Purple phototrophic bacteria release crotonate as metabolic overflow pathway to complement other redox-balancing routes

Purple phototrophic bacteria release crotonate as metabolic overflow pathway to complement other redox-balancing routes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Purple phototrophic bacteria release crotonate as metabolic overflow pathway to complement other redox-balancing routes Daniel Puyol, Luis Allegue, Maria Ventura, Siegfried Vlaeminck, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7242062/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Purple phototrophic bacteria (PPB) are renowned for versatile metabolic strategies to maintain redox balance, including CO₂ fixation, H₂ production, and polymer storage. Here we report a previously unknow redox-balancing mechanism in PPB: the extracellular release of crotonate as a metabolic overflow. In batch photoheterotrophic cultures of enriched PPB, crotonate accumulated transiently under conditions of carbon excess. Crotonate excretion coincided with the depletion of other electron sinks (polyhydroxybutyrate and H₂) and was reversed when an alternate electron acceptor (DMSO) was provided, indicating a regulated overflow rather than irreversible fermentation. Using metaproteomics, we found that dominant Rhodopseudomonas species redirect metabolism toward crotonyl-CoA production when conventional acetyl-CoA assimilation routes are limited. Key enzymes of the glyoxylate cycle were suppressed, while enzymes converting acetyl-CoA to crotonyl-CoA were up-regulated, leading to crotonyl-CoA accumulation. Notably, a CoA-transferase enzyme was identified as a candidate responsible for converting crotonyl-CoA to free crotonate, enabling excretion. These results reveal crotonate as an “escape valve” metabolite that PPB naturally deploy to dissipate excess reductant. The discovery of crotonate overflow expands our understanding of microbial redox homeostasis and highlights a novel facet of PPB metabolism with potential implications for optimizing biotechnological processes such as bioplastic production Biological sciences/Biotechnology/Environmental biotechnology Biological sciences/Microbiology/Applied microbiology Biological sciences/Molecular biology/Proteomics Figures Figure 1 Figure 2 Introduction Purple phototrophic bacteria (PPB) are metabolically versatile microorganisms used in biotechnologies ranging from wastewater valorization to biopolymer production. Central to their versatility is a complex redox-balancing machinery that enables them to thrive under diverse environmental conditions. Their defining feature is their ability to grow photoheterotrophically (particularly in the case of the subset of purple non-sulfur bacteria), using light as an energy source while metabolizing organic compounds as both carbon and electron sources ( 1 ). This phototroph metabolism enables PPB to achieve remarkably high efficiencies, with carbon conversion yields reaching up to 100% under optimal conditions ( 2 ). These characteristics have generated substantial interest in harnessing PPB for environmental applications, particularly in wastewater treatment and resource recovery. PPB offer a sustainable alternative for the production of bulk commodities, including microbial protein and bioplastics, outperforming conventional processes in terms of resource efficiency and environmental impact. ( 1 , 3 ). Central to the adaptability of PPB is their highly evolved redox-balancing machinery, which enables them to maintain intracellular redox homeostasis under varying environmental conditions. Redox balance in anaerobic photoheterotrophs is achieved through a hierarchy of strategies that modulate intracellular reducing equivalents (e.g., NADH/NAD⁺) to prevent the buildup of excess reducing power, which could otherwise disrupt cellular metabolism ( 4 – 6 ). The Calvin-Benson-Bassham (CBB) cycle is a primary electron sink, fixing CO₂ into organic intermediates that are integrated into central metabolic pathways such as glycolysis. In CBB cycle-positive (IC⁺) purple phototrophic bacteria, such as Rhodopseudomonas (Rps.) palustris , the CBB cycle acts as a key electron sink, fixing CO₂ and contributing up to 40–60% of redox flux during growth on reduced substrates like acetate ( 7 ). Its activity is tightly correlated with the oxidation state of the substrate, as CO₂ fixation offsets excess reducing equivalents, ensuring metabolic equilibrium. In contrast, CBB cycle-negative (IC⁻) strains like Rhodospirillum (Rsp.) rubrum rely on the ethylmalonyl-CoA (EMC) pathway for both carbon assimilation and CO₂ fixation ( 8 ), illustrating alternative strategies for redox balance and the metabolic flexibility of PPB. Another key redox-balancing strategy in PPB is hydrogen (H₂) production, catalyzed by one or more nitrogenases. Although primarily involved in nitrogen fixation, nitrogenases can divert excess electrons toward H₂ evolution. This process occurs through nitrogenase's intrinsic hydrogen-evolving activity, which requires ATP. Additionally, under certain conditions, H₂ can be produced via a reverse electron transfer mechanism involving hydrogenase, but without ATP consumption ( 9 ). However, the presence of ammonium (NH₃) represses nitrogenase expression, thereby limiting its contribution to redox balancing under such conditions ( 10 ). The synthesis of polyhydroxyalkanoates (PHA) provides an additional mechanism for redox management, particularly under nitrogen-limited conditions. These intracellular carbon and electron storage polymers buffer the flow of reducing equivalents and ensure redox homeostasis by temporarily sequestering excess electrons. Since PHA production is environmentally regulated, its role becomes particularly important during organic carbon overload, when managing redox flux is critical ( 6 ). Beyond these broad-spectrum strategies, some PPB species exhibit specialized pathways for redox balancing. For instance, in Rsp. rubrum , alternative pathways have been identified that involve running parts of the tricarboxylic acid (TCA) cycle in reverse to produce alpha-ketoglutarate, a precursor for amino acid biosynthesis ( 11 ). This reversal is particularly relevant under conditions where conventional carbon fixation pathways are impaired or absent, necessitating alternative routes to redistribute reducing equivalents and sustain biosynthetic needs. Similarly, the biosynthesis of branched-chain amino acids, such as isoleucine, has been directly linked to redox balancing ( 12 ). When Rsp. rubrum grows photoheterotrophically on reduced carbon sources like acetate, the synthesis of isoleucine provides an effective electron sink, consuming excess reducing power and preventing an imbalance in intracellular redox homeostasis. However, the contribution of these pathways is limited by the cellular demand for the specific amino acids they produce, making them auxiliary rather than primary strategies for redox management. Crotonyl-CoA is a central intermediate in metabolic pathways such as PHA synthesis and degradation, β-oxidation and the EMC pathway. Despite crotonyl-CoA’s central role, the extracellular release of crotonate by wild-type organisms has never been observed. Crotonate possesses a degree of reduction of 4.5 electrons per mol carbon (e⁻/C), making it more reduced than typical microbial biomass (C₅H₇O₂N ~ 4.2 e⁻/C) and similar to PHA, whose monomeric units (e.g., 3-hydroxybutyrate) share the same redox value (4.5 e⁻/C). This high degree of reduction suggests that crotonate could serve as an electron sink, providing a potential strategy for redox balancing under conditions of carbon and electron excess. To date, extracellular crotonate production has been only observed in genetically engineered organisms. For instance, Escherichia coli has been modified to produce crotonate through a dedicated thioesterase ( 13 ). Similarly, in Methylobacterium extorquens , deletion of the gene encoding crotonyl-CoA carboxylase/reductase redirected metabolic flux through the EMC pathway, leading to crotonate secretion ( 14 ). We hypothesized that under conditions of carbon and redox excess, PPB excrete crotonate as a novel electron sink. To test this, we combined controlled photoheterotrophic cultivations with metaproteomic profiling to determine the regulatory and metabolic basis of crotonate excretion. This study reveals, for the first time, that wild-type PPB can release crotonate as a redox overflow metabolite, expanding our understanding of microbial electron sink strategies and offering new tools for metabolic monitoring in bioprocesses Results Identification of crotonate release To investigate the conditions associated with crotonate excretion, the biomass growth dynamics, substrate consumption, PHA accumulation, and byproduct formation were analyzed. Figure 1 presents the key trends observed in the initial test, conducted under high organic carbon conditions (HighOC). The goal was to assess how metabolic constraints influence carbon distribution and whether crotonate emerges as a relevant intermediate in PPB metabolism. The simultaneous consumption of acetate and butyrate, where butyrate was fully depleted within 60 hours and 15% of the initial acetate (146 mg COD L⁻¹) remained, suggests a glyoxylate shunt-based metabolism ( 15 ). The depletion of NH₄⁺ after 60 hours imposed a nitrogen limitation, halting further biomass growth and restricting acetate assimilation. PHA accumulation during the test was limited to PHB, consistent with the use of even-carbon substrates (acetate and butyrate). The absence of odd-carbon substrates, such as propionate or valerate, prevented the synthesis of polyhydroxyvalerate (PHV), as described in ( 16 ). PHB content peaked at 11 % dry wight basis (dwb) (15% of total biomass COD), with this maximum occurring at the end of the exponential growth phase, in agreement with prior studies ( 17 , 18 ). As growth ceased, PHB was depolymerized, and H₂ production increased, supporting earlier hypotheses regarding the anaerobic phototrophic metabolism ( 6 ). Interestingly, crotonate was detected in the culture supernatant, marking the first reported instance of its excretion by anaerobic phototrophic bacteria. This identification was confirmed via GC-MS/MS analysis, which matched the retention time and fragmentation pattern of the crotonate standard (Fig. 1 B), ensuring its unambiguous detection. A maximum concentration of 3.3 mg COD L⁻¹ was observed, accounting for 0.23% of the total COD present. This finding suggests that crotonate may play an unrecognized role in the metabolism of PPB, warranting further investigation. Metabolic analysis of the crotonate release process We conducted a series of tests under varying conditions to better understand the factors triggering crotonate production. First, we investigated the impact of non-limiting N levels on crotonate production by setting a COD/N ratio of 6 (HighN, Fig. 2 a). In this condition, all available COD was consumed (both acetate and butyrate). PHB accumulation was limited to 6% dwb (8.2% of total biomass COD). Crotonate production was observed but was delayed until PHB depolymerization began. H₂ was not produced under these conditions, as NH₄⁺ was not depleted, with 55 mg N L⁻¹ remaining at the end of the experiment. In the second test, the conditions of the N-limitation condition were maintained, but DMSO was added after the second sampling point, when crotonate was already present in the medium but before nitrogen depletion (DMSO, Fig. 2 b). While other parameters followed trends similar to the first test, the addition of DMSO halted crotonate release and led to its reassimilation, as crotonate was no longer detectable in the supernatant after this point. The maximum concentration of crotonate reached before reassimilation was 2.1 mg COD L⁻¹. Concurrently, 60% of the added DMSO (16 mmol L − 1 or 153.6 mg COD L⁻¹) was consumed. PHA and H₂ exhibit similar trends to HighOC, but show a slight increase following DMSO addition. This is somewhat counterintuitive, as DMSO also functions as an electron acceptor. The third condition tested was the absence of inorganic carbon in the medium (NoIC, Fig. 2 c). This limitation significantly restricted biomass growth, with a final biomass concentration of 712 mg COD L⁻¹, equivalent to 49% of the biomass concentration achieved in the initial test. Only 25% of the butyrate was consumed, while acetate consumption reached 43%, indicating a higher redox imbalance due to the lack of CO 2 for electron sinking. PHB accumulation was lower, with a maximum of 5.4% dwb, and H₂ production was minimal, probably due to limited carbon assimilation and decreased requirement for N 2 fixation. Notably, crotonate release was the highest under these conditions, reaching 12.9 mg COD L⁻¹ (0.8% of all COD present, and 1.8% of the biomass COD equivalent). These results indicate that the absence of inorganic carbon which supposedly exacerbates redox stress, strongly correlating with increased crotonate release. Figure 2 d presents the relative abundance of microbial communities at the end of each test. All tests were under open mixed conditions, which were consistently enriched in PPB, comprising