Redox Sink Research Articles

Growth-coupled anaerobic production of isobutanol from glucose in minimal medium with Escherichia coli

BackgroundThe microbial production of isobutanol holds promise to become a sustainable alternative to fossil-based synthesis routes for this important chemical. Escherichia coli has been considered as one production host, however, due to redox imbalance, growth-coupled anaerobic production of isobutanol from glucose in E. coli is only possible if complex media additives or small amounts of oxygen are provided. These strategies have a negative impact on product yield, productivity, reproducibility, and production costs.ResultsIn this study, we propose a strategy based on acetate as co-substrate for resolving the redox imbalance. We constructed the E. coli background strain SB001 (ΔldhA ΔfrdA ΔpflB) with blocked pathways from glucose to alternative fermentation products but with an enabled pathway for acetate uptake and subsequent conversion to ethanol via acetyl-CoA. This strain, if equipped with the isobutanol production plasmid pIBA4, showed robust exponential growth (µ = 0.05 h−1) under anaerobic conditions in minimal glucose medium supplemented with small amounts of acetate. In small-scale batch cultivations, the strain reached a glucose uptake rate of 4.8 mmol gDW−1 h−1, a titer of 74 mM and 89% of the theoretical maximal isobutanol/glucose yield, while secreting only small amounts of ethanol synthesized from acetate. Furthermore, we show that the strain keeps a high metabolic activity also in a pulsed fed-batch bioreactor cultivation, even if cell growth is impaired by the accumulation of isobutanol in the medium.ConclusionsThis study showcases the beneficial utilization of acetate as a co-substrate and redox sink to facilitate growth-coupled production of isobutanol under anaerobic conditions. This approach holds potential for other applications with different production hosts and/or substrate–product combinations.

Enabling anaerobic growth of Escherichia coli on glycerol in defined minimal medium using acetate as redox sink

Glycerol has become an attractive substrate for bio-based production processes. However, Escherichia coli, an established production organism in the biotech industry, is not able to grow on glycerol under strictly anaerobic conditions in defined minimal medium due to redox imbalance. Despite extensive research efforts aiming to overcome these limitations, anaerobic growth of wild-type E. coli on glycerol always required either the addition of electron acceptors for anaerobic respiration (e.g. fumarate) or the supplementation with complex and relatively expensive additives (tryptone or yeast extract). In the present work, driven by model-based calculations, we propose and validate a novel and simple strategy to enable fermentative growth of E. coli on glycerol in defined minimal medium. We show that redox balance could be achieved by uptake of small amounts of acetate with subsequent reduction to ethanol via acetyl-CoA. Using a directed laboratory evolution approach, we were able to confirm this hypothesis and to generate an E. coli strain that shows, under anaerobic conditions with glycerol as the main substrate and acetate as co-substrate, robust growth (μ = 0.06 h−1), a high specific glycerol uptake rate (10.2 mmol/gDW/h) and an ethanol yield close to the theoretical maximum (0.92 mol per mol glycerol). Using further stoichiometric calculations, we also clarify why complex additives such as tryptone used in previous studies enable E. coli to achieve redox balance. Our results provide new biological insights regarding the fermentative metabolism of E. coli and offer new perspectives for sustainable production processes based on glycerol.

