Phototrophic Lactate Utilization by Rhodopseudomonas palustris Is Stimulated by Coutilization with Additional Substrates

Bacterial carbon source utilization is frequently assessed using cultures provided single carbon sources. However, the utilization of carbon mixtures by bacteria (i.e., mixed-substrate utilization) is of both fundamental and practical importance; it is central to bacterial physiology and ecology, and it influences the utility of bacteria as biotechnology. Here we investigated mixed-substrate utilization by the model organism Rhodopseudomonas palustris. Using mixtures of organic acids and glycerol, we show that R. palustris exhibits an expanded range of usable carbon substrates when provided substrates in mixtures. Specifically, coutilization enabled the prompt consumption of lactate, a substrate that is otherwise not readily used by R. palustris. Additionally, we found that R. palustris utilizes acetate and glycerol sequentially, revealing that this species has the capacity to use some substrates in a preferential order. These results provide insights into R. palustris physiology that will aid the use of R. palustris for industrial and commercial applications.

ABSTRACT

The phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris is known for its metabolic versatility and is of interest for various industrial and environmental applications. Despite decades of research on R. palustris growth under diverse conditions, patterns of R. palustris growth and carbon utilization with mixtures of carbon substrates remain largely unknown. R. palustris readily utilizes most short-chain organic acids but cannot readily use lactate as a sole carbon source. Here we investigated the influence of mixed-substrate utilization on phototrophic lactate consumption by R. palustris. We found that lactate was simultaneously utilized with a variety of other organic acids and glycerol in time frames that were insufficient for R. palustris growth on lactate alone. Thus, lactate utilization by R. palustris was expedited by its coutilization with additional substrates. Separately, experiments using carbon pairs that did not contain lactate revealed acetate-mediated inhibition of glycerol utilization in R. palustris. This inhibition was specific to the acetate-glycerol pair, as R. palustris simultaneously utilized acetate or glycerol when either was paired with succinate or lactate. Overall, our results demonstrate that (i) R. palustris commonly employs simultaneous mixed-substrate utilization, (ii) mixed-substrate utilization expands the spectrum of readily utilized organic acids in this species, and (iii) R. palustris has the capacity to exert carbon catabolite control in a substrate-specific manner.

IMPORTANCE Bacterial carbon source utilization is frequently assessed using cultures provided single carbon sources. However, the utilization of carbon mixtures by bacteria (i.e., mixed-substrate utilization) is of both fundamental and practical importance; it is central to bacterial physiology and ecology, and it influences the utility of bacteria as biotechnology. Here we investigated mixed-substrate utilization by the model organism Rhodopseudomonas palustris. Using mixtures of organic acids and glycerol, we show that R. palustris exhibits an expanded range of usable carbon substrates when provided substrates in mixtures. Specifically, coutilization enabled the prompt consumption of lactate, a substrate that is otherwise not readily used by R. palustris. Additionally, we found that R. palustris utilizes acetate and glycerol sequentially, revealing that this species has the capacity to use some substrates in a preferential order. These results provide insights into R. palustris physiology that will aid the use of R. palustris for industrial and commercial applications.

INTRODUCTION

Many bacteria in natural environments likely consume multiple carbon sources simultaneously (1, 2). However, bacterial substrate utilization is most often studied using bacteria in isolation with single carbon sources (3). Data on mixed-substrate utilization in diverse bacteria is crucial for both the understanding of nutrient acquisition, metabolism, and community dynamics within microbial ecosystems (1, 4) and the rational application of bacteria as biotechnology (5–8).

When encountering multiple carbon sources (i.e., substrates), a bacterium will utilize the substrates either simultaneously (i.e., coutilization) or sequentially depending on the identity of the substrates. Sequential utilization typically results in a diauxic growth pattern characterized by two or more exponential phases that are each separated by a lag phase (2, 9); however, sequential utilization can also occur without an intervening lag phase (2, 4, 10), a pattern often referred to as “biphasic growth.” It generally holds true that during sequential utilization, bacteria preferentially use the carbon source that supports the highest growth rate during the first phase of growth while utilization of the other carbon source(s) is limited until the preferred carbon source is no longer available (2); this process is commonly referred to as carbon catabolite repression (CCR) (11). A classic example of CCR involves the lac operon in Escherichia coli, which directs the preferential consumption of glucose prior to lactose when cells are provided a mix of the two sugars (11). Although sequential carbon utilization was once thought to predominate among bacteria, particularly at high substrate concentrations (2–4), it has become clear that simultaneous utilization of carbon substrates is common (1). For example, coutilization occurs in Pseudomonas putida grown with glucose and aromatic compounds (12), in E. coli grown with various organic acid pairs (13), and in Lactococcus brevis grown with glucose paired with other sugars (14).

The phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris is a model organism for investigating metabolic flexibility in response to environmental conditions (15, 16) and is of interest for various commercial applications, including the production of hydrogen gas (17–19) and the biodegradation of aromatic inhibitors in biofuel feedstocks (20). However, there is limited information regarding carbon preference, mixed-substrate utilization, or carbon catabolite control in this species. We recently showed that R. palustris utilizes multiple products of E. coli mixed-acid fermentation when grown in a synthetic coculture (21). One of these products, lactate, is not readily utilized as a sole carbon source by phototrophically grown R. palustris (18). Taken together, these observations raised the question of how coculturing enables R. palustris lactate consumption. Using mixtures of different carbon substrates, here we show that R. palustris simultaneously consumes lactate with several other organic acids and glycerol. Importantly, this coutilization endowed R. palustris with the ability to promptly utilize lactate, in spite of the fact that lactate alone did not support growth in the same time frame. Hence, in coculture, the presence of mixed-acid fermentation products enables the coutilization of lactate by R. palustris. Separately, experiments with additional carbon pairs revealed acetate-mediated inhibition of glycerol catabolism, establishing that R. palustris can exert hierarchical regulation of carbon utilization.

RESULTS

Acetate and succinate prompt the expedited and simultaneous utilization of lactate.

We previously observed that when grown anaerobically in a mutualistic coculture with E. coli, R. palustris simultaneously consumed the acetate, succinate, and lactate excreted as fermentation products by E. coli during the 200-h culturing period (21). Whereas acetate and succinate are both known to be readily utilized by R. palustris (19), R. palustris requires long-term incubation with lactate before it will utilize lactate as a sole carbon source (18). Indeed, we observed no growth of R. palustris with lactate alone within the time frames sufficient for growth on other organic acids (≤300 h; see below). As the utilization of a given carbon source can be influenced by the presence of an additional carbon source (1, 12, 13), we hypothesized that the presence of mixed fermentation products facilitated lactate utilization by R. palustris in coculture. We reasoned that the most likely mediators of this effect would be succinate and/or acetate, given that they were the other carbon substrates consumed by R. palustris in coculture. To test if the presence of acetate and succinate could stimulate lactate consumption, we grew R. palustris Nx, the strain we had used in coculture with E. coli, in monoculture with a mixture of succinate, acetate, and lactate (5 mM each). Cultures grown with this mix exhibited a single exponential phase with a specific growth rate of 0.074 ± 0.001 h⁻¹ (± standard deviation [SD]), and growth plateaued within 150 h (Fig. 1A). To determine which substrate(s) in the mix had been consumed, we analyzed culture supernatants using high-performance liquid chromatography (HPLC). The data showed that all three compounds had been partially consumed by mid-log phase and were fully consumed by stationary phase (Fig. 1B). Thus, acetate and succinate were sufficient to expedite lactate consumption by R. palustris.

FIG 1.

R. palustris Nx simultaneously utilizes succinate, acetate, and lactate when provided a mix of the three substrates. (A) Representative growth curve of R. palustris Nx grow in MDC with 5 mM (each) succinate, acetate, and lactate (45 mM carbon total). Similar trends were observed for three other biological replicates. (B) Amount (%) of succinate (circles), acetate (triangles), and lactate (squares) remaining in culture supernatants at the indicated cell densities. Each of the four shades of gray indicates an independent biological replicate.