over 50% of the community in all cases. The test with the highest PPB abundance was observed under HighN conditions, where PPB reached 65% of the total community. This was dominated by Rhodopseudomonas (42%) and Rhodobacter (23%). In contrast, during the test with maximum crotonate production (NoIC), the microbial community was dominated by Rhodopseudomonas (62%), with Rhodobacter making up only 2%. Metaproteomic analysis of the crotonate release process Metaproteomic analysis was performed to identify metabolic pathways associated with crotonate production in mixed purple phototrophic bacterial (PPB) cultures. Protein expression profiles were evaluated across all experimental conditions, but the NoIC test was selected as the primary focus due to its higher crotonate accumulation and the highest number of significantly differentially expressed proteins, as illustrated in the volcano plots (Fig. 2 c, right). In addition to the broader set of expression changes, the NoIC condition revealed functionally relevant proteins associated with PHA metabolism and central carbon pathways. Data from the other conditions are provided in the Supplementary Information. To capture the early molecular response associated with crotonate release, samples for metaproteomic analysis were collected at 25 and 41 hours, corresponding to the onset of extracellular crotonate detection. Taxonomic profiling indicated that Rhodopseudomonas palustris and R. faecalis were the most abundant species in the consortium under NoIC conditions and accounted for the majority of significant protein-level changes. Protein abundance data were normalized to correct for compositional shifts across conditions and shown in Table 2 . Table 2 Differentially regulated proteins in Rhodopseudomonas palustris and Rhodopseudomonas faecalis under NoIC conditions. The table shows significantly regulated proteins (FC: fold change and p-value) linked to PHB metabolism and the TCA–glyoxylate bypass. Protein IDs, functions, and statistical values are provided for both species. Accession N FC ρ value Description Rhodopseudomonas palustris PHB Pathway Q21BD0 1,53 7,5E-03 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) A0A0D7E6U6 2,15 1,5E-02 Enoyl-CoA hydratase (EC 4.2.1.17) Q07Q61 1,52 3,4E-02 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) Q07QY2 2,52 2,9E-03 Enoyl-CoA hydratase (EC 4.2.1.17) Q2J2U0, Q13EE5 2,34 2,1E-04 Acetyl-CoA acetyltransferase thiolase (EC 2.3.1.9) Q07VA5 2,42 1,2E-04 Acetyl-CoA acetyltransferase (EC 2.3.1.9) Q07QY2 2,70 6,9E-04 Enoyl-CoA hydratase (EC 4.2.1.17) Q217M3 2,75 2,1E-03 Formyl-CoA:oxalate CoA-transferase (FCOCT) (EC 2.8.3.16) TCA - related Q131G7, E6VCA6, Q6N1L1 0,39 1,6E-04 Isocitrate lyase (EC 4.1.3.1) Q07QP5 2,04 7,2E-04 Phosphoenolpyruvate carboxylase (EC 4.1.1.31) Rhodopseudomonas faecalis PHB pathway A0A318TA66 1,51 5,3E-05 acetoacetyl-CoA reductase A0A318T9I4 1,54 5,0E-04 Acetolactate synthase small subunit (AHAS) (ALS) (EC 2.2.1.6) A0A318T9B0 2,30 1,8E-03 Putative polyhydroxyalkanoic system A0A318TLU6 1,97 1,9E-03 Enoyl-CoA hydratase A0A318U0I7 2,49 1,3E-03 Phasin family protein A0A318TJM4 4,16 3,6E-06 Phasin TCA - related A0A318TC46 0,62 4,0E-02 Succinate–CoA ligase (EC 6.2.1.5) A0A318T7Y9 0,64 1,6E-02 Acetyl-coenzyme A carboxylase carboxyl transferase (EC 2.1.3.15) A0A318TM82 2,18 9,4E-05 Formyl-CoA:oxalate CoA-transferase (EC 2.8.3.16 A0A318TJL3 0,52 2,8E-03 Methylmalonyl-CoA mutase A0A318TF87 0,59 9,9E-03 propionyl-CoA carboxylase (EC 6.4.1.3) A0A318TB01 0,38 2,6E-04 Isocitrate lyase (EC 4.1.3.1) A0A318TI25 0,31 5,7E-03 Succinyl-CoA A0A318TYZ0 1,72 5,2E-03 Phosphoenolpyruvate carboxylase A consistent response observed in both R. palustris and R. faecalis was the downregulation of isocitrate lyase (ICL; EC 4.1.3.1), with fold changes of ~ 0.39–0.41 (p < 0.01), indicating a repression of the glyoxylate shunt. This pathway typically enables the assimilation of acetyl-CoA without carbon loss via CO₂. Its suppression under IC-limiting conditions suggests a reduction in acetate assimilation capacity, consistent with the observed accumulation of acetyl-CoA precursors. Both species also exhibited upregulation of enoyl-CoA hydratase (EC 4.2.1.17), with fold changes of 2.5 and 1.9 in R. palustris and R. faecalis , respectively. This enzyme catalyzes the dehydration of S-hydroxybutyryl-CoA to crotonyl-CoA. Additional enzymes associated with β-oxidation and crotonyl-CoA formation were also upregulated in both strains, indicating an increased metabolic flux toward this intermediate. No R-specific enoyl-CoA hydratase, which would convert crotonyl-CoA into the R-3-hydroxybutyryl-CoA monomer required for PHA biosynthesis, was detected in the proteomes or found in available Rhodopseudomonas genomes. Acetoacetyl-CoA reductase (EC 1.1.1.36), the canonical enzyme for producing R-hydroxybutyryl-CoA from acetoacetyl-CoA, was not detected in either species. However, R. faecalis expressed a closely related enzyme annotated as oxoacyl-CoA reductase, which was significantly upregulated. This enzyme was also detected in R. palustris but without significant regulation. In both species, formyl-CoA:oxalate CoA-transferase (FCOCT; EC 2.8.3.16) was significantly upregulated. Although this enzyme is not annotated for crotonyl-CoA specificity, it belongs to a class known for broad substrate tolerance and may contribute to the cleavage of crotonyl-CoA, enabling crotonate excretion. In contrast, other genera detected in the microbial community, including Rhodobacter capsulatus , Dysgonomonas , and Citrobacter spp., did not show significant changes in expression of proteins associated with the TCA cycle, PHA metabolism, or SCFA processing (p < 0.05; fold change thresholds of ≥ 1.5 or ≤ 0.66). Combined with their lower relative abundance, these data indicate that these organisms are unlikely to contribute substantially to crotonate metabolism under the tested conditions. Discussion Identification of crotonate release This study identifies crotonate excretion as a previously unreported metabolic response in a photoheterotrophic enrichment of purple bacteria. Rather than functioning as a primary redox homeostasis mechanism, our findings suggest that crotonate production represents a form of secondary, overflow metabolism in Rhodopseudomonas species. We demonstrated reproducible crotonate excretion in open mixed cultures of PPB when assimilating organic carbon sources with an electron density exceeding that of biomass (4.40 mol e⁻ per mol C in the substrate compared to the theoretical 4 mol e⁻ per mol C in biomass). Crotonate presence in the supernatant was confirmed via GC-MS and subsequently quantified using GC with an external standard. This excretion was observed under conditions of excess reduced organic carbon and was closely associated with substantial PHA accumulation (Fig. 1 ), reinforcing the idea that crotonate may emerge as a metabolic overflow product when other electron sinks, such as PHA synthesis, are insufficient to fully dissipate excess reducing power. The metabolic versatility of PPB, including alternative electron sinks such as inorganic carbon fixation via the CBB cycle, H₂ production, and PHA accumulation, makes this phenomenon particularly relevant in understanding the broader metabolic plasticity of these bacteria. Recognizing crotonate as a secondary metabolite linked to carbon and redox imbalances provides new insights into PPB physiology and its implications for biotechnological applications. The polymerization and depolymerization of PHA in PPB is well documented ( 19 , 20 ). We hypothesize that PHA depolymerization leads to the overaccumulation of acetoacetyl-CoA, which cannot reenter the TCA cycle and is instead redirected to the EMP, resulting in crotonyl-Coa B-oxidation into crotonate excretion (Posible Fig. 3). While the EMC pathway has received limited research attention in PPB, its role in dissipating reductive power is pivotal ( 11 ), facilitating inorganic carbon fixation and contributing to the reoxidation of reduced cofactors ( 4 ). Moreover, it may compete with carbohydrate production from IC via the CBB cycle, presenting a dynamic balance between metabolic pathways that manage energy and carbon flow. Metabolic analysis of the crotonate release process To investigate the mechanisms underlying crotonate excretion, three experimental conditions were tested to evaluate key hypotheses. First, the COD/N ratio was adjusted to the theoretical optimum for PPB assimilation to assess whether crotonate production is linked to nutrient stress. The results showed a significant decrease in crotonate excretion under higher nutrient availability, suggesting a strong relationship with nitrogen limitation. This reduction can be explained by two main factors: ( 1 ) excess carbon was redirected towards biomass synthesis instead of PHA polymerization, reducing the need for PHA depolymerization as a redox-balancing mechanism; and ( 2 ) increased inorganic carbon fixation via the CBB cycle, which likely served as an alternative electron sink, further mitigating redox stress. Despite these changes, crotonate excretion persisted at later stages of the experiment, when PHA levels dropped to approximately 6% of the dry biomass, indicating that residual metabolic imbalances still required additional electron dissipation pathways. Second, the role of external electron acceptors in crotonate metabolism was tested by supplementing the medium with DMSO. DMSO functions as a terminal electron acceptor through its reduction to dimethyl sulfide (DMS) via dimethyl sulfoxide reductase (DMSOR), effectively bypassing endogenous redox-balancing pathways. Upon DMSO addition, crotonate was rapidly re-assimilated from the medium, and no further excretion was detected. This strongly suggests that crotonate production is not a direct metabolic necessity but rather a compensatory response to excess reducing power. The availability of an alternative electron sink through DMSO reduction relieved this imbalance, reinforcing the hypothesis that crotonate excretion serves as a redox-balancing mechanism in the absence of more efficient electron disposal routes. Finally, the effect of removing inorganic carbon from the medium was investigated, hypothesizing that the elimination of the primary electron sink would exacerbate the redox imbalance, leading to increased crotonate excretion. This hypothesis was confirmed, with significantly higher crotonate production observed under these conditions. Previous studies have shown that PPB recycle approximately 46% of CO₂ when acetate is used as a substrate, indicating that the CBB cycle, although reduced, remains active ( 7 ). Additionally, the EMP pathway requires CO₂ fixation to convert crotonyl-CoA into ethylmalonyl-CoA. The absence of CO₂ likely exacerbates metabolic imbalances, driving increased crotonate excretion as an alternative redox-balancing strategy. Notably, PEP-carboxylase, a key anaplerotic enzyme, was upregulated under these conditions, suggesting a compensatory mechanism to re-oxidize reduced cofactors through CO₂ fixation. Given that CO₂ fixation already accounts for nearly 50% of reducing power utilization when PPB grow on acetate ( 7 ), its further upregulation under CO₂-limited conditions indicates a critical role in maintaining carbon flux and redox homeostasis. These findings, supported by macroscopic trends in Figs. 1 and 2 , reveal a maximum relative crotonate production of 6% COD under the most extreme conditions tested, underscoring the strong interplay between CO₂ availability and redox-balancing mechanisms in PPB. The relative abundance data show that PPB consistently dominated the microbial community, maintaining at least 50% relative abundance across all conditions and reaching a peak of 65% in both the HighN and NoIC batches. Notably, the Rhodopseudomonas genus displayed a higher relative abundance in the NoIC batch (62%) compared to the HighN batch (42%). However, no significant correlations (P > 0.05) were found between Rhodopseudomonas abundance and crotonate concentrations, suggesting that the intricate community shifts cannot be attributed to the activity of a single genus or metabolite. Anaerobic fermentative bacteria were consistently present across all conditions, likely engaging in commensal interactions within the microbial community. Rather than solely degrading dead biomass ( 21 ), these species may play a cooperative role by metabolizing organic acids such as crotonate into simpler compounds like acetate and H₂, which could, in turn, be utilized by purple phototrophic bacteria ( 22 ). A similar syntrophic relationship has been previously described between Wolinella and Rhodopseudomonas ( 23 ), and the recurrent detection of Dysgonomonas in our systems, including in previous studies( 6 , 23 ) further supports the potential metabolic interdependence within the consortium. Their ubiquitous presence suggests an important secondary ecological function: recycling excreted metabolites, maintaining community stability, and mitigating the accumulation of inhibitory intermediates. This highlights the complex interplay within the microbial community, where metabolic cross-feeding and interdependence enhance