Cross Talk Between Cellular Redox State and the Antiapoptotic Protein Bcl-2

B cell lymphoma-2 (Bcl-2) was discovered over three decades ago and is the prototype antiapoptotic member of the Bcl-2 family that comprises proteins with contrasting effects on cell fate. First identified as a consequence of chromosomal translocation (t 14:18) in human lymphoma, subsequent studies have revealed mutations and/or gene copy number alterations as well as post-translational modifications of Bcl-2 in a variety of human cancers. The canonical function of Bcl-2 is linked to its ability to inhibit mitochondrial membrane permeabilization, thereby regulating apoptosome assembly and activation by blocking the cytosolic translocation of death amplification factors. Of note, the identification of specific domains within the Bcl-2 family of proteins (Bcl-2 homology domains; BH domains) has not only provided a mechanistic insight into the various interactions between the member proteins but has also been the impetus behind the design and development of small molecule inhibitors and BH3 mimetics for clinical use. Recent Advances: Aside from its role in maintaining mitochondrial integrity, recent evidence provides testimony to a novel facet in the biology of Bcl-2 that involves an intricate cross talk with cellular redox state. Bcl-2 overexpression modulates mitochondrial redox metabolism to create a "pro-oxidant" milieu, conducive for cell survival. However, under states of oxidative stress, overexpression of Bcl-2 functions as a redox sink to prevent excessive buildup of reactive oxygen species, thereby inhibiting execution signals. Emerging evidence indicates various redox-dependent transcriptional changes and post-translational modifications with different functional outcomes. Understanding the complex interplay between Bcl-2 and the cellular redox milieu from the standpoint of cell fate signaling remains vital for a better understanding of pathological states associated with altered redox metabolism and/or aberrant Bcl-2 expression. Based on its canonical functions, Bcl-2 has emerged as a potential druggable target. Small molecule inhibitors of Bcl-2 and/or other family members with similar function, as well as BH3 mimetics, are showing promise in the clinic. The emerging evidence for the noncanonical activity linked to cellular redox metabolism provides a novel avenue for the design and development of diagnostic and therapeutic strategies against cancers refractory to conventional chemotherapy by the overexpression of this prosurvival protein.

Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness

Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC-MS) analytics to define the molecular loci by which two yield-enhancing feeds improve recombinant antibody yields from a model GS-CHO cell line. With data across core metabolic pathways, that report on metabolism within several cellular compartments, these data identify key metabolites and events associated with increased cell survival and specific productivity of cells. Of particular importance, increased process efficiency was linked to the functional activity of the mitochondria, with the amount and time course of use/production of intermediates of the citric acid cycle, for uses such as lipid biosynthesis, precursor generation and energy production, providing direct indicators of cellular status with respect to productivity. The data provide clear association between specific cellular metabolic indicators and cell process efficiency, extending from prior indications of the relevance of lactate metabolic balance to other redox sinks (glycerol, sorbitol and threitol). The information, and its interpretation, identifies targets for engineering cell culture efficiency, either from genetic or environmental perspectives, and greater understanding of the significance of specific medium components towards overall CHO cell bioprocessing.

Dekkera bruxellensis--spoilage yeast with biotechnological potential, and a model for yeast evolution, physiology and competitiveness.

Dekkera bruxellensis is a non-conventional yeast normally considered a spoilage organism in wine (off-flavours) and in the bioethanol industry. But it also has potential as production yeast. The species diverged from Saccharomyces cerevisiae 200 mya, before the whole genome duplication. However, it displays similar characteristics such as being Crabtree- and petite positive, and the ability to grow anaerobically. Partial increases in ploidy and promoter rewiring may have enabled evolution of the fermentative lifestyle in D. bruxellensis. On the other hand, it has genes typical for respiratory yeasts, such as for complex I or the alternative oxidase AOX1. Dekkera bruxellensis grows more slowly than S. cerevisiae, but produces similar or greater amounts of ethanol, and very low amounts of glycerol. Glycerol production represents a loss of energy but also functions as a redox sink for NADH formed during synthesis of amino acids and other compounds. Accordingly, anaerobic growth required addition of certain amino acids. In spite of its slow growth, D. bruxellensis outcompeted S. cerevisiae in glucose-limited cultures, indicating a more efficient energy metabolism and/or higher affinity for glucose. This review tries to summarize the latest discoveries about evolution, physiology and metabolism, and biotechnological potential of D. bruxellensis.

Kinetic of Azo Dyes Decolourization by Enterobacteriaceae Species in the Intact Cell Assay System

