Introduction
Lactic acid bacteria are key species in many fermentative processes (Axelsson and Ahrné, 2000), such as biogas production and food-related fermentations (Leroy and De Vuyst, 2004). They also are essential in promoting human health, e.g. a healthy human infant microbiome (Solís et al., 2010). In an industrial biotechnology setting, these microorganisms are applied in the production of lactic acid, which is used to preserve food and to produce the biobased and biodegradable plastic polylactic acid (Straathof, 2014). The lactic acid market is expected to reach 9.8 billion US dollars by 2025 which shows the economic significance of lactic acid.
Mixed culture biotechnology (Kleerebezem and van Loosdrecht, 2007) can aid to the development of more sustainable and energy-efficient bioprocesses. Such processes rely on “ecology-based design” of bioprocesses to perform a desired conversion, which contrasts with the traditional pure culture approach. Typically, enrichment cultures are used to function as a model system to develop such ecology-based bioprocesses. Compared to pure culture processes, ecology-based processes offer the advantage of (semi)-continuous bioprocessing and omit the need for sterilisation of the feedstock and equipment (Kleerebezem and van Loosdrecht, 2007). Examples of successful ecology-based bioprocesses are PHA production from VFAs (Johnson et al., 2009) or biological phosphorous removal (reviewed by Bunce et al., 2018). To create a stable ecology-based process, its design needs to be based on the competitive advantage of the concerned type of conversion. In the case of lactic acid to be produced from carbohydrates, the ecological question is which environmental conditions provide lactic acid bacteria with a competitive advantage over other carbohydrate fermenting microorganisms?
Lactic acid bacteria tend to dominate in anaerobic, carbohydrate containing environments characterised by acidic pH and an abundant availability of compounds required for anabolism, such as in fermented milk, meats and vegetables (Axelsson and Ahrné, 2000). Most well-studied lactic acid bacteria are part of the Bacilli class, such asStreptococcus , Lactococcus , Bacillus andLactobacillus species. Lactic acid bacteria have high maximal biomass specific growth rates (µmax), e.g. Streptococcus salivarius shows a µmax of 2.8 h-1 in a complex medium at 37°C, at neutral pH (Roger et al., 2011). This can be compared to µmax forEscherichia coli strain K12 of around 0.98 h-1 at similar conditions (Kim et al., 2007). Lactic acid bacteria seem to have a kinetic advantage over other species and have quite extraordinary growth rates while being anaerobic microorganisms.
Lactic acid bacteria only display fast growth when sufficient B vitamins and peptides are supplied to their medium environment. For example,Lactococcus lactis strains are auxotrophic for 14 of the 20 amino acids (Cocaign-Bousquet et al., 1995). A comparative genome study predicted that of the 46 Lactobacillus species analysed all are auxotrophic for biotin, folate, pantothenate and thiamine (Magnúsdóttir et al., 2015). Lactic acid bacteria grow poorly or do not grow at all in environments where such B vitamins or peptides are not available. We therefore suggest that auxotrophies are common among lactic acid bacteria, certainly under conditions of high growth rates.
Prototrophic fermentative microorganisms in general have lower µmax-values when compared to lactic acid bacteria. These organisms can be found in the genus of Clostridium and the families of Enterobacteriaceae and Ruminococcaceae . The extensively studied Enterobacteriaceae species E. coli is a prototroph, and is reported to have a µmax of 0.31 h-1 at 37 °C and a pH of 7 in a mineral medium with glucose (Hasona et al., 2004). E. coli here produced a mixture of acetate, ethanol and formate. We hypothesise that lactic acid bacteria will outcompete prototrophic fermenters by achieving a higher µmax in complex environments where there is an abundance of peptides and B vitamins.
The switch between lactate production on the one hand, and acetate and ethanol production on the other hand, has been reported for a single species under complex medium conditions. Lactococcus lactis(formerly known as Streptococcus lactis ), switches its catabolism from lactate production to acetate, ethanol and formate or H2 production at lower dilution rates, i.e. lower growth rates (Thomas et al., 1979). Lactate is produced from pyruvate with one enzyme and delivers and acetate and ethanol with five enzymes. Lactate delivers 2 ATP by substrate level phosphorylation, while acetate and ethanol deliver 3 ATP. This switch is thought to be caused by resource allocation, which essentially describes that a cell has a certain amount of functional protein available, and shorter catabolic pathways can evoke a higher biomass specific substrate uptake rate, qsmax, (de Groot et al., 2018; Molenaar et al., 2009), often at the expense of less energy harvesting per unit of substrate.
Here, we tested the hypothesis that lactic acid producing enrichment cultures can be obtained by providing a complex medium and selecting on high growth rate. We compared two parallel anaerobic non-axenic or open mixed culture sequencing batch reactors (SBRs) operated under mesophilic (T = 30°) and slightly acidic conditions (pH = 5), with either mineral or complex cultivation media. The mineral medium was replicated from the work of Temudo (2008) and containing 4 g L-1 of glucose. The complex medium consisted of the mineral medium, and also 0.8 g L-1 of tryptone and 9 B vitamins. The cultures were characterised for their stoichiometric, kinetic and thermodynamic properties and the microbial community structure was analysed.