the system's overall resilience and efficiency. Further studies integrating functional metagenomics, isotope tracing, and flux metabolic analysis are essential to unravel the specific metabolic roles of fermentative bacteria in crotonate turnover. These approaches will provide a comprehensive understanding of electron flow and carbon flux, enabling the closure of mass balances and the stoichiometric characterization of PPB-driven processes. Metaproteomic analysis of the crotonate release process Metaproteomic analyses under the HighN and DMSO conditions (Supplementary Materials) yielded limited insights due to low peptide assignment confidence and insufficient statistical resolution. These limitations arose from the high diversity of expressed proteins, low abundance of crotonate-related enzymes, and signal overlap with dominant central metabolic pathways. In contrast, the NoIC condition revealed clear and functionally relevant shifts in protein expression, especially within Rhodopseudomonas palustris and R. faecalis , allowing deeper interpretation. A common feature in both strains was the strong downregulation of isocitrate lyase (ICL), a key enzyme in the glyoxylate shunt. Since this pathway enables acetate assimilation without CO₂ loss, its suppression under IC-limiting conditions effectively blocks the main route for acetyl-CoA assimilation in Rhodopseudomonas . When combined with inactive Calvin cycle metabolism, this leads to a buildup of acetyl-CoA originating from short-chain fatty acid assimilation and PHB depolymerization. To mitigate redox and carbon overflow, both species responded by upregulating enzymes that direct flux toward crotonyl-CoA formation. Enoyl-CoA hydratase (EC 4.2.1.17), which catalyzes the conversion of S-hydroxybutyryl-CoA to crotonyl-CoA, was significantly upregulated in both species. This indicates an active conversion route for acetyl-CoA derivatives under stress. However, canonical progression to PHA requires R-3-hydroxybutyryl-CoA, which is formed either via an R-specific enoyl-CoA hydratase (not detected) or by acetoacetyl-CoA reductase (EC 1.1.1.36), also absent in R. palustris . Interestingly, a homologous enzyme—acetoacetyl-CoA reductase—was detected and upregulated in R. faecalis , alongside high expression of phasin proteins, suggesting active PHA accumulation in this strain. This pattern contrasts with R. palustris , which showed a more pronounced expression of PHB depolymerization enzymes but no phasins, pointing toward catabolic activity rather than storage. Thus, while both species accumulate crotonyl-CoA under stress, only R. faecalis appears to divert a portion into PHA, whereas R. palustris likely relies on crotonate excretion to resolve the metabolic bottleneck. Supporting this, both strains significantly upregulated a formyl-CoA:oxalate CoA-transferase (EC 2.8.3.16), a promiscuous enzyme class capable of catalyzing CoA removal from various acyl-CoAs. Though not annotated for crotonyl-CoA specifically, its consistent upregulation under redox stress suggests a functional role in crotonate release. Crotonate has a reduction level between acetate and butyrate, making it a fermentation-like redox sink, releasing electrons while sparing energy and maintaining carbon balance. This supports the interpretation of crotonate as a conditionally produced fermentation product, emerging when acetyl-CoA cannot be assimilated or stored. The shared upregulation of crotonyl-CoA-producing enzymes and a plausible CoA transferase in both strains suggests that crotonate production is a general overflow mechanism in the Rhodopseudomonas group, not species-specific, but rather dictated by intracellular conditions and enzymatic completeness. Although crotonate levels remained low in absolute terms (< 1.8% of biomass COD equivalent), its presence signals a stress-responsive shift in carbon and electron management. The consistent upregulation of key enzymes across both Rhodopseudomonas strains, and particularly the stronger depolymerization signature in R. palustris , suggest that crotonate overflow is a shared, non-species-specific mechanism, with R. palustris likely playing the leading role under high-stress conditions. This discovery not only expands our understanding of PPB metabolic plasticity but also introduces crotonate as a potential biosensing metabolite. Its close coupling with PHB depolymerization and redox overflow makes it a candidate marker for metabolic stress. In photobioreactor systems, crotonate could serve as an early-warning indicator for redox imbalance or process instability, enabling timely interventions. Looking ahead, our results open new questions about the prevalence and genetic basis of this overflow pathway. We anticipate that targeted experiments, such as isotopic tracing of carbon flow and knockout or overexpression of the putative crotonate-releasing enzyme, will confirm the precise biochemical route of crotonate formation. Additionally, exploring crotonate production in pure cultures and diverse PPB strains will establish how widespread this trait is beyond our mixed culture. By illuminating an unrecognized aspect of PPB metabolism, our work lays a foundation for both improved metabolic models of these bacteria and innovative strategies to control and exploit their unique redox economy in industrial applications. Materials and Methods Preparation of synthetic medium and PPB enrichment Acetate and butyrate were selected as substrates due to their differing degrees of reduction, carrying electron densities of 4.0 and 5.0 e⁻/C, respectively. In terms of chemical oxygen demand (COD), a total of 1.5 g COD L⁻¹ was fixed (1 g COD equivalent of acetate and 0.5 COD g equivalent of butyrate), corresponding to an average electron contribution of 4.40 e⁻/C. This value exceeds the electron density of biomass (C₅H₇O₂N), which is approximately 4 mol e⁻ per mol of carbon ( 4 ), indicating that the supplied substrate was more reduced than the assimilated biomass, thereby necessitating alternative redox-balancing mechanisms. Macro- and micronutrients were prepared following the Ormerod concentration as described in Allegue et al., (2020). All chemical compounds came from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The initial culture was taken out from a photoheterotrophic continuous reactor feed with a fermented broth described in Allegue et al. (2022). Specific phototrophic activity test (SPA) for crotonate identification The activity of the phototrophic biomass was determined by Specific Phototrophic Activity (SPA) batch tests following the indications in Puyol et al., (2017). Experiments were performed in sextuplicate, with triplicates designated for liquid sample analysis and gas sample analysis to mitigate errors due to potential volume changes. The tests were conducted in 200 mL anaerobic serum bottles with a working volume of 160 mL. These bottles were placed in a temperature-controlled incubator set to 30°C, with an initial pH of 6.5. Oxygen was removed from the gas phase by purging with argon, and the bottles were hermetically sealed. Illumination was provided by infrared (IR) lamps (Philips, BR125 IR, Spain) delivering approximately 45 W m − 2 , measured with a JASCO V-630 Spectrometer, with the setup covered by UV/VIS filtering foil to block UV wavelengths. The inoculum used in the tests consisted of 10 mg volatile suspended solids (VSS) L − 1 . We initially set out to identify extracellular metabolites excreted by PPB under nitrogen-limiting conditions such as at a COD/N ratio of 40, a condition far above the theoretical COD/N value of ~ 12 derived from the elemental composition of microbial biomass (C₅H₇O₂N). Under these electron-rich, nitrogen-poor conditions, we unexpectedly detected extracellular crotonate, a reduced four-carbon organic acid not previously reported as a secretion product in PPB. To validate this observation, an initial test replicated these high organic carbon conditions to confirm extracellular crotonate production (HighOC). Subsequent experiments investigated potential regulatory factors by testing (i) nitrogen sufficient conditions with a COD/N ratio of 12 to eliminate nutrient limitation (HighN), (ii) the addition of 0.25 gCOD L⁻¹ dimethyl sulfoxide (DMSO) as an alternative electron acceptor, and (iii) inorganic carbon limitation by restricting inorganic carbon (NaHCO₃; NoIC). The conditions tested are summarized in Table 1 . Table 1 Summary of parameter variations across experiments. COD – chemical oxygen demand; DMSO – dimethyl sulfoxide. Parameter HighOC HighN DMSO NoIC Acetate and butyrate (g COD L − 1 ) 1.5 1.5 1.5 1.5 Ammonium (mg N L − 1 ) 38 125 38 38 DMSO (g COD L − 1 ) 0 0 0.25 0 NaHCO 3 (mg IC L − 1 ) 143 143 143 0 Paste your materials and methods section here. Analytical methods Samples were collected through the gas-tight septum, with 20 mL taken during the first four samplings and 10 mL thereafter. Half of each sample was filtered through a 0.45 µm cellulose-ester filter (Advantech) prior to analysis. The unfiltered portion was used to measure optical density (OD), pH, temperature, PHA (specifically polyhydroxybutyrate [PHB]), and total COD (tCOD). Additionally, 1 mL of unfiltered sample was centrifuged, and the resulting pellet was stored at -20°C for proteomic analysis. Filtered samples were analysed for acetate, butyrate, crotonate, soluble COD (sCOD), and ammonium (NH 4 + ​). Measurements of total and volatile suspended solids (TSS and VSS) were performed on the first and last samples. The OD at 660 nm and 805 nm was determined using a UV-VIS spectrophotometer (Shimadzu, Japan). Measurements of pH, COD, NH 4 + ​, TSS, and VSS followed standard protocols. Acetate and butyrate were quantified using an ion-exclusion Rezex™ ROA-Organic Acid H + + HPLC column (Phenomenex), coupled to a refractive index detector (Agilent), operating at 65°C with a flow rate of 1 mL min − 1 . The mobile phase consisted of 0.005 M H 2 ​SO 4 ​ as the eluent. PHB samples were fixed with 0.2% (v/v) formaldehyde and freeze-dried overnight. Quantification of PHB was performed following established digestion, extraction, and gas chromatography protocols using flame ionization detection (GC-FID). Calibration curves were generated using commercial PHB standards (Sigma-Aldrich, USA). Crotonate was quantified using the same analytical approach, based on filtered culture supernatants and commercial standards (Sigma-Aldrich, USA). To confirm crotonate identity, gas chromatography–mass spectrometry (GC-MS) was conducted with a Restek Rxi-5Sil MS column (30 m × 0.25 mm, 0.25 µm film thickness). The oven temperature program began at 50°C (3 min hold), ramped to 180°C at 12°C/min, then to 250°C at 7°C/min, with a final hold of 5 minutes. In the triplicate samples designated for gas analysis, the concentrations of H 2 ​ and CO 2 ​ in the headspace were monitored using a gas chromatograph equipped with a thermal conductivity detector (GC-TCD, Agilent Technologies, USA). Argon was employed as the carrier gas at a flow rate of 5 mL min − 1 . The system was operated with the oven and detector temperatures set at 45°C and 220°C, respectively, ensuring optimal separation and detection of the target gases. To account for changes in headspace pressure, a manual manometer (Baumer, Germany) was used to measure the pressure in the flasks. Gas production within the closed system was subsequently calculated using the ideal gas law, allowing for precise quantification of volumetric gas production under the experimental conditions. Statistical significance was analyzed by calculating the confidence intervals (at 95%) (CI95) for all the experimental data and the estimated parameters. Variation intervals in all tables and error bars in all figures represent CI95. COD calculations To enable a consistent comparison of chemical energy flow across metabolic products, the concentrations of biomass, PHB, acetate, butyrate, crotonate, and H₂ were converted into chemical oxygen demand (COD) equivalents. COD serves as a proxy for the number of electrons required to fully oxidize a compound to CO₂ and H₂O, thus providing a unified metric to trace electron distribution and redox balance throughout the system. For context, 1 mole of electrons corresponds to 8 grams of O₂ in COD units, allowing a direct translation from COD values to electron equivalents. The COD associated with biomass, representing the enriched PPB culture, was estimated by subtracting the soluble COD (sCOD) contributions of individual components from the total COD (tCOD) measured in each sample. The COD values for specific compounds were determined based on their theoretical oxygen demand from stoichiometric oxidation reactions to CO₂ and H₂O: acetate (1.07 gCOD g⁻¹), butyrate (1.82 gCOD g⁻¹), crotonate (1.67 gCOD g⁻¹), PHB (1.32 gCOD g⁻¹), H₂ (8.00 gCOD g⁻¹), and biomass (C₅H₇O₂N) at 1.42 gCOD g⁻¹. This approach simplifies the assessment of electron distribution and redox balance across the system. 