To ascertain if lactate consumption was stimulated by succinate and acetate together, by only one of the cosubstrates, or by either cosubstrate, we also examined growth and carbon utilization in cultures pairing lactate with either acetate or succinate. Cultures reached stationary phase with each of these mixtures within 150 to 200 h (Fig. 2A). For comparison, no growth was detected during the same time period when 10 mM lactate was provided as the sole carbon source (Fig. 2A). All cultures consumed all substrates provided (Fig. 2B), indicating that lactate consumption was stimulated by either cosubstrate. These data also indicated that the coutilization of lactate did not diminish consumption of succinate or acetate. As the disappearance of substrate from culture supernatants could be due to either substrate transformation (e.g., degradation) or assimilation, we also compared the growth yields of the cultures. To account for the different amounts of total carbon provided to the cultures (see Fig. 2 legend), we calculated growth yields as the net cell carbon (derived from the final cell optical density less that of the inoculum) per mole of carbon consumed. All final biomass yields were near the theoretical maximum of 1 mole of biomass per mole of carbon consumed (Fig. 2C), signifying that all substrates were being assimilated in these cultures. Such high growth yields are consistent with those previously observed for R. palustris (16, 19).

FIG 2.

Both succinate and acetate individually stimulate expedited coutilization of lactate by R. palustris Nx. (A) Representative growth curves of R. palustris Nx in MDC with 10 mM individual substrates (acetate [ace; 20 mM carbon], lactate [lac; 30 mM carbon], or succinate [succ; 40 mM carbon]) or 5 mM (each) paired substrates (ace/lac [25 mM carbon total]; succ/lac [35 mM carbon total]). (B, C) Amount (%) of each substrate consumed at stationary phase (B) and growth yields (C) of cultures provided 10 mM acetate (20 mM carbon), 5 mM each acetate and lactate (25 mM carbon total), or 5 mM each succinate, acetate, and lactate (45 mM carbon total). Error bars indicate SDs; n ≥ 3. (C) Growth yields were derived from the change in OD₆₆₀ divided by the change in substrate carbon concentration between the initial and final time points. OD₆₆₀ values were converted into molar carbon using conversion factors of 625 mg dry cell weight/liter/OD₆₆₀ (43) and the molecular weight for a biomass of 22.426 g/mol based on the elemental composition of R. palustris 42OL, CH_1.8N_0.18O_0.38 (44). The theoretical maximum growth yield is 1 mol biomass C per mol C consumed. (D) Representative growth curves of R. palustris Nx in MDC with 5 mM acetate alone, 5 mM lactate alone, or 5 mM lactate supplemented with the indicated concentrations of acetate. (A, D) Similar trends were observed for three other biological replicates for each condition. (E) Growth curve of R. palustris Nx that was subcultured from stationary-phase ace/lac cultures (orange diamonds in panel A) into MDC with 10 mM lactate alone. Error bars indicate SDs; n = 4.

To assess the sensitivity of R. palustris lactate utilization to the presence of a cosubstrate, we next examined growth in cultures containing 5 mM lactate with various concentrations of acetate (0.5 mM to 5 mM). Although the growth rates varied slightly with acetate concentration, stimulation of lactate utilization occurred with all acetate concentrations tested (Fig. 2D). Thus, lactate utilization can be stimulated by a range of cosubstrate concentrations and different lactate/cosubstrate ratios. Notably, cells from cultures grown with acetate plus lactate, which presumably contained the necessary enzymes for lactate catabolism, failed to grow when transferred to fresh medium with lactate as the sole carbon source (Fig. 2E); this suggests that coutilization itself is necessary for expedited lactate utilization and that cosubstrates are inadequate to prime cell physiology for growth on lactate as the sole carbon source. Based on these data, we conclude that utilization of acetate or succinate is sufficient to stimulate the expedited and simultaneous coutilization of lactate by R. palustris.

Mixed-substrate utilization stimulates lactate consumption in diverse R. palustris strains.