The method Intact cell was adopted to assess the influencing factors on the process of decolourization ability of the enterobacteriaceaea specie isolates under predisposed environment. Taken into account several ingredient are added individually as well in combinations in the assay reaction. Values of dye decolourized in µg ml -1 with Multi vitanmins solution (MVS) with glucose 42.53; MVS 36.00; Riboflavin (RF) 39.00; Yeast extract 36.00 and B- complex 23.00 from the initial dye of 46.00µg ml -1 were observed , where as glucose, Ascorbic acid, Cysteine, Cetyl-trimethyl-ammonium bromide (CTAB), Sodium molybdate, Biotin, KNO3, NaNO2, folic acid and 1-amino-2- naphtho-4-sulfonic acid (ANSA) does not shows influence on to the process, but, some of them showed inhibitory effect toward the decolourization. It was observed that the riboflavin addition at 19.95 n moles ml -1 in the reaction mixture, rate of decolourization was suddenly change from 0.019 ∆A/minutes to 0.20 ∆A/minutes, which is extremely high by 10 fold fast and subsequently remains faster i.e 0.2 ∆A/minutes without further additional RF in the same assay mixture. Rate of decolourization with different concentrations of riboflavin i.e. 13.0 n moles ml -1 to 59.9 n moles ml -1 showed second order kinetic. This indicates that the minimum amount of RF is essential to trigger the process of decolourization by the intact cells under the assay condition. While five different azo dyes were subjected, showed diverse behavior on to the rate of decolourization. Results of entire study incite on role of riboflavin could to a certain extent act as a redox mediator in the reaction(s) process and electron mediator between intracellular pool to the dye available at periplasmic redox sink @ JASEM

Formation of glycerol from glucose in rat brain and cultured brain cells. Augmentation with kainate or ischemia

An increase in the concentration of glycerol in the ischemic brain is assumed to reflect degradation of phospholipids of plasma membranes. However, glycerol could, theoretically, be formed from glucose, which after glycolytic conversion to dihydroxyacetone phosphate, could be converted to glycerol-3-phosphate and hence to glycerol. We show here that (13)C-labeled glycerol accumulate in incubation media of cultured cerebellar granule cells and astrocytes incubated with [(13)C]glucose, 3 mmol/L, demonstrating the formation of glycerol from glucose. Co-incubation of cerebellar granule cells with kainate, 50 micromol/L, led to increased glucose metabolism and increased accumulation of [(13)C]glycerol. Accumulation of [(13)C]glycerol and its precursor, [(13)C]glycerol-3-phosphate, was evident in brain, but not in serum, of kainate-treated rats that received [U-(13)C]glucose, 5 micromol/g bodyweight, intravenously and survived for 5 min. Global ischemia induced by decapitation also caused accumulation of [(13)C]glycerol and [(13)C]glycerol-3-phosphate. These results show that glycerol can be formed from glucose in brain; they also demonstrate the existence of a cerebral glycerol-3-phosphatase activity. Ischemia-induced increases in brain glycerol may, in part, reflect an altered metabolism of glucose, in which glycerol formation, like lactate formation, acts as a redox sink.

NADH-reductive stress in Saccharomyces cerevisiae induces the expression of the minor isoform of glyceraldehyde-3-phosphate dehydrogenase (TDH1).

A strain of Saccharomyces cerevisiae lacking the GPD2 gene, encoding one of the glycerol-3-phosphate dehydrogenases, grows slowly under anaerobic conditions, due to reductive stress caused by the accumulation of cytoplasmic NADH. We used 2D-PAGE to study the effect on global protein expression of reductive stress in the anaerobically grown gpd2Delta strain. The most striking response was a strongly elevated expression of Tdh1p, the minor isoform of glyceraldehyde-3-phosphate dehydrogenase. This increased expression could be reversed by the addition of acetoin, a NADH-specific redox sink, which furthermore largely restored anaerobic growth of the gpd2Delta strain. Additional deletion of the TDH1 gene (but not of TDH2 or TDH3) improved anaerobic growth of the gpd2Delta strain. We therefore propose that TDH1 has properties not displayed by the other TDH isogenes and that its expression is regulated by reductive stress caused by an excess of cytoplasmic NADH.

Effects of furfural on anaerobic continuous cultivation of Saccharomyces cerevisiae.

