Many heterotrophs can produce ATP through both respiratory and fermentative pathways, allowing them to survive with or without oxygen. Since the molar ATP yield (molar ATP yield: mole of ATP produced/mole of substrate consumed) from respiration is about 15-fold higher than that from fermentation, ATP production via respiration is more efficient. Surprisingly, at high catabolic rate, many facultative aerobic organisms employ fermentative pathways simultaneously with respiration, even in the presence of abundant oxygen to produce ATP (Pfeiffer et al, 2001; Vemuri et al, 2006, 2007; Veit et al, 2007; MacLean, 2008; Molenaar et al, 2009). This leads to an observable tradeoff between the ATP yield and the catabolic rate (Pfeiffer et al, 2001; Vemuri et al, 2006). This respiro-fermentation physiology is commonly observed in microorganisms, including Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae (Molenaar et al, 2009), as well as cancer cells (Vander Heiden et al, 2009). Despite extensive research, the biochemical basis for this phenomenon remains obscure.
One influential theory attributed the utilization of the fermentative pathways to a hypothetical limitation on the respiratory capacity (Sonnleitner and Kappeli, 1986; Majewski and Domach, 1990). This theory suggests that as the respiratory pathway becomes saturated at high substrate influx, the organism may choose to satisfy its ATP demand by fermenting additional substrates, a strategy that offers a fitness advantage at the cost of lowering the ATP yield (Majewski and Domach, 1990; Varma and Palsson, 1994; Pfeiffer et al, 2001). However, overexpressing the genes encoding for the rate-limiting enzymes did not increase the respiratory capacity (Cupp and McAlister-Henn, 1991; Repetto and Tzagoloff, 1991). Furthermore, it is puzzling why the respiratory capacity varies with different substrates. Despite this caveat, metabolic models (Palsson, 2000) such as the flux balance analysis (FBA) (Varma and Palsson, 1994; Edwards et al, 2001; Feist et al, 2007) commonly adopt the ‘respiratory capacity limitation’ theory through the introduction of an empirically measured cap on maximal oxygen uptake rate (OUR) (Figure 1A and B). In addition to respiration, the tricarboxylic acid (TCA) cycle is actively downregulated in E. coli, B. subtilis, and S. cerevisiae during respiro-fermentation (Vemuri et al, 2006, 2007; Sonenshein, 2007); this implies that the OURs of these organisms at higher catabolic rates are perhaps regulated to be lower than their respective maximal OURs, possibly reflecting an unexplained evolutionary advantage for lowered respiration (Molenaar et al, 2009).
Challenging the conventional assumption that aerobic respiration is always preferred over fermentation (Majewski and Domach, 1990; Varma and Palsson, 1994), a recent theory (Schuster et al, 2008) proposed that while the cellular metabolism maximizes the ATP yield in nutrient-poor environments, it maximizes the catabolic rate and the rate of energy dissipation in nutrient-rich environments. The biochemical basis for this switch in metabolic objective is the prohibitively expensive synthesis costs of respiratory enzymes, particularly during high catabolic rate (Pfeiffer and Bonhoeffer, 2004; Molenaar et al, 2009). This line of reasoning leads to the conclusion that pure fermentation be accompanied with high growth rate. Yet, rapidly growing facultative aerobes also respire. Furthermore, if the catabolic rate is indeed maximized during unlimited growth, it is unclear why the maximum substrate uptake is slower under aerobic condition than anaerobic conditions (Portnoy et al, 2008). Another theory proposed that the tradeoff between ATP yield and catabolic rate is dependent on the fraction of intracellular volume occupied by respiratory enzymes and glycolytic enzymes, respectively (Vazquez et al, 2008). While the FBA with ‘molecular crowding constraint’ (FBAwMC) (Beg et al, 2007; Vazquez et al, 2008) can predict acetate production to a certain extent, it could not predict the experimentally observed changes in growth rate and yield (Supplementary information). Furthermore, FBAwMC cannot predict the production of acetate if the electron transport chain enzymes—membrane-bound enzymes that consumes little intracellular volume—are removed from its formulation (Supplementary information). Despite these shortcomings, these theories highlight that the rate of metabolic processes must be accounted for in addition to the metabolic stoichiometry in understanding respiro-fermentative metabolism.
Finally, these aforementioned theories assume that the observed tradeoff between the ATP yield and the catabolic rate is solely caused by the utilization of fermentative pathways. However, experimental evidence (Supplementary information) suggests that the efficiency of the respiratory pathway itself may be compromised due to the utilization of less-efficient dehydrogenases and cytochromes. Given that there exists a thermodynamic tradeoff between the turnover rate and the energetic efficiency of an enzyme (Meyer and Jones, 1973; Waddell et al, 1997; Pfeiffer and Bonhoeffer, 2002), less-efficient enzymes may be preferred for their increased turnover rate. Based on these observations, we propose a simple, alternative explanation of the respiro-fermentation phenomenon by considering membrane occupancy, which provides a mechanistic explanation to all the observed physiological changes during the transition from respiratory to respiro-fermentative metabolism.