16S rRNA gene amplicon sequencing For the DNA extraction and microbial community analysis by PacBio sequencing, representative samples were taken twice a week to analyze the development of the bacterial communities, ensuring that each phase studied had at least three samples each, except for the S0 acclimation phase, where only one sample was collected. Samples were stored at − 4°C until use. Total DNA was extracted using a Microbial DNA isolation kit (Soil DNA Isolation Kit, CANVAX, Cordoba, Spain) to perform the molecular identification of prokaryotes and frozen to − 20°C for 4 days until use. The quantification, amplification (PCR), 16S rRNA gene measurements, and the amplicon taxonomic annotation and comparative analysis were outsourced to Instituto ISABIAL-FISABIO, Hospital General Universitario de Alicante, Alicante, Spain. SMRT sequencing was performed on a PacBio RS II instrument. Metaproteomic analyses Protein extraction was performed using a buffer containing 6 M guanidine HCl and 50 mM K₂HPO₄, followed by sonication (3 × 10 s at 20% amplitude). Tryptic peptides were analyzed in Data-Dependent Acquisition (DDA) mode using a TripleTOF 6600 mass spectrometer (Sciex). The resulting data were processed using Protein Pilot 4.2 and searched against a Uniprot-derived proteome database containing the primary species and genera identified in microbial community analysis, including Rps. palustris , Rps. faecalis, Rhodobacter (Rb.) capsulatus , other Rhodobacter species, Dysgonomonas and Citrobacter . Methionine oxidation and cysteine carbamidomethylation were set as variable and fixed modifications, respectively. Quantification of proteins was performed using Skyline software in "precursor" mode, focusing solely on unmodified peptides and excluding those with missed cleavages. To ensure accurate taxonomic assignment, only unique peptides were considered, and shared peptides belonging to multiple taxa were excluded from the analysis. Areas under the curve (AUCs) were exported to Excel for statistical evaluation using Student’s t-test. Key enzymes of interest were identified by manually searching for proteins involved in specific metabolic pathways, including the TCA, PHA synthesis and degradation, EMP, and anaplerotic CO₂ fixation. Protein abundance data were normalized to mitigate potential biases. Normalization was performed by median equalization across replicates, enabling comparison of protein abundance changes between conditions. However, this method does not account for relative abundance shifts due to changes in microbial community composition. To address this, a second normalization strategy was employed to analyze taxon-specific regulation, focusing on the four most abundant species in the consortium. For each species, normalization was performed by median equalization across replicates, isolating taxon-level abundance modifications from species-level compositional changes. The fold-change (FC) parameter was calculated as the ratio of protein abundance between the samples before crotonate production (Sample 3 – S3) and after (Sample 4 S4), expressed as: $$\:FC=\frac{{Area}_{S3}}{{Area}_{S4}}$$ Analyses were performed in triplicate, and average values were used to estimate FC. Proteins with FC > 1.5 or < 0.66 and p-values < 0.05 (reactor-level) or < 0.1 (taxon-level) were considered significant. Areas were further normalized to the sum of all peptide areas in each sample to study taxon-level changes. Declarations Acknowledgments This work was funded by the Madrid’s Community through the project BIVALIA-CM [TEC-2024/BIO-177] and by the Project VALPIG4FOOD (TED2021-129595B-I00), funded by the Spanish Ministry of Science, Innovation and Universities and the European Union’s NextGenerationEU. The work has received funding support by the European Project Purple4Life (Grant Agreement No. 101212806), funded by the Circular Bio-based Europe Joint Undertaking (CBE JU) under the EU’s Horizon Europe programme. This work was supported by research funding from the Research Foundation–Flanders (FWO) and the Fonds National de la Recherche Scientifique (FNRS) under the Weave inter-federal research programme, Project Redoxome [G096623N]. L.D.A. is supported by a Junior Postdoctoral Fellowship from the Research Foundation – Flanders (FWO-Vlaanderen) [12A2925N]. This work is based upon work from COST Action PurpleGain (CA21146), supported by COST (European Cooperation in Science and Technology). Author Contributions: L.D.A. designed the study, performed the experiments, curated the data, carried out formal analysis, and wrote the manuscript. M.V. contributed to the investigation and formal analysis. S.E.V. supervised the work and contributed to manuscript review. J.A.M. contributed to the conceptualization, reviewed the manuscript, supervised the research, and secured funding. B.L. contributed to the conceptualization, developed methodology, performed formal analysis, and reviewed the manuscript. D.P. contributed to the conceptualization, supervised the project, acquired funding, and performed the final review of the manuscript. Competing Interest Statement: The authors declare no competing interests. Materials & Correspondence: Daniel Puyol - [email protected] References Capson-Tojo, G., et al.: Purple phototrophic bacteria for resource recovery: Challenges and opportunities. Biotechnol. Adv. 43 , 107567 (2020) Allegue, L.D., Puyol, D., Melero, J.A.: Novel approach for the treatment of the organic fraction of municipal solid waste: Coupling thermal hydrolysis with anaerobic digestion and photo-fermentation. Sci. Total Environ. 714 , 136845 (2020) Martin-Gamboa, M., Allegue, L.D., Puyol, D., Melero, J.A., Dufour, J.: Environmental life cycle assessment of polyhydroxyalkanoates production by purple phototrophic bacteria mixed cultures. J. Clean. Prod. 428 , 139421 (2023) Alloul, A., et al.: Dehazing redox homeostasis to foster purple bacteria biotechnology. Trends Biotechnol. 41 , 106–119 (2023) Hädicke, O., Grammel, H., Klamt, S.: Metabolic network modeling of redox balancing and biohydrogen production in purple nonsulfur bacteria. BMC Syst. Biol. 5 , 150 (2011) Allegue, L.D., Ventura, M., Melero, J.A., Puyol, D.: Unraveling PHA production from urban organic waste with purple phototrophic bacteria via organic overload. Renew. Sustain. Energy Rev. 166 , 112687 (2022) McKinlay, J.B., Harwood, C.S.: Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria. Proceedings of the National Academy of Sciences 107, 11669–11675 (2010) Gordon, G.C., McKinlay, J.B.: Calvin Cycle Mutants of Photoheterotrophic Purple Nonsulfur Bacteria Fail To Grow Due to an Electron Imbalance Rather than Toxic Metabolite Accumulation. J. Bacteriol. 196 , 1231–1237 (2014) Madigan, M., Cox, S.S., Stegeman, R.A.: Nitrogen fixation and nitrogenase activities in members of the family Rhodospirillaceae. J. Bacteriol. 157 , 73–78 (1984) Chowdhury, N.B., Alsiyabi, A., Saha, R.: Characterizing the Interplay of Rubisco and Nitrogenase Enzymes in Anaerobic-Photoheterotrophically Grown Rhodopseudomonas palustris CGA009 through a Genome-Scale Metabolic and Expression Model. Microbiol Spectr 10 (2022) Carius, L., Hädicke, O., Grammel, H.: Stepwise reduction of the culture redox potential allows the analysis of microaerobic metabolism and photosynthetic membrane synthesis in Rhodospirillum rubrum . Biotechnol. Bioeng. 110 , 573–585 (2013) Bayon-Vicente, G., et al.: Analysis of the Involvement of the Isoleucine Biosynthesis Pathway in Photoheterotrophic Metabolism of Rhodospirillum rubrum. Front. Microbiol. 12 (2021) Kim, S., Cheong, S., Gonzalez, R.: Engineering Escherichia coli for the synthesis of short- and medium-chain α,β-unsaturated carboxylic acids. Metab. Eng. 36 , 90–98 (2016) Schada, L., von Borzyskowski, et al.: Replacing the Ethylmalonyl-CoA Pathway with the Glyoxylate Shunt Provides Metabolic Flexibility in the Central Carbon Metabolism of Methylobacterium extorquens AM1. ACS Synth. Biol. 7 , 86–97 (2018) Cabecas Segura, P., et al.: Preferential photoassimilation of volatile fatty acids by purple non-sulfur bacteria: Experimental kinetics and dynamic modelling. Biochem. Eng. J. 186 , 108547 (2022) Fradinho, J.C., Oehmen, A., Reis, M.A.M.: Photosynthetic mixed culture polyhydroxyalkanoate (PHA) production from individual and mixed volatile fatty acids (VFAs): Substrate preferences and co-substrate uptake. J. Biotechnol. 185 , 19–27 (2014) Cabecas Segura, P., et al.: Effects of Mixing Volatile Fatty Acids as Carbon Sources on Rhodospirillum rubrum Carbon Metabolism and Redox Balance Mechanisms. Microorganisms 9, (2021). (1996) Allegue, L.D., Ventura, M., Melero, J.A., Puyol, D.: Integrated sustainable process for polyhydroxyalkanoates production from lignocellulosic waste by purple phototrophic bacteria. GCB Bioenergy. 13 , 862–875 (2021) Bayon-Vicente, G., Wattiez, R., Leroy, B.: Global Proteomic Analysis Reveals High Light Intensity Adaptation Strategies and Polyhydroxyalkanoate Production in Rhodospirillum rubrum Cultivated With Acetate as Carbon Source. Front. Microbiol. 11 (2020) Fuller, R.C.: Polyesters and Photosynthetic Bacteria. In: Anoxygenic Photosynthetic Bacteria. Advances in Photosynthesis and Respiration, pp. 1245–1256. Kluwer Academic (1995) Hirakata, Y., et al.: Identification and cultivation of anaerobic bacterial scavengers of dead cells. ISME J. 17 , 2279–2289 (2023) Hallenbeck, P.C.: Fundamentals of the fermentative production of hydrogen. Water Sci. Technol. 52 , 21–29 (2005) Díaz-Rullo, S., Edreira, et al.: Elucidating metabolic tuning of mixed purple phototrophic bacteria biofilms in photoheterotrophic conditions through microbial photo-electrosynthesis. Commun. Biol. 7 , 1526 (2024) Puyol, D., Barry, E.M., Hülsen, T., Batstone, D.J.: A mechanistic model for anaerobic phototrophs in domestic wastewater applications: Photo-anaerobic model (PAnM). Water Res. 116 , 241–253 (2017) Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMetaproteomicsdata.xlsx Supplementary Dataset 1 SupplementaryMetaproteomicsICperspecie.xlsx Supplementary Dataset 2 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7242062","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":504355026,"identity":"4f7e23db-306b-4f73-8461-bc3d818599a2","order_by":0,"name":"Daniel Puyol","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie2RMQrCMBSGnwR0Ka1jeosHgiCoZ3lScNIiFMRREOrSA3gMj5CQoUvVtZMIgpNDwcXBQY12DRkF85HhG/KRHwLgcPwi4n1mWhuiAmCWCX5cbmwTqBPl2ezy850UFUKMZSTUID22giVTlSkJi5jkBiHBckxqmiaMi+aYmxIUE1QewmhbvmSaEkPwusZheLiietRJTyfBzTgM3zehThqfV8A4LCyvKDPkSVhcSGZ7Ylw1u8bEP0w61X3Rj/08kqf7nKJgvTobh33hBG3SFtl8poYgEFqGloHD4XD8EU+R7U9zOK9N3QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2152-4095","institution":"University Rey Juan Carlos","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Puyol","suffix":""},{"id":504355027,"identity":"1a8a1ccf-8c83-417d-8d1f-8d0cfae25047","order_by":1,"name":"Luis Allegue","email":"","orcid":"","institution":"University of Antwerp","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"","lastName":"Allegue","suffix":""},{"id":504355028,"identity":"53f228e8-fc36-4fb1-b701-7329b22bc44d","order_by":2,"name":"Maria Ventura","email":"","orcid":"","institution":"University Rey Juan Carlos","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Ventura","suffix":""},{"id":504355029,"identity":"0fbb0bd4-511b-4f93-8f34-9a6adbca03b9","order_by":3,"name":"Siegfried Vlaeminck","email":"","orcid":"https://orcid.org/0000-0002-2596-8857","institution":"University of Antwerp","correspondingAuthor":false,"prefix":"","firstName":"Siegfried","middleName":"","lastName":"Vlaeminck","suffix":""},{"id":504355030,"identity":"bee19f1b-3478-49f1-8839-18a7f3ce95ae","order_by":4,"name":"Juan Melero","email":"","orcid":"","institution":"Universidad Rey Juan Carlos de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Melero","suffix":""},{"id":504355031,"identity":"b1d01581-527b-4b64-94c1-7e99fc631c40","order_by":5,"name":"Baptiste Leroy","email":"","orcid":"","institution":"University of Mons","correspondingAuthor":false,"prefix":"","firstName":"Baptiste","middleName":"","lastName":"Leroy","suffix":""}],"badges":[],"createdAt":"2025-07-29 10:15:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7242062/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7242062/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90421522,"identity":"bf60da9b-56cd-4d22-9d1d-4d78d9512749","added_by":"auto","created_at":"2025-09-02 14:02:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":642179,"visible":true,"origin":"","legend":"\u003cp\u003eCrotonate production under nitrogen-limiting conditions (HighOC), with acetate and butyrate as carbon sources. (a) Temporal dynamics of biomass growth, sCOD, and substrate consumption (acetate and butyrate) and (b) polyhydroxybutyrate (PHB), H₂, and crotonate production. The experiment was conducted under anaerobic photoheterotrophic conditions at 30°C using an enriched purple phototrophic bacteria inoculum. (c) Gas chromatography–mass spectrometry (GC-MS) chromatogram of the culture supernatant at 75h (top) compared to a crotonate standard (bottom), confirming crotonate accumulation. Error bars represent 95% confidence intervals.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-7242062/v1/a254ccc04c1e474be28b1be1.png"},{"id":90421523,"identity":"630cc28f-f99a-439e-aa59-41654c1bf245","added_by":"auto","created_at":"2025-09-02 14:02:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":952629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eImpact of experimental conditions on crotonate production and microbial community composition.