Furfural is an important inhibitor of yeast metabolism in lignocellulose-derived substrates. The effect of furfural on the physiology of Saccharomyces cerevisiae CBS 8066 was investigated using anaerobic continuous cultivations. Experiments were performed with furfural in the feed medium (up to 8.3 g/L) using three different dilution rates (0.095, 0.190, and 0.315 h(-1)). The measured concentration of furfural was low (< 0.1 g/L) at all steady states obtained. However, it was not possible to achieve a steady state at a specific conversion rate of furfural, q(f), higher than approximately 0.15 g/g.h. An increased furfural concentration in the feed caused a decrease in the steady-state glycerol yield. This agreed well with the decreased need for glycerol production as a way to regenerate NAD+, i.e., to function as a redox sink because furfural was reduced to furfuryl alcohol. Transient experiments were also performed by pulse addition of furfural directly into the fermentor. In contrast to the situation at steady-state conditions, both glycerol and furfuryl alcohol yields increased after pulse addition of furfural to the culture. Furthermore, the maximum specific conversion rate of furfural (0.6 g/g.h) in dynamic experiments was significantly higher than what was attainable in the chemostat experiments. The dynamic furfural conversion could be described by the use of a simple Michaelis-Menten-type kinetic model. Also furfural conversion under steady-state conditions could be explained by a Michaelis-Menten-type kinetic model, but with a higher affinity and a lower maximum conversion rate. This indicated the presence of an additional component with a higher affinity, but lower maximum capacity, either in the transport system or in the conversion system of furfural.

Branched-chain alpha-keto acid catabolism via the gene products of the bkd operon in Enterococcus faecalis: a new, secreted metabolite serving as a temporary redox sink.

Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain alpha-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the bkd gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the bkd gene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain alpha-keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the alpha-keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of alpha-keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD(+) from the NADH produced by the branched-chain alpha-keto acid dehydrogenase complex was required for complete conversion of alpha-ketoisocaproate. Interestingly, during the conversion of the branched-chain alpha-keto acids an intermediate was always detected extracellularly. With alpha-ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1, 1-dihydroxy-4-methyl-2-pentanone. This reduced form of alpha-ketoisocaproic acid was found to serve as a temporary redox sink.

Microaerobic glycerol formation in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae produces large amounts of glycerol as an osmoregulator during hyperosmotic stress and as a redox sink at low oxygen availability. NAD(+)-dependent glycerol-3-phosphate dehydrogenase in S. cerevisiae is present in two isoforms, coded for by two different genes, GPD1 and GPD2. Mutants for either one or both of these genes were investigated under carefully controlled static and dynamic conditions in continuous cultures at low oxygen transfer rates. Our results show that S. cerevisiae controls the production of glycerol in response to hypoxic conditions by regulating the expression of several genes. At high demand for NADH reoxidation, a strong induction was seen not only of the GPD2 gene, but also of GPP1, encoding one of the molecular forms of glycerol-3-phosphatase. Induction of the GPP1 gene appears to play a decisive role at elevated growth rates. At low demand for NADH reoxidation via glycerol formation, the GPD1, GPD2, GPP1, and GPP2 genes were all expressed at basal levels. The dynamics of the gene induction and the glycerol formation at low demand for NADH reoxidation point to an important role of the Gpd1p; deletion of the GPD1 gene strongly altered the expression patterns of the GPD2 and GPP1 genes under such conditions. Furthermore, our results indicate that GCY1 and DAK1, tentatively encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, may be involved in the redox regulation of S. cerevisiae.

Influence of furfural on anaerobic glycolytic kinetics of Saccharomyces cerevisiae in batch culture.

Furfural was reduced to furfuryl alcohol by Saccaromyces cerevisiae with a yield of 0.97 +/- 0.01 mmol/ mmol causing a lag phase in cell growth. In the presence of 29 mM, furfural the cell-mass yield on glucose decreased from 11.0 +/- 0.1 mg/mmol (0.06 +/- 0.0006 g/g) in a reference fermentation without furfural to 9.7 +/- 0.07 mg/ mmol (0.05 +/- 0.0004 g/g), whereas the ethanol yield on glucose increased from 1.6 +/- 0.03 to 1.7 +/- 0.03 mmol/mmol. No glycerol was excreted during furfural reduction, and the lag phase in acetate production was extended from 1 h in the reference fermentation to 5 h in the presence of furfural. Acetaldehyde and pyruvate were excreted during the furfural reduction phase. Cell growth and cell maintenance were proportional to glucose consumption during the entire fermentation, whereas the cell-mass yield on ATP produced was low during furfural reduction. These observations indicate that furfural addition to a batch culture decreased cell replication without inhibiting cell activity (designated as replicative inactivation). The absence of glycerol production during furfural reduction suggests that furfural acted as an alternative redox sink oxidizing excess NADH formed in biosynthesis. A mechanistic mathematical model was developed that described accurately the fermentation in the absence and presence of furfural. The model was based on the assumptions that: (i) furfural reduction to furfuryl alcohol by NADH-dependent dehydrogenases had a higher priority than reduction of dihydroxyacetone phosphate to glycerol; and (ii) furfural caused inactivation of cell replication. The effect of furfural on cell replication is discussed in relation to acetaldehyde formation. Inactivation of cell replication was modeled by considering two populations within the cell culture, both metabolically active, but only one replicating. The kinetic description was developed as a tool to estimate transient fluxes of carbon, NADH/NAD+ and ATP/ADP.