The above-described experiments examined lactate utilization under conditions that mimicked coculture conditions, wherein lactate coutilization was first observed, in the following two regards. First, we used the engineered Nx strain of R. palustris, which harbors a mutation in nifA resulting in constitutive N₂ fixation, deletion of hupS to prevent H₂ oxidation, and deletion of hfsE to prevent cell aggregation (21, 22). Second, the cultures were grown in a minimal medium (MDC) with N₂ as the sole nitrogen source (21). To assess if the engineered mutations and/or N₂-fixing conditions contributed to the coutilization of lactate with acetate and succinate, we examined carbon utilization in CGA009, the wild-type parent strain of R. palustris Nx, grown with the acetate, succinate, and lactate mixture or with lactate alone in either MDC or in an NH₄⁺-containing minimal medium, PM. The presence of NH₄⁺ in PM represses N₂ fixation in CGA009 (23). Lactate utilization patterns in R. palustris CGA009 were similar to those in R. palustris Nx, regardless of the medium: CGA009 consumed all three compounds when provided as a mixture within 150 h and failed to grow with lactate alone in the same time frame (Fig. 3). Thus, the observed lactate consumption patterns were not due to either the engineered mutations in the Nx strain or the N₂-fixing conditions.

FIG 3.

Stimulation of lactate consumption via coutilization of acetate and succinate also occurs in wild-type R. palustris CGA009 and is independent of N₂ fixation. (A) Representative growth curves of R. palustris CGA009 in MDC or PM with either 5 mM (each) succinate, acetate, and lactate (ace/succ/lac; 45 mM carbon total) or 10 mM lactate alone (30 mM carbon). Similar trends were observed for three other biological replicates under each condition. (B) Amount (%) of each substrate consumed at stationary phase in cultures of R. palustris CGA009 in MDC or PM with 5 mM concentrations each of succinate, acetate, and lactate (45 mM carbon total). Error bars indicate SDs; n = 4.

We also investigated if acetate and succinate stimulated lactate utilization in environmental R. palustris strains. Environmental isolates of R. palustris have large genetic differences and exhibit unique metabolic characteristics that are thought to aid in nutrient acquisition, anaerobic fermentation, and/or light harvesting (24–26). Thus, it was conceivable that other R. palustris strains behave differently with regard to lactate utilization, either readily using lactate as a sole carbon source or failing to use lactate even in the presence of additional organic acids. However, these potential alternatives were refuted for two environmental isolates, namely, BisB5 and DX-1. When BisB5 and DX-1 were grown with acetate, succinate, and lactate in PM (Fig. 4A), all three compounds were consumed within 120 h (Fig. 4B). In contrast, little or no growth was observed with lactate as the sole carbon source within the same time frame (Fig. 4A). These results indicate that stimulation of lactate catabolism via mixed-substrate utilization is conserved among diverse R. palustris strains.

FIG 4.

Stimulation of lactate consumption via coutilization of acetate and succinate also occurs in environmental R. palustris isolates. (A) Representative growth curves of R. palustris strains BisB5 or DX-1 in PM with either 5 mM (each) succinate, acetate, and lactate (45 mM carbon total) or 10 mM lactate alone (30 mM carbon). Similar trends were observed for three other biological replicates for each strain under each condition. (B) Amount (%) of each substrate consumed at stationary phase in cultures of R. palustris BisB5 or DX-1 in PM with 5 mM each succinate, acetate, and lactate (45 mM carbon total). Error bars indicate SDs; n = 4.

R. palustris Nx lactate utilization is stimulated by diverse carbon cosubstrates.

The carbon substrates available to R. palustris in natural environments are presumably more diverse than E. coli fermentation products. Therefore, we investigated if lactate utilization by R. palustris was stimulated by coconsumption of carbon substrates other than acetate and succinate. Specifically, we grew R. palustris with malate, butyrate, or glycerol, either as the sole carbon source or paired with lactate. Glucose was not tested because R. palustris cannot consume sugars (27). It warrants mentioning that R. palustris cannot grow phototrophically on butyrate alone unless it can dispose of the excess electrons associated with this substrate; CO₂ fixation is perhaps the best-known mechanism by which R. palustris will dispose of excess electrons, but N₂ fixation can also fill this role (16). Because the R. palustris Nx strain harbors a NifA* mutation resulting in constitutive nitrogenase activity (16), and the MDC medium used in these cultures necessitates N₂ fixation for growth (21), R. palustris Nx is readily able to achieve electron balance and grow with butyrate alone. Similar to the results with acetate and succinate, when we grew R. palustris with lactate paired with malate, butyrate, or glycerol, we observed that lactate was utilized simultaneously with each of the three substrates (Fig. 5A to C). These data demonstrate that lactate utilization can be stimulated by diverse cosubstrates.