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u003cbr\u003e\n(\u003c/strong\u003ea–c) Profiles under HighN (a), DMSO (b), and NoIC (c) conditions showing: biomass growth and organic carbon consumption (sCOD, acetate, and butyrate) (left panels); production of PHB, H₂, and crotonate (middle panels); and volcano plots of metaproteomic data comparing protein abundance before and after crotonate production (right panels). In the volcano plots, black points represent proteins with non-significant changes (p \u0026gt; 0.05), while red and green points indicate significantly downregulated and upregulated proteins, respectively (p \u0026lt; 0.05).\u003cbr\u003e\n(d) Genus-level microbial community composition at the end of each experiment, illustrating the relative abundance of dominant taxa across conditions. All experiments were performed under anaerobic photoheterotrophic conditions at 30 °C using an enriched PPB culture.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-7242062/v1/b94b68acc5776e3c8a7d2b13.png"},{"id":90424101,"identity":"462d86a1-8f59-4b15-95c8-ccd81cffde50","added_by":"auto","created_at":"2025-09-02 14:26:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2326763,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7242062/v1/74fb6d8f-0213-44b0-997c-9e61aa60200e.pdf"},{"id":90422524,"identity":"a856cb92-2a2a-490c-8226-ac0aa7fb85f7","added_by":"auto","created_at":"2025-09-02 14:10:38","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12303119,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Dataset 1\u003c/p\u003e","description":"","filename":"SupplementaryMetaproteomicsdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7242062/v1/9767e624966b946853d6df5d.xlsx"},{"id":90421535,"identity":"67030e9e-95be-4ded-af0b-26dcce05c691","added_by":"auto","created_at":"2025-09-02 14:02:38","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3413357,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Dataset 2\u003c/p\u003e","description":"","filename":"SupplementaryMetaproteomicsICperspecie.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7242062/v1/3bebbe712f8350d0c197f49c.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Purple phototrophic bacteria release crotonate as metabolic overflow pathway to complement other redox-balancing routes","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePurple phototrophic bacteria (PPB) are metabolically versatile microorganisms used in biotechnologies ranging from wastewater valorization to biopolymer production. Central to their versatility is a complex redox-balancing machinery that enables them to thrive under diverse environmental conditions. Their defining feature is their ability to grow photoheterotrophically (particularly in the case of the subset of purple non-sulfur bacteria), using light as an energy source while metabolizing organic compounds as both carbon and electron sources (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). This phototroph metabolism enables PPB to achieve remarkably high efficiencies, with carbon conversion yields reaching up to 100% under optimal conditions (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). These characteristics have generated substantial interest in harnessing PPB for environmental applications, particularly in wastewater treatment and resource recovery. PPB offer a sustainable alternative for the production of bulk commodities, including microbial protein and bioplastics, outperforming conventional processes in terms of resource efficiency and environmental impact. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCentral to the adaptability of PPB is their highly evolved redox-balancing machinery, which enables them to maintain intracellular redox homeostasis under varying environmental conditions. Redox balance in anaerobic photoheterotrophs is achieved through a hierarchy of strategies that modulate intracellular reducing equivalents (e.g., NADH/NAD⁺) to prevent the buildup of excess reducing power, which could otherwise disrupt cellular metabolism (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The Calvin-Benson-Bassham (CBB) cycle is a primary electron sink, fixing CO₂ into organic intermediates that are integrated into central metabolic pathways such as glycolysis. In CBB cycle-positive (IC⁺) purple phototrophic bacteria, such as \u003cem\u003eRhodopseudomonas (Rps.) palustris\u003c/em\u003e, the CBB cycle acts as a key electron sink, fixing CO₂ and contributing up to 40\u0026ndash;60% of redox flux during growth on reduced substrates like acetate (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Its activity is tightly correlated with the oxidation state of the substrate, as CO₂ fixation offsets excess reducing equivalents, ensuring metabolic equilibrium. In contrast, CBB cycle-negative (IC⁻) strains like \u003cem\u003eRhodospirillum (Rsp.) rubrum\u003c/em\u003e rely on the ethylmalonyl-CoA (EMC) pathway for both carbon assimilation and CO₂ fixation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), illustrating alternative strategies for redox balance and the metabolic flexibility of PPB.\u003c/p\u003e\u003cp\u003eAnother key redox-balancing strategy in PPB is hydrogen (H₂) production, catalyzed by one or more nitrogenases. Although primarily involved in nitrogen fixation, nitrogenases can divert excess electrons toward H₂ evolution. This process occurs through nitrogenase's intrinsic hydrogen-evolving activity, which requires ATP. Additionally, under certain conditions, H₂ can be produced via a reverse electron transfer mechanism involving hydrogenase, but without ATP consumption (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, the presence of ammonium (NH₃) represses nitrogenase expression, thereby limiting its contribution to redox balancing under such conditions (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The synthesis of polyhydroxyalkanoates (PHA) provides an additional mechanism for redox management, particularly under nitrogen-limited conditions. These intracellular carbon and electron storage polymers buffer the flow of reducing equivalents and ensure redox homeostasis by temporarily sequestering excess electrons. Since PHA production is environmentally regulated, its role becomes particularly important during organic carbon overload, when managing redox flux is critical (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBeyond these broad-spectrum strategies, some PPB species exhibit specialized pathways for redox balancing. For instance, in \u003cem\u003eRsp. rubrum\u003c/em\u003e, alternative pathways have been identified that involve running parts of the tricarboxylic acid (TCA) cycle in reverse to produce alpha-ketoglutarate, a precursor for amino acid biosynthesis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This reversal is particularly relevant under conditions where conventional carbon fixation pathways are impaired or absent, necessitating alternative routes to redistribute reducing equivalents and sustain biosynthetic needs. Similarly, the biosynthesis of branched-chain amino acids, such as isoleucine, has been directly linked to redox balancing (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). When \u003cem\u003eRsp. rubrum\u003c/em\u003e grows photoheterotrophically on reduced carbon sources like acetate, the synthesis of isoleucine provides an effective electron sink, consuming excess reducing power and preventing an imbalance in intracellular redox homeostasis. However, the contribution of these pathways is limited by the cellular demand for the specific amino acids they produce, making them auxiliary rather than primary strategies for redox management.\u003c/p\u003e\u003cp\u003eCrotonyl-CoA is a central intermediate in metabolic pathways such as PHA synthesis and degradation, β-oxidation and the EMC pathway. Despite crotonyl-CoA\u0026rsquo;s central role, the extracellular release of crotonate by wild-type organisms has never been observed. Crotonate possesses a degree of reduction of 4.5 electrons per mol carbon (e⁻/C), making it more reduced than typical microbial biomass (C₅H₇O₂N\u0026thinsp;~\u0026thinsp;4.2 e⁻/C) and similar to PHA, whose monomeric units (e.g., 3-hydroxybutyrate) share the same redox value (4.5 e⁻/C). This high degree of reduction suggests that crotonate could serve as an electron sink, providing a potential strategy for redox balancing under conditions of carbon and electron excess. To date, extracellular crotonate production has been only observed in genetically engineered organisms. For instance, \u003cem\u003eEscherichia coli\u003c/em\u003e has been modified to produce crotonate through a dedicated thioesterase (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Similarly, in \u003cem\u003eMethylobacterium extorquens\u003c/em\u003e, deletion of the gene encoding crotonyl-CoA carboxylase/reductase redirected metabolic flux through the EMC pathway, leading to crotonate secretion (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe hypothesized that under conditions of carbon and redox excess, PPB excrete crotonate as a novel electron sink. To test this, we combined controlled photoheterotrophic cultivations with metaproteomic profiling to determine the regulatory and metabolic basis of crotonate excretion. This study reveals, for the first time, that wild-type PPB can release crotonate as a redox overflow metabolite, expanding our understanding of microbial electron sink strategies and offering new tools for metabolic monitoring in bioprocesses\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of crotonate release\u003c/h2\u003e\u003cp\u003eTo investigate the conditions associated with crotonate excretion, the biomass growth dynamics, substrate consumption, PHA accumulation, and byproduct formation were analyzed. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the key trends observed in the initial test, conducted under high organic carbon conditions (HighOC). The goal was to assess how metabolic constraints influence carbon distribution and whether crotonate emerges as a relevant intermediate in PPB metabolism. The simultaneous consumption of acetate and butyrate, where butyrate was fully depleted within 60 hours and 15% of the initial acetate (146 mg COD L⁻\u0026sup1;) remained, suggests a glyoxylate shunt-based metabolism (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The depletion of NH₄⁺ after 60 hours imposed a nitrogen limitation, halting further biomass growth and restricting acetate assimilation. PHA accumulation during the test was limited to PHB, consistent with the use of even-carbon substrates (acetate and butyrate). The absence of odd-carbon substrates, such as propionate or valerate, prevented the synthesis of polyhydroxyvalerate (PHV), as described in (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). PHB content peaked at 11 % dry wight basis (dwb) (15% of total biomass COD), with this maximum occurring at the end of the exponential growth phase, in agreement with prior studies (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs growth ceased, PHB was depolymerized, and H₂ production increased, supporting earlier hypotheses regarding the anaerobic phototrophic metabolism (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Interestingly, crotonate was detected in the culture supernatant, marking the first reported instance of its excretion by anaerobic phototrophic bacteria. This identification was confirmed via GC-MS/MS analysis, which matched the retention time and fragmentation pattern of the crotonate standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), ensuring its unambiguous detection. A maximum concentration of 3.3 mg COD L⁻\u0026sup1; was observed, accounting for 0.23% of the total COD present. This finding suggests that crotonate may play an unrecognized role in the metabolism of PPB, warranting further investigation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMetabolic analysis of the crotonate release process\u003c/h3\u003e\n\u003cp\u003eWe conducted a series of tests under varying conditions to better understand the factors triggering crotonate production. First, we investigated the impact of non-limiting N levels on crotonate production by setting a COD/N ratio of 6 (HighN, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In this condition, all available COD was consumed (both acetate and butyrate). PHB accumulation was limited to 6% dwb (8.2% of total biomass COD). Crotonate production was observed but was delayed until PHB depolymerization began. H₂ was not produced under these conditions, as NH₄⁺ was not depleted, with 55 mg N L⁻\u0026sup1; remaining at the end of the experiment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the second test, the conditions of the N-limitation condition were maintained, but DMSO was added after the second sampling point, when crotonate was already present in the medium but before nitrogen depletion (DMSO, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). While other parameters followed trends similar to the first test, the addition of DMSO halted crotonate release and led to its reassimilation, as crotonate was no longer detectable in the supernatant after this point. The maximum concentration of crotonate reached before reassimilation was 2.1 mg COD L⁻\u0026sup1;. Concurrently, 60% of the added DMSO (16 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e or 153.6 mg COD L⁻\u0026sup1;) was consumed. PHA and H₂ exhibit similar trends to HighOC, but show a slight increase following DMSO addition. This is somewhat counterintuitive, as DMSO also functions as an electron acceptor.