The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation.

The two homologous genes GPD1 and GPD2 encode the isoenzymes of NAD-dependent glycerol 3-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae. Previous studies showed that GPD1 plays a role in osmoadaptation since its expression is induced by osmotic stress and gpd1 delta mutants are osmosensitive. Here we report that GPD2 has an entirely different physiological role. Expression of GPD2 is not affected by changes in external osmolarity, but is stimulated by anoxic conditions. Mutants lacking GPD2 show poor growth under anaerobic conditions. Mutants deleted for both GPD1 and GPD2 do not produce detectable glycerol, are highly osmosensitive and fail to grow under anoxic conditions. This growth inhibition, which is accompanied by a strong intracellular accumulation of NADH, is relieved by external addition of acetaldehyde, an effective oxidizer of NADH. Thus, glycerol formation is strictly required as a redox sink for excess cytosolic NADH during anaerobic metabolism. The anaerobic induction of GPD2 is independent of the HOG pathway which controls the osmotic induction of GPD1. Expression of GPD2 is also unaffected by ROX1 and ROX3, encoding putative regulators of hypoxic and stress-controlled gene expression. In addition, GPD2 is induced under aerobic conditions by the addition of bisulfite which causes NADH accumulation by inhibiting the final, reductive step in ethanol fermentation and this induction is reversed by addition of acetaldehyde. We conclude that expression of GPD2 is controlled by a novel, oxygen-independent, signalling pathway which is required to regulate metabolism under anoxic conditions.

Physiological response to anaerobicity of glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae, in which one or both of the genes encoding the two isoforms of NAD-dependent glycerol-3-phosphate dehydrogenase had been deleted, were studied in aerobic batch cultures and in aerobic-anaerobic step change experiments. The respirofermentative growth rates under aerobic conditions with semisynthetic medium (20 g of glucose per liter) of two single mutants, gpd1 delta and gpd2 delta, and the parental strain (mu = 0.5 h-1) were almost identical, whereas the growth rate of a double mutant, gpd1 delta gpd2 delta, was approximately half that of the parental strain. Upon a step change from aerobic to anaerobic conditions in the exponential growth phase, the specific carbon dioxide evolution rates (CER) of the wild-type strain and the gpd1 delta strain were almost unchanged. The gpd2 delta mutant showed an immediate, large (> 50%) decrease in CER upon a change to anaerobic conditions. However, after about 45 min the CER increased again, although not to the same level as under aerobic conditions. The gpd1 delta gpd2 delta mutant showed a drastic fermentation rate decrease upon a transition to anaerobic conditions. However, the CER values increased to and even exceeded the aerobic levels after the addition of acetoin. High-pressure liquid chromatographic analyses demonstrated that the added acetoin served as an acceptor of reducing equivalents by being reduced to butanediol. The results clearly show the necessity of glycerol formation as a redox sink for S. cerevisiae under anaerobic conditions.

A glycerol-3-phosphate dehydrogenase-deficient mutant of Saccharomyces cerevisiae expressing the heterologous XYL1 gene.

The gene XYL1, encoding a xylose reductase, from Pichia stipitis was transformed into a mutant of Saccharomyces cerevisiae incapable of glycerol production because of deletion of the genes GPD1 and GPD2. The transformed strain was capable of anaerobic glucose conversion in the presence of added xylose, indicating that the xylose reductase reaction can fulfill the role of the glycerol-3-phosphate dehydrogenase reaction as a redox sink. The specific xylitol production rate obtained was 0.38 g g-1 h-1.