FIG 5.

R. palustris coutilizes many, but not all, carbon substrate pairs. (A to C) Representative growth curves and amount (%) of substrates remaining at early log phase for R. palustris Nx in MDC with 5 mM (each) paired substrates, as follows: malate plus lactate (mal/lac; 35 mM carbon total) (A), butyrate plus lactate (buty/lac; 35 mM carbon total) (B), and glycerol plus lactate (gly/lac; 30 mM carbon total) (C). Representative growth curves for R. palustris Nx in MDC with 10 mM nonlactate substrates alone (malate [40 mM carbon], butyrate [40 mM carbon], glycerol [30 mM carbon]) are included for comparison. Similar trends were observed for two or more additional biological replicates for each condition. (D) Specific growth rates of R. palustris Nx in MDC with the indicated carbon substrates. NG, no growth. Error bars indicate SDs; n ≥ 3. Different letters indicate statistically significant differences between groups (P < 0.05; one-way analysis of variance with Tukey's multiple-comparison test). (E to G) Representative growth curves and amount (%) of substrates remaining at log phase and stationary phase for R. palustris Nx in MDC with 5 mM (each) nonlactate paired substrates, as follows: succinate plus acetate (succ/ace; 30 mM carbon total) (E), succinate plus glycerol (succ/gly; 35 mM carbon total) (F), and acetate plus glycerol (ace/gly; 25 mM carbon total) (G). Similar trends were observed for two or more additional biological replicates. In panel G, the numbered brackets indicate the two exponential growth phases (see panel D). (H) Amount (%) of acetate and glycerol remaining in supernatants of ace/gly cultures at the indicated cell densities. Each of the four shades of green (acetate) or purple (glycerol) indicates an independent biological replicate.

Differential effects of mixed-substrate utilization on R. palustris Nx growth.

In working with different substrate mixtures containing lactate, we noticed that coutilization sometimes resulted in growth rates that differed from those on single carbon sources. Specific growth rates (μ) during coutilization could be categorized in comparison to the growth rates on the constituent substrates alone, as follows: (i) the mixed-substrate μ was higher than that on either substrate alone (i.e., enhanced μ); (ii) the mixed-substrate μ approximated that when grown on the individual substrate allowing the fastest growth (i.e., equivalent μ); or (iii) the mixed-substrate μ was between the μ’s on the individual substrates (i.e., intermediate μ). We considered cultures with lactate as the sole carbon source to have a growth rate of 0 h⁻¹, as no growth was observed in these cultures within experimental time frames (≤300 h). We observed enhanced μ in cultures pairing lactate with glycerol, equivalent μ in cultures pairing lactate with succinate or malate, and intermediate μ in cultures pairing lactate with acetate or butyrate (Fig. 5D). Akin to growth patterns in other species (13), there was no evident correlation between the effect of mixed-substrate utilization on growth rate and either the metabolic entry point of the cosubstrate or the growth rate on the cosubstrate alone.

To investigate if the changes in mixed-substrate growth rates were contingent on the cosubstrate rather than on lactate itself, we examined growth of R. palustris with three substrate pairs that did not contain lactate: succinate with acetate, succinate with glycerol, and acetate with glycerol. When acetate was paired with succinate, the compounds were utilized simultaneously and the growth rate matched that of cultures with acetate alone (equivalent μ) (Fig. 5D and E). Similar results were seen in cultures containing succinate paired with glycerol, with growth rates approximating those of succinate, the “preferred” carbon source (equivalent μ) (Fig. 5D and F). However, pairing acetate with glycerol resulted in two distinct exponential growth phases, with the first and second phases having growth rates that approximated those with acetate alone and glycerol alone, respectively (Fig. 5D and G). This pattern suggested that acetate and glycerol were being consumed sequentially rather than simultaneously. HPLC results confirmed that acetate consumption occurred during the first exponential phase whereas glycerol consumption did not occur until acetate had been depleted from the medium (Fig. 5G and H). From these data, we conclude that whereas R. palustris can simultaneously consume a wide range of substrates when provided in mixtures of two and three, acetate and glycerol are consumed sequentially by R. palustris.