\u003c/p\u003e\u003cp\u003eThe third condition tested was the absence of inorganic carbon in the medium (NoIC, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This limitation significantly restricted biomass growth, with a final biomass concentration of 712 mg COD L⁻\u0026sup1;, equivalent to 49% of the biomass concentration achieved in the initial test. Only 25% of the butyrate was consumed, while acetate consumption reached 43%, indicating a higher redox imbalance due to the lack of CO\u003csub\u003e2\u003c/sub\u003e for electron sinking. PHB accumulation was lower, with a maximum of 5.4% dwb, and H₂ production was minimal, probably due to limited carbon assimilation and decreased requirement for N\u003csub\u003e2\u003c/sub\u003e fixation. Notably, crotonate release was the highest under these conditions, reaching 12.9 mg COD L⁻\u0026sup1; (0.8% of all COD present, and 1.8% of the biomass COD equivalent). These results indicate that the absence of inorganic carbon which supposedly exacerbates redox stress, strongly correlating with increased crotonate release.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed presents the relative abundance of microbial communities at the end of each test. All tests were under open mixed conditions, which were consistently enriched in PPB, comprising over 50% of the community in all cases. The test with the highest PPB abundance was observed under HighN conditions, where PPB reached 65% of the total community. This was dominated by \u003cem\u003eRhodopseudomonas\u003c/em\u003e (42%) and \u003cem\u003eRhodobacter\u003c/em\u003e (23%). In contrast, during the test with maximum crotonate production (NoIC), the microbial community was dominated by \u003cem\u003eRhodopseudomonas\u003c/em\u003e (62%), with \u003cem\u003eRhodobacter\u003c/em\u003e making up only 2%.\u003c/p\u003e\n\u003ch3\u003eMetaproteomic analysis of the crotonate release process\u003c/h3\u003e\n\u003cp\u003eMetaproteomic analysis was performed to identify metabolic pathways associated with crotonate production in mixed purple phototrophic bacterial (PPB) cultures. Protein expression profiles were evaluated across all experimental conditions, but the NoIC test was selected as the primary focus due to its higher crotonate accumulation and the highest number of significantly differentially expressed proteins, as illustrated in the volcano plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, right). In addition to the broader set of expression changes, the NoIC condition revealed functionally relevant proteins associated with PHA metabolism and central carbon pathways. Data from the other conditions are provided in the Supplementary Information.\u003c/p\u003e\u003cp\u003eTo capture the early molecular response associated with crotonate release, samples for metaproteomic analysis were collected at 25 and 41 hours, corresponding to the onset of extracellular crotonate detection. Taxonomic profiling indicated that \u003cem\u003eRhodopseudomonas palustris\u003c/em\u003e and \u003cem\u003eR. faecalis\u003c/em\u003e were the most abundant species in the consortium under NoIC conditions and accounted for the majority of significant protein-level changes. Protein abundance data were normalized to correct for compositional shifts across conditions and shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferentially regulated proteins in \u003cem\u003eRhodopseudomonas palustris\u003c/em\u003e and \u003cem\u003eRhodopseudomonas faecalis\u003c/em\u003e under NoIC conditions. The table shows significantly regulated proteins (FC: fold change and p-value) linked to PHB metabolism and the TCA\u0026ndash;glyoxylate bypass. Protein IDs, functions, and statistical values are provided for both species.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAccession N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eρ value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eRhodopseudomonas palustris\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003ePHB Pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQ21BD0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7,5E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A0D7E6U6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,5E-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEnoyl-CoA hydratase (EC 4.2.1.17)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQ07Q61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3,4E-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQ07QY2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2,9E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEnoyl-CoA hydratase (EC 4.2.1.17)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQ2J2U0, Q13EE5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2,1E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAcetyl-CoA acetyltransferase thiolase (EC 2.3.1.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQ07VA5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,2E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAcetyl-CoA acetyltransferase (EC 2.3.1.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQ07QY2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6,9E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEnoyl-CoA hydratase (EC 4.2.1.17)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQ217M3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2,1E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFormyl-CoA:oxalate CoA-transferase (FCOCT) (EC 2.8.3.16)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTCA - related\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQ131G7, E6VCA6, Q6N1L1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,6E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIsocitrate lyase (EC 4.1.3.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQ07QP5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7,2E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePhosphoenolpyruvate carboxylase (EC 4.1.1.31)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eRhodopseudomonas faecalis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003ePHB\u003c/p\u003e\u003cp\u003epathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TA66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5,3E-05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eacetoacetyl-CoA reductase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318T9I4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5,0E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAcetolactate synthase small subunit (AHAS) (ALS) (EC 2.2.1.6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318T9B0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,8E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePutative polyhydroxyalkanoic system\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TLU6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,9E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEnoyl-CoA hydratase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318U0I7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,3E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePhasin family protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TJM4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4,16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3,6E-06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePhasin\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eTCA - related\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TC46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4,0E-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSuccinate\u0026ndash;CoA ligase (EC 6.2.1.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318T7Y9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,6E-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAcetyl-coenzyme A carboxylase carboxyl transferase (EC 2.1.3.15)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TM82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9,4E-05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFormyl-CoA:oxalate CoA-transferase (EC 2.8.3.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TJL3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2,8E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMethylmalonyl-CoA mutase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TF87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9,9E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003epropionyl-CoA carboxylase (EC 6.4.1.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TB01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2,6E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIsocitrate lyase (EC 4.1.3.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TI25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5,7E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSuccinyl-CoA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA0A318TYZ0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5,2E-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePhosphoenolpyruvate carboxylase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA consistent response observed in both \u003cem\u003eR. palustris\u003c/em\u003e and \u003cem\u003eR. faecalis\u003c/em\u003e was the downregulation of isocitrate lyase (ICL; EC 4.1.3.1), with fold changes of ~\u0026thinsp;0.39\u0026ndash;0.41 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating a repression of the glyoxylate shunt. This pathway typically enables the assimilation of acetyl-CoA without carbon loss via CO₂. Its suppression under IC-limiting conditions suggests a reduction in acetate assimilation capacity, consistent with the observed accumulation of acetyl-CoA precursors.\u003c/p\u003e\u003cp\u003eBoth species also exhibited upregulation of enoyl-CoA hydratase (EC 4.2.1.17), with fold changes of 2.5 and 1.9 in \u003cem\u003eR. palustris\u003c/em\u003e and \u003cem\u003eR. faecalis\u003c/em\u003e, respectively. This enzyme catalyzes the dehydration of S-hydroxybutyryl-CoA to crotonyl-CoA. Additional enzymes associated with β-oxidation and crotonyl-CoA formation were also upregulated in both strains, indicating an increased metabolic flux toward this intermediate. No R-specific enoyl-CoA hydratase, which would convert crotonyl-CoA into the R-3-hydroxybutyryl-CoA monomer required for PHA biosynthesis, was detected in the proteomes or found in available \u003cem\u003eRhodopseudomonas\u003c/em\u003e genomes.\u003c/p\u003e\u003cp\u003eAcetoacetyl-CoA reductase (EC 1.1.1.36), the canonical enzyme for producing R-hydroxybutyryl-CoA from acetoacetyl-CoA, was not detected in either species. However, \u003cem\u003eR. faecalis\u003c/em\u003e expressed a closely related enzyme annotated as oxoacyl-CoA reductase, which was significantly upregulated. This enzyme was also detected in \u003cem\u003eR. palustris\u003c/em\u003e but without significant regulation. In both species, formyl-CoA:oxalate CoA-transferase (FCOCT; EC 2.8.3.16) was significantly upregulated. Although this enzyme is not annotated for crotonyl-CoA specificity, it belongs to a class known for broad substrate tolerance and may contribute to the cleavage of crotonyl-CoA, enabling crotonate excretion.\u003c/p\u003e\u003cp\u003eIn contrast, other genera detected in the microbial community, including \u003cem\u003eRhodobacter capsulatus\u003c/em\u003e, \u003cem\u003eDysgonomonas\u003c/em\u003e, and \u003cem\u003eCitrobacter\u003c/em\u003e spp., did not show significant changes in expression of proteins associated with the TCA cycle, PHA metabolism, or SCFA processing (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; fold change thresholds of \u0026ge;\u0026thinsp;1.5 or \u0026le;\u0026thinsp;0.66). Combined with their lower relative abundance, these data indicate that these organisms are unlikely to contribute substantially to crotonate metabolism under the tested conditions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of crotonate release\u003c/h2\u003e\u003cp\u003eThis study identifies crotonate excretion as a previously unreported metabolic response in a photoheterotrophic enrichment of purple bacteria. Rather than functioning as a primary redox homeostasis mechanism, our findings suggest that crotonate production represents a form of secondary, overflow metabolism in \u003cem\u003eRhodopseudomonas\u003c/em\u003e species. We demonstrated reproducible crotonate excretion in open mixed cultures of PPB when assimilating organic carbon sources with an electron density exceeding that of biomass (4.40 mol e⁻ per mol C in the substrate compared to the theoretical 4 mol e⁻ per mol C in biomass). Crotonate presence in the supernatant was confirmed via GC-MS and subsequently quantified using GC with an external standard. This excretion was observed under conditions of excess reduced organic carbon and was closely associated with substantial PHA accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), reinforcing the idea that crotonate may emerge as a metabolic overflow product when other electron sinks, such as PHA synthesis, are insufficient to fully dissipate excess reducing power.