Existence of Cyanide-Insensitive Respiration in the Yeast Pichia stipitis and Its Possible Influence on Product Formation during Xylose Utilization

A cyanide-insensitive and salicyl hydroxamic acid-sensitive respiration (CIR) was found in the yeast Pichia stipitis in contrast to Candida utilis, Pachysolen tannophilus, and Saccharomyces cerevisiae. During xylose utilization in the presence of either salicyl hydroxamic acid or cyanide, P. stipitis formed xylitol, arabitol, and ribitol. The existence of CIR is discussed in terms of a redox sink preventing xylitol formation in P. stipitis.

Mitochondrial DNA mutation and the ageing process: bioenergy and pharmacological intervention

A comprehensive hypothesis concerning the contribution of mitochondrial DNA (mtDNA) mutations to the human ageing process is reviewed and the implications for cellular bioenergy loss and pharmacological therapy are considered. The central idea is that random mutations in the population of mtDNA molecules of each cell occur throughout life, and that this is a major contributor to the gradual loss of cellular bioenergy capacity within tissues and organs, associated with general senescence and diseases of ageing. An elaboration of four major aspects of the general proposition, together with relevant supporting data, is presented. (1) An extensive array of deletions in mtDNA of many tissues of humans and other mammals has been observed to occur in an age-related manner. (2) The preservation and selection of fully functional mtDNA molecules in the female germ line cells is proposed to occur via a human mtDNA cycle, in which selective amplification of a limited number of mtDNA templates occurs during oocyte development. This proposal explains the endowment of normal neonates with a mtDNA complement minimally contaminated by damaged mtDNA molecules. The phenomena of maternal inheritance and rapid fixation of sequence variants of mtDNA in mammals, as well as selection of cells based on mitochondrial function, are taken into account. (3) Tissue bioenergy mosaics result from accumulated mtDNA damage during ageing, representing different rates of cellular bioenergy loss within individual cells of a tissue. The random segregation of mtDNA during cell division will also further contribute to the tissue energy mosaic. Cells unable to meet their particular bioenergy demand will become non-functional, leading to cell death; the bioenergy threshold is different for the various cell types in the tissues of the body. (4) In order to bioenergetically resuscitate cells and tissues suffering from impaired mitochondrial functions as a result of the ageing process, we propose that redox compounds may be used therapeutically in the pharmacological configurations of a by-pass strategy or as a redox sink therapy. The role of these compounds is to maintain at least part of the mitochondrial respiratory chain function (by-pass) as well as to maintain adequate levels of cellular NAD+ (redox sink) for ATP synthesis, predominantly by the cytosolic glycolytic pathway, with some contribution from mitochondrial oxidative phosphorylation.

Anaerobic degradation of trans-cinnamate and omega-phenylalkane carboxylic acids by the photosynthetic bacterium Rhodopseudomonas palustris: evidence for a beta-oxidation mechanism.

The mechanism responsible for the initial steps in the anaerobic degradation of trans-cinnamate and omega-phenylalkane carboxylates by the purple non-sulphur photosynthetic bacterium Rhodopseudomonas palustris was investigated. Phenylacetate did not support growth and there was a marked CO2 dependence for growth on acids with greater side-chain lengths. Here, CO2 was presumably acting as a redox sink for the disposal of excess reducing equivalents. Growth on benzoate did not require the addition of exogenous CO2. Aromatic acids with an odd number of side-chain carbon atoms (3-phenylpropionate, 5-phenylvalerate, 7-phenylheptanoate) gave greater apparent molar growth yields than those with an even number of side-chain carbon atoms (4-phenylbutyrate, 6-phenylhexanoate, 8-phenyloctanoate). HPLC analysis revealed that phenylacetate accumulated and persisted in the culture medium during growth on these latter compounds. Cinnamate and benzoate transiently accumulated in the culture medium during growth on 3-phenylpropionate, and benzoate alone accumulated transiently during the course of trans-cinnamate degradation. The transient accumulation of 4-phenyl-2-butenoic acid occurred during growth on 4-phenylbutyrate, and phenylacetate accumulated to a 1:1 molar stoichiometry with the initial 4-phenylbutyrate concentration. It is proposed that the initial steps in the anaerobic degradation of trans-cinnamate and the group of acids from 3-phenylpropionate to 8-phenyloctanoate involves beta-oxidation of the side-chain.