DISCUSSION

Here we revealed that lactate can be readily catabolized by R. palustris in the presence of various other organic acids and glycerol (Fig. 2 and 5), despite that lactate did not support growth as the sole carbon source in the same time frames. Mixed-substrate-mediated stimulation of lactate consumption occurred in both lab-adapted and environmental wild-type R. palustris strains that are genetically distinct (Fig. 3 and 4). Thus, this phenomenon appears to be broadly conserved despite the high degree of genetic diversity that exists among isolates of this species (24–26).

It is tempting to speculate how coutilization expedites lactate consumption. The fact that we observed a similar induction effect with diverse substrates that enter central metabolism at different points of both glycolysis/gluconeogenesis and the tricarboxylic acid (TCA) cycle makes it difficult to predict the underlying mechanism(s). However, we believe that several mechanisms can be excluded. First, there are instances where cosubstrates enable anaerobic growth by acting as alternative electron acceptors and thereby contributing to cellular redox balance (28–30). The contribution of cosubstrates to electron balance during lactate coutilization is unlikely because (i) the same pattern of lactate utilization was observed under two conditions that differentially allow N₂ fixation (Fig. 3), a process known to satisfy electron balance in R. palustris (16), and (ii) lactate utilization was stimulated equivalently by carbon substrates that were more oxidized or less oxidized than lactate (Table 1). Second, cotransport is unlikely to be responsible, as coconsumption was not strictly dependent on lactate/cosubstrate stoichiometry (Fig. 2D) and induction occurred with diverse cosubstrates that presumably do not all utilize the same transporter (Fig. 2 and 5). Finally, in some instances, cosubstrates can have an “auxiliary” effect by providing energy during the catabolism of energy-deficit substrates (1, 31). The need for supplemental energy generation is unlikely in the case presented herein, given that R. palustris was grown under phototrophic conditions where energy is derived from light. Although outside the scope of this study, we hope that future work identifies the mechanism(s) by which mixed-substrate utilization expedites lactate consumption by R. palustris.

TABLE 1.

Oxidation states of tested R. palustris growth substrates

Substrate	Formula	Oxidation statea
Malate	C4H6O5	+1
Succinate	C4H6O4	+0.5
Lactate	C3H6O3	0
Acetate	C2H3O2	0
Glycerol	C3H8O3	–0.67
Butyrate	C4H8O2	–1

^aOxidation states were calculated as previously described (19).

This study was initiated to investigate the potential coutilization of lactate with other carbon substrates. However, our results also revealed acetate inhibition of glycerol catabolism in R. palustris (Fig. 5G and H). R. palustris is well known for its strict control of nitrogen utilization, wherein the presence of ammonium strictly inhibits expression of the nitrogenase enzyme that catalyzes N₂ fixation (23, 32). However, we are unaware of any report of CCR in this species. There was no evident lag phase between the two exponential growth phases in R. palustris cultures containing acetate paired with glycerol (Fig. 5G). This direct transition between exponential phases could indicate that acetate-mediated inhibition of glycerol assimilation in R. palustris occurs at the level of protein activity (e.g., transport or catabolic enzyme activity), rather than the level of protein expression (2, 4, 33). However, it is also possible that new proteins required for glycerol consumption in the second phase can be synthesized upon acetate depletion in a comparatively short time frame relative to the long R. palustris doubling time, such that an intervening lag phase is not observed. Future studies are needed to determine the mechanism by which acetate represses glycerol consumption. R. palustris CGA009 has more than 400 genes predicted to be involved in regulation and signal transduction (27). Among these are genes encoding Crp- and Hpr-like proteins (27, 34). Crp and Hpr homologues regulate diverse biological functions that include CCR in certain species (34, 35). As such, the Hpr- and Crp-like proteins seem logical initial targets for mutagenesis in the endeavor to characterize catabolite control mechanisms in R. palustris. Identifying the transporters used for different carbon substrates in R. palustris will likely also be important for elucidating such mechanisms. As the genome of R. palustris encodes more than 300 different transport systems (27), and results from a large-scale study of ABC transporter proteins indicate that sequence-based homology is unreliable for predicting ligand specificity (36), this will not be a trivial task.