\u003c/p\u003e\u003cp\u003eThe metabolic versatility of PPB, including alternative electron sinks such as inorganic carbon fixation via the CBB cycle, H₂ production, and PHA accumulation, makes this phenomenon particularly relevant in understanding the broader metabolic plasticity of these bacteria. Recognizing crotonate as a secondary metabolite linked to carbon and redox imbalances provides new insights into PPB physiology and its implications for biotechnological applications.\u003c/p\u003e\u003cp\u003eThe polymerization and depolymerization of PHA in PPB is well documented (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). We hypothesize that PHA depolymerization leads to the overaccumulation of acetoacetyl-CoA, which cannot reenter the TCA cycle and is instead redirected to the EMP, resulting in crotonyl-Coa B-oxidation into crotonate excretion (Posible Fig.\u0026nbsp;3). While the EMC pathway has received limited research attention in PPB, its role in dissipating reductive power is pivotal (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), facilitating inorganic carbon fixation and contributing to the reoxidation of reduced cofactors (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Moreover, it may compete with carbohydrate production from IC via the CBB cycle, presenting a dynamic balance between metabolic pathways that manage energy and carbon flow.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMetabolic analysis of the crotonate release process\u003c/h2\u003e\u003cp\u003eTo investigate the mechanisms underlying crotonate excretion, three experimental conditions were tested to evaluate key hypotheses. First, the COD/N ratio was adjusted to the theoretical optimum for PPB assimilation to assess whether crotonate production is linked to nutrient stress. The results showed a significant decrease in crotonate excretion under higher nutrient availability, suggesting a strong relationship with nitrogen limitation. This reduction can be explained by two main factors: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) excess carbon was redirected towards biomass synthesis instead of PHA polymerization, reducing the need for PHA depolymerization as a redox-balancing mechanism; and (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) increased inorganic carbon fixation via the CBB cycle, which likely served as an alternative electron sink, further mitigating redox stress. Despite these changes, crotonate excretion persisted at later stages of the experiment, when PHA levels dropped to approximately 6% of the dry biomass, indicating that residual metabolic imbalances still required additional electron dissipation pathways.\u003c/p\u003e\u003cp\u003eSecond, the role of external electron acceptors in crotonate metabolism was tested by supplementing the medium with DMSO. DMSO functions as a terminal electron acceptor through its reduction to dimethyl sulfide (DMS) via dimethyl sulfoxide reductase (DMSOR), effectively bypassing endogenous redox-balancing pathways. Upon DMSO addition, crotonate was rapidly re-assimilated from the medium, and no further excretion was detected. This strongly suggests that crotonate production is not a direct metabolic necessity but rather a compensatory response to excess reducing power. The availability of an alternative electron sink through DMSO reduction relieved this imbalance, reinforcing the hypothesis that crotonate excretion serves as a redox-balancing mechanism in the absence of more efficient electron disposal routes.\u003c/p\u003e\u003cp\u003eFinally, the effect of removing inorganic carbon from the medium was investigated, hypothesizing that the elimination of the primary electron sink would exacerbate the redox imbalance, leading to increased crotonate excretion. This hypothesis was confirmed, with significantly higher crotonate production observed under these conditions. Previous studies have shown that PPB recycle approximately 46% of CO₂ when acetate is used as a substrate, indicating that the CBB cycle, although reduced, remains active (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Additionally, the EMP pathway requires CO₂ fixation to convert crotonyl-CoA into ethylmalonyl-CoA. The absence of CO₂ likely exacerbates metabolic imbalances, driving increased crotonate excretion as an alternative redox-balancing strategy. Notably, PEP-carboxylase, a key anaplerotic enzyme, was upregulated under these conditions, suggesting a compensatory mechanism to re-oxidize reduced cofactors through CO₂ fixation. Given that CO₂ fixation already accounts for nearly 50% of reducing power utilization when PPB grow on acetate (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), its further upregulation under CO₂-limited conditions indicates a critical role in maintaining carbon flux and redox homeostasis. These findings, supported by macroscopic trends in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, reveal a maximum relative crotonate production of 6% COD under the most extreme conditions tested, underscoring the strong interplay between CO₂ availability and redox-balancing mechanisms in PPB.\u003c/p\u003e\u003cp\u003eThe relative abundance data show that PPB consistently dominated the microbial community, maintaining at least 50% relative abundance across all conditions and reaching a peak of 65% in both the HighN and NoIC batches. Notably, the \u003cem\u003eRhodopseudomonas\u003c/em\u003e genus displayed a higher relative abundance in the NoIC batch (62%) compared to the HighN batch (42%). However, no significant correlations (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were found between \u003cem\u003eRhodopseudomonas\u003c/em\u003e abundance and crotonate concentrations, suggesting that the intricate community shifts cannot be attributed to the activity of a single genus or metabolite. Anaerobic fermentative bacteria were consistently present across all conditions, likely engaging in commensal interactions within the microbial community. Rather than solely degrading dead biomass (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), these species may play a cooperative role by metabolizing organic acids such as crotonate into simpler compounds like acetate and H₂, which could, in turn, be utilized by purple phototrophic bacteria (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). A similar syntrophic relationship has been previously described between \u003cem\u003eWolinella\u003c/em\u003e and \u003cem\u003eRhodopseudomonas\u003c/em\u003e (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), and the recurrent detection of \u003cem\u003eDysgonomonas\u003c/em\u003e in our systems, including in previous studies(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) further supports the potential metabolic interdependence within the consortium. Their ubiquitous presence suggests an important secondary ecological function: recycling excreted metabolites, maintaining community stability, and mitigating the accumulation of inhibitory intermediates. This highlights the complex interplay within the microbial community, where metabolic cross-feeding and interdependence enhance the system's overall resilience and efficiency. Further studies integrating functional metagenomics, isotope tracing, and flux metabolic analysis are essential to unravel the specific metabolic roles of fermentative bacteria in crotonate turnover. These approaches will provide a comprehensive understanding of electron flow and carbon flux, enabling the closure of mass balances and the stoichiometric characterization of PPB-driven processes.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMetaproteomic analysis of the crotonate release process\u003c/h3\u003e\n\u003cp\u003eMetaproteomic analyses under the HighN and DMSO conditions (Supplementary Materials) yielded limited insights due to low peptide assignment confidence and insufficient statistical resolution. These limitations arose from the high diversity of expressed proteins, low abundance of crotonate-related enzymes, and signal overlap with dominant central metabolic pathways. In contrast, the NoIC condition revealed clear and functionally relevant shifts in protein expression, especially within \u003cem\u003eRhodopseudomonas palustris\u003c/em\u003e and \u003cem\u003eR. faecalis\u003c/em\u003e, allowing deeper interpretation.\u003c/p\u003e\u003cp\u003eA common feature in both strains was the strong downregulation of isocitrate lyase (ICL), a key enzyme in the glyoxylate shunt. Since this pathway enables acetate assimilation without CO₂ loss, its suppression under IC-limiting conditions effectively blocks the main route for acetyl-CoA assimilation in \u003cem\u003eRhodopseudomonas\u003c/em\u003e. When combined with inactive Calvin cycle metabolism, this leads to a buildup of acetyl-CoA originating from short-chain fatty acid assimilation and PHB depolymerization. To mitigate redox and carbon overflow, both species responded by upregulating enzymes that direct flux toward crotonyl-CoA formation.\u003c/p\u003e\u003cp\u003eEnoyl-CoA hydratase (EC 4.2.1.17), which catalyzes the conversion of S-hydroxybutyryl-CoA to crotonyl-CoA, was significantly upregulated in both species. This indicates an active conversion route for acetyl-CoA derivatives under stress. However, canonical progression to PHA requires R-3-hydroxybutyryl-CoA, which is formed either via an R-specific enoyl-CoA hydratase (not detected) or by acetoacetyl-CoA reductase (EC 1.1.1.36), also absent in \u003cem\u003eR. palustris\u003c/em\u003e. Interestingly, a homologous enzyme\u0026mdash;acetoacetyl-CoA reductase\u0026mdash;was detected and upregulated in \u003cem\u003eR. faecalis\u003c/em\u003e, alongside high expression of phasin proteins, suggesting active PHA accumulation in this strain. This pattern contrasts with \u003cem\u003eR. palustris\u003c/em\u003e, which showed a more pronounced expression of PHB depolymerization enzymes but no phasins, pointing toward catabolic activity rather than storage.\u003c/p\u003e\u003cp\u003eThus, while both species accumulate crotonyl-CoA under stress, only \u003cem\u003eR. faecalis\u003c/em\u003e appears to divert a portion into PHA, whereas \u003cem\u003eR. palustris\u003c/em\u003e likely relies on crotonate excretion to resolve the metabolic bottleneck. Supporting this, both strains significantly upregulated a formyl-CoA:oxalate CoA-transferase (EC 2.8.3.16), a promiscuous enzyme class capable of catalyzing CoA removal from various acyl-CoAs. Though not annotated for crotonyl-CoA specifically, its consistent upregulation under redox stress suggests a functional role in crotonate release.\u003c/p\u003e\u003cp\u003eCrotonate has a reduction level between acetate and butyrate, making it a fermentation-like redox sink, releasing electrons while sparing energy and maintaining carbon balance. This supports the interpretation of crotonate as a conditionally produced fermentation product, emerging when acetyl-CoA cannot be assimilated or stored. The shared upregulation of crotonyl-CoA-producing enzymes and a plausible CoA transferase in both strains suggests that crotonate production is a general overflow mechanism in the \u003cem\u003eRhodopseudomonas\u003c/em\u003e group, not species-specific, but rather dictated by intracellular conditions and enzymatic completeness.\u003c/p\u003e\u003cp\u003eAlthough crotonate levels remained low in absolute terms (\u0026lt;\u0026thinsp;1.8% of biomass COD equivalent), its presence signals a stress-responsive shift in carbon and electron management. The consistent upregulation of key enzymes across both \u003cem\u003eRhodopseudomonas\u003c/em\u003e strains, and particularly the stronger depolymerization signature in \u003cem\u003eR. palustris\u003c/em\u003e, suggest that crotonate overflow is a shared, non-species-specific mechanism, with \u003cem\u003eR. palustris\u003c/em\u003e likely playing the leading role under high-stress conditions.\u003c/p\u003e\u003cp\u003eThis discovery not only expands our understanding of PPB metabolic plasticity but also introduces crotonate as a potential biosensing metabolite. Its close coupling with PHB depolymerization and redox overflow makes it a candidate marker for metabolic stress. In photobioreactor systems, crotonate could serve as an early-warning indicator for redox imbalance or process instability, enabling timely interventions.