high specific surface to assimilate sugar faster, which, together with the low energy-coupling ED pathway, forms a catabolic pathway for carbon metabolism to produce ethanol faster (Rutkis et al., 2016). Moreover, Z. mobilis can be engineered with pentose metabolism through the isomerase pathway without cofactor imbalance for intermediate accumulation (Zhang et al., 1995), which is an intrinsic drawback for engineering S. cerevisiae with the redox pathway for the same purpose (Gopinarayanan et al., 2019). These merits make Z. mobilis suitable for engineering to produce not only cellulosic ethanol but also other bulk products from lignocellulosic biomass (He et al., 2014).
ZM401, a mutant developed from ZM4, a unicellular model strain ofZ. mobilis , self-flocculates with advantages for industrial production (Cao et al., 2022). When self-flocculated, bacteria can be conveniently immobilized within bioreactors for high cell density to improve productivity, as highlighted previously in ethanol fermentation with self-flocculating yeast (Zhao et al., 2019). In addition, bacterial flocs can be recovered through cost-effective gravity sedimentation instead of centrifugation, a regular practice for harvesting unicellular cells with high capital investment in centrifuges, as well as intensive energy consumption during their operation, needless to say cost with frequent maintenance.
Tolerance to environmental stresses is a prerequisite for the robust production of microbial strains because various stresses are present under industrial conditions (Gong et al., 2017). Product inhibition is one of these stresses, because a high product titer has been pursued endlessly in industry to save energy consumption on product recovery and reduce the discharge of wastewater, which has been highlighted in high-gravity ethanol fermentation (Puligundla et al., 2019). Toxicity from by-products is another stress, one of the biggest challenges for lignocellulose biorefineries to produce biofuels and bio-based chemicals, as toxic byproducts, including furfural, 5-hydroxymethylfurfural, and acetic acid, are inevitably generated during the pretreatment of lignocellulosic biomass (Ling et al., 2014).
Although various technologies, such as physical adsorption, chemical treatment, and biological degradation, have been developed for detoxifying the hydrolysate of lignocellulosic biomass, none of them is economically feasible for industrial applications (Nogueira et al., 2021). Meanwhile, tolerance to individual stresses such as ethanol, acetic acid, and high temperature has been studied for Z. mobilis(Carreón-Rodríguez et al., 2019; Yang et al., 2020; Li et al., 2021), but the progress is less significant because multiple stresses always co-exist under industrial production conditions, and general stress responses are preferred (Guan et al., 2017). Self-flocculation with Z. mobilis bacterial cells of Z. mobilis can make them more tolerant to elevated ethanol and inhibitors present in the hydrolysate of lignocellulosic biomass (Zhao et al., 2014).
Microbes can develop multicellular morphologies, such as biofilms and activated sludge, under stressful conditions (Ciofu et al., 2022; Wilén et al., 2018). However, self-flocculation with Z. mobilis bacterial cells of Z. mobilis presents a unique morphology. Compared to amorphous biofilms, which generally require abiotic surfaces for development with a life cycle (Rumbaugh et al., 2020), no surface is needed for the bacterial cells to self-flocculate, since the process is mediated by cellulose fibers that are self-synthesized (Xia et al., 2018). Furthermore, a dynamic balance can develop between the breakup of large flocs and the re-flocculation of small flocs under specific hydrodynamic conditions that are developed within bioreactors, which can renew the inside time for the bacterial flocs to sustain viability and perform production efficiently. Unlike activated sludge, which is formed naturally during mixed cultures with abundant microbes as a core community for more efficient syntrophy, such as bacteria for the degradation of short-chain fatty acids and methanogens for methane production in anaerobic digestion (Saunders et al., 2016; Hao et al., 2020), self-flocculation of bacterial cells occurs under pure culture conditions.
The chemical basis for self-flocculation with the bacterial cells of ZM401 was experimentally validated to be cellulose fibrils (Xia et al., 2018), which are synthesized in the mutant more efficiently by the bacterial cellulose synthase (Bcs) complex due to single nucleotide polymorphism (SNP) mutations occurring in genes ZMO1082 and ZMO1055 (Cao et al., 2022). As a second messenger, cyclic diguanosine monophosphate (c-di-GMP) regulates intracellular processes through a dynamic balance between its biosynthesis and degradation, which are catalyzed by diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively (Ute et al., 2006; Jenal et al., 2012). Since bacterial cellulose biosynthesis is regulated by c-di-GMP (Ross et al., 1987; Morgan et al., 2014), we hypothesized that the intracellular accumulation of c-di-GMP in Z. mobilis could impact the self-flocculation of the bacterial cells through its regulation of the biosynthesis of cellulose fibrils.
In this study, we explored genes encoding enzymes related to c-di-GMP metabolism in Z. mobilis and studied the intracellular accumulation of this signal molecule as well as its impact on the self-flocculation of bacterial cells. This progress is significant not only for engineering unicellular strains fromZ. mobilis with such a multicellular morphology for robust production but also for understanding the mechanism underlying c-di-GMP metabolism through intracellular biosynthesis and degradation in the bacterium.