Although simultaneous utilization of carbon substrates is most commonly described under nutrient-limited conditions (2–4), examples are accumulating, including for R. palustris as shown here, wherein bacteria simultaneously utilize substrates even at high concentrations (1, 13). R. palustris simultaneously consumed seven of the eight substrate pairs tested in this study, and published data suggest that this behavior may extend beyond organic acids and glycerol. For example, data from a recent study indicated that R. palustris simultaneously utilizes acetate and various aromatic compounds when grown in corn stover hydrolysate (20), though it was not determined which compounds were being assimilated into biomass. The same study reported simultaneous biological transformation of several aromatic compounds that are not readily utilized as sole carbon sources (15, 20), perhaps indicating that mixed-substrate utilization influences the aromatic utilization spectrum of R. palustris as well. It is possible that assessment of bacterial nutritional repertoires using single substrates underestimates the catabolic capabilities of some bacteria. From an ecological perspective, it would not necessarily be surprising if R. palustris coutilizes a large range of carbon sources. Such a strategy could allow R. palustris to take full advantage of the diverse carbon sources it encounters within the numerous environments it inhabits (27, 37). It has been proposed that carbon source preference reflects the likelihood of encountering various substrates in the environment (38). Thus, to speculate further, the disparity between lactate utilization in the presence and absence of a cosubstrate could indicate that lactate is rarely encountered as the sole carbon source in natural environments. In this case, the inability to readily use lactate as the sole carbon source would not be of consequence to R. palustris. Finally, beyond these potential ecological implications, substrate coutilization, particularly at high substrate concentrations, is preferable for industrial and commercial applications (8, 39). Specifically, such behavior is crucial for developing bioprocesses that utilize inexpensive, renewable waste materials, such as industrial effluents, lignocellulosic biomass, and food waste, as feedstocks for the production of biofuels and value-added products. We believe the proclivity to coutilize carbon substrates enhances the potential biotechnological value of R. palustris.

MATERIALS AND METHODS

Chemicals, strains, and growth conditions.

The R. palustris strains used in this study are listed in Table 2. R. palustris was routinely cultivated on defined mineral (PM) (40) agar supplemented with 10 mM succinate. All cultures were grown in 27-ml anaerobic test tubes containing 10 ml of either defined M9-derived coculture medium (MDC) (21) or PM medium. MDC or PM was bubbled with 100% N₂ or Ar, respectively, and tubes were sealed with rubber stoppers and aluminum crimps prior to autoclaving.

TABLE 2.

R. palustris strains used in this study

Strain	Description	Reference
CGA009	Wild-type strain; spontaneous Cmr derivative of CGA001	40
CGA4005	CGA009 nifA* ΔhupS Δrpa2750; called Nx in this study	21
BisB5	Environmental isolate	24
DX-1	Environmental isolate	42

For starter cultures, single colonies were used to inoculate MDC with limiting (3 mM) acetate. For experimental cultures, 100-μl aliquots of replicate stationary-phase starter cultures were used to inoculate MDC or PM supplemented with either 10 mM single carbon substrate or 5 mM (each) multiple carbon substrates, unless indicated otherwise in the figure legends. Carbon sources were added to the desired final concentrations from 1 M stock solutions of glycerol and sodium salts of l-lactate, acetate, succinate, l-malate, and butyrate. All cultures were incubated horizontally at 30°C under a 43-W A19 halogen bulb (750 lumens) with shaking at 150 rpm. At least three independent biological replicates were performed for each culture condition.

Analytical procedures.

R. palustris growth was monitored via optical density at 660 nm (OD₆₆₀) using a Genesys 20 spectrophotometer (Thermo-Fisher, Waltham, MA, USA). Growth readings were measured in culture tubes without sampling. Specific growth rates were calculated using OD₆₆₀ values between 0.1 and 1.0 where cell density and OD₆₆₀ are linearly correlated. Final cell densities were measured in cuvettes with samples diluted as needed to achieve an OD₆₆₀ within the linear range. Organic acids and glycerol were quantified using a Shimadzu high-performance liquid chromatograph, as previously described (41).