\u003c/p\u003e\u003cp\u003eLooking ahead, our results open new questions about the prevalence and genetic basis of this overflow pathway. We anticipate that targeted experiments, such as isotopic tracing of carbon flow and knockout or overexpression of the putative crotonate-releasing enzyme, will confirm the precise biochemical route of crotonate formation. Additionally, exploring crotonate production in pure cultures and diverse PPB strains will establish how widespread this trait is beyond our mixed culture. By illuminating an unrecognized aspect of PPB metabolism, our work lays a foundation for both improved metabolic models of these bacteria and innovative strategies to control and exploit their unique redox economy in industrial applications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of synthetic medium and PPB enrichment\u003c/h2\u003e\u003cp\u003eAcetate and butyrate were selected as substrates due to their differing degrees of reduction, carrying electron densities of 4.0 and 5.0 e⁻/C, respectively. In terms of chemical oxygen demand (COD), a total of 1.5 g COD L⁻\u0026sup1; was fixed (1 g COD equivalent of acetate and 0.5 COD g equivalent of butyrate), corresponding to an average electron contribution of 4.40 e⁻/C. This value exceeds the electron density of biomass (C₅H₇O₂N), which is approximately 4 mol e⁻ per mol of carbon (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), indicating that the supplied substrate was more reduced than the assimilated biomass, thereby necessitating alternative redox-balancing mechanisms. Macro- and micronutrients were prepared following the Ormerod concentration as described in Allegue et al., (2020). All chemical compounds came from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The initial culture was taken out from a photoheterotrophic continuous reactor feed with a fermented broth described in Allegue et al. (2022).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSpecific phototrophic activity test (SPA) for crotonate identification\u003c/h2\u003e\u003cp\u003eThe activity of the phototrophic biomass was determined by Specific Phototrophic Activity (SPA) batch tests following the indications in Puyol et al., (2017). Experiments were performed in sextuplicate, with triplicates designated for liquid sample analysis and gas sample analysis to mitigate errors due to potential volume changes. The tests were conducted in 200 mL anaerobic serum bottles with a working volume of 160 mL. These bottles were placed in a temperature-controlled incubator set to 30\u0026deg;C, with an initial pH of 6.5. Oxygen was removed from the gas phase by purging with argon, and the bottles were hermetically sealed. Illumination was provided by infrared (IR) lamps (Philips, BR125 IR, Spain) delivering approximately 45 W m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, measured with a JASCO V-630 Spectrometer, with the setup covered by UV/VIS filtering foil to block UV wavelengths. The inoculum used in the tests consisted of 10 mg volatile suspended solids (VSS) L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe initially set out to identify extracellular metabolites excreted by PPB under nitrogen-limiting conditions such as at a COD/N ratio of 40, a condition far above the theoretical COD/N value of ~\u0026thinsp;12 derived from the elemental composition of microbial biomass (C₅H₇O₂N). Under these electron-rich, nitrogen-poor conditions, we unexpectedly detected extracellular crotonate, a reduced four-carbon organic acid not previously reported as a secretion product in PPB. To validate this observation, an initial test replicated these high organic carbon conditions to confirm extracellular crotonate production (HighOC). Subsequent experiments investigated potential regulatory factors by testing (i) nitrogen sufficient conditions with a COD/N ratio of 12 to eliminate nutrient limitation (HighN), (ii) the addition of 0.25 gCOD L⁻\u0026sup1; dimethyl sulfoxide (DMSO) as an alternative electron acceptor, and (iii) inorganic carbon limitation by restricting inorganic carbon (NaHCO₃; NoIC). The conditions tested are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of parameter variations across experiments. COD \u0026ndash; chemical oxygen demand; DMSO \u0026ndash; dimethyl sulfoxide.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHighOC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHighN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDMSO\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNoIC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcetate and butyrate (g COD L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmmonium (mg N L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDMSO (g COD L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaHCO\u003csub\u003e3\u003c/sub\u003e (mg IC L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePaste your materials and methods section here.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAnalytical methods\u003c/h2\u003e\u003cp\u003eSamples were collected through the gas-tight septum, with 20 mL taken during the first four samplings and 10 mL thereafter. Half of each sample was filtered through a 0.45 \u0026micro;m cellulose-ester filter (Advantech) prior to analysis. The unfiltered portion was used to measure optical density (OD), pH, temperature, PHA (specifically polyhydroxybutyrate [PHB]), and total COD (tCOD). Additionally, 1 mL of unfiltered sample was centrifuged, and the resulting pellet was stored at -20\u0026deg;C for proteomic analysis. Filtered samples were analysed for acetate, butyrate, crotonate, soluble COD (sCOD), and ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e​). Measurements of total and volatile suspended solids (TSS and VSS) were performed on the first and last samples.\u003c/p\u003e\u003cp\u003eThe OD at 660 nm and 805 nm was determined using a UV-VIS spectrophotometer (Shimadzu, Japan). Measurements of pH, COD, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e​, TSS, and VSS followed standard protocols. Acetate and butyrate were quantified using an ion-exclusion Rezex\u0026trade; ROA-Organic Acid H\u0026thinsp;+\u0026thinsp;+\u0026thinsp;HPLC column (Phenomenex), coupled to a refractive index detector (Agilent), operating at 65\u0026deg;C with a flow rate of 1 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The mobile phase consisted of 0.005 M H\u003csub\u003e2\u003c/sub\u003e​SO\u003csub\u003e4\u003c/sub\u003e​ as the eluent. PHB samples were fixed with 0.2% (v/v) formaldehyde and freeze-dried overnight. Quantification of PHB was performed following established digestion, extraction, and gas chromatography protocols using flame ionization detection (GC-FID). Calibration curves were generated using commercial PHB standards (Sigma-Aldrich, USA). Crotonate was quantified using the same analytical approach, based on filtered culture supernatants and commercial standards (Sigma-Aldrich, USA). To confirm crotonate identity, gas chromatography\u0026ndash;mass spectrometry (GC-MS) was conducted with a Restek Rxi-5Sil MS column (30 m \u0026times; 0.25 mm, 0.25 \u0026micro;m film thickness). The oven temperature program began at 50\u0026deg;C (3 min hold), ramped to 180\u0026deg;C at 12\u0026deg;C/min, then to 250\u0026deg;C at 7\u0026deg;C/min, with a final hold of 5 minutes.\u003c/p\u003e\u003cp\u003eIn the triplicate samples designated for gas analysis, the concentrations of H\u003csub\u003e2\u003c/sub\u003e​ and CO\u003csub\u003e2\u003c/sub\u003e​ in the headspace were monitored using a gas chromatograph equipped with a thermal conductivity detector (GC-TCD, Agilent Technologies, USA). Argon was employed as the carrier gas at a flow rate of 5 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The system was operated with the oven and detector temperatures set at 45\u0026deg;C and 220\u0026deg;C, respectively, ensuring optimal separation and detection of the target gases. To account for changes in headspace pressure, a manual manometer (Baumer, Germany) was used to measure the pressure in the flasks. Gas production within the closed system was subsequently calculated using the ideal gas law, allowing for precise quantification of volumetric gas production under the experimental conditions.\u003c/p\u003e\u003cp\u003eStatistical significance was analyzed by calculating the confidence intervals (at 95%) (CI95) for all the experimental data and the estimated parameters. Variation intervals in all tables and error bars in all figures represent CI95.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCOD calculations\u003c/h2\u003e\u003cp\u003eTo enable a consistent comparison of chemical energy flow across metabolic products, the concentrations of biomass, PHB, acetate, butyrate, crotonate, and H₂ were converted into chemical oxygen demand (COD) equivalents. COD serves as a proxy for the number of electrons required to fully oxidize a compound to CO₂ and H₂O, thus providing a unified metric to trace electron distribution and redox balance throughout the system. For context, 1 mole of electrons corresponds to 8 grams of O₂ in COD units, allowing a direct translation from COD values to electron equivalents. The COD associated with biomass, representing the enriched PPB culture, was estimated by subtracting the soluble COD (sCOD) contributions of individual components from the total COD (tCOD) measured in each sample.\u003c/p\u003e\u003cp\u003eThe COD values for specific compounds were determined based on their theoretical oxygen demand from stoichiometric oxidation reactions to CO₂ and H₂O: acetate (1.07 gCOD g⁻\u0026sup1;), butyrate (1.82 gCOD g⁻\u0026sup1;), crotonate (1.67 gCOD g⁻\u0026sup1;), PHB (1.32 gCOD g⁻\u0026sup1;), H₂ (8.00 gCOD g⁻\u0026sup1;), and biomass (C₅H₇O₂N) at 1.42 gCOD g⁻\u0026sup1;. This approach simplifies the assessment of electron distribution and redox balance across the system.\u003c/p\u003e\u003cp\u003e\u003cb\u003e16S rRNA gene amplicon sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the DNA extraction and microbial community analysis by PacBio sequencing, representative samples were taken twice a week to analyze the development of the bacterial communities, ensuring that each phase studied had at least three samples each, except for the S0 acclimation phase, where only one sample was collected. Samples were stored at \u0026minus;\u0026thinsp;4\u0026deg;C until use. Total DNA was extracted using a Microbial DNA isolation kit (Soil DNA Isolation Kit, CANVAX, Cordoba, Spain) to perform the molecular identification of prokaryotes and frozen to \u0026minus;\u0026thinsp;20\u0026deg;C for 4 days until use. The quantification, amplification (PCR), 16S rRNA gene measurements, and the amplicon taxonomic annotation and comparative analysis were outsourced to Instituto ISABIAL-FISABIO, Hospital General Universitario de Alicante, Alicante, Spain. SMRT sequencing was performed on a PacBio RS II instrument.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMetaproteomic analyses\u003c/h2\u003e\u003cp\u003eProtein extraction was performed using a buffer containing 6 M guanidine HCl and 50 mM K₂HPO₄, followed by sonication (3 \u0026times; 10 s at 20% amplitude). Tryptic peptides were analyzed in Data-Dependent Acquisition (DDA) mode using a TripleTOF 6600 mass spectrometer (Sciex). The resulting data were processed using Protein Pilot 4.2 and searched against a Uniprot-derived proteome database containing the primary species and genera identified in microbial community analysis, including \u003cem\u003eRps. palustris\u003c/em\u003e, \u003cem\u003eRps. faecalis, Rhodobacter (Rb.) capsulatus\u003c/em\u003e, other \u003cem\u003eRhodobacter\u003c/em\u003e species, \u003cem\u003eDysgonomonas\u003c/em\u003e and \u003cem\u003eCitrobacter\u003c/em\u003e. Methionine oxidation and cysteine carbamidomethylation were set as variable and fixed modifications, respectively.\u003c/p\u003e\u003cp\u003eQuantification of proteins was performed using Skyline software in \"precursor\" mode, focusing solely on unmodified peptides and excluding those with missed cleavages. To ensure accurate taxonomic assignment, only unique peptides were considered, and shared peptides belonging to multiple taxa were excluded from the analysis. Areas under the curve (AUCs) were exported to Excel for statistical evaluation using Student\u0026rsquo;s t-test. Key enzymes of interest were identified by manually searching for proteins involved in specific metabolic pathways, including the TCA, PHA synthesis and degradation, EMP, and anaplerotic CO₂ fixation.\u003c/p\u003e\u003cp\u003eProtein abundance data were normalized to mitigate potential biases. Normalization was performed by median equalization across replicates, enabling comparison of protein abundance changes between conditions. However, this method does not account for relative abundance shifts due to changes in microbial community composition. To address this, a second normalization strategy was employed to analyze taxon-specific regulation, focusing on the four most abundant species in the consortium. For each species, normalization was performed by median equalization across replicates, isolating taxon-level abundance modifications from species-level compositional changes.\u003c/p\u003e\u003cp\u003eThe fold-change (FC) parameter was calculated as the ratio of protein abundance between the samples before crotonate production (Sample 3 \u0026ndash; S3) and after (Sample 4 S4), expressed as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:FC=\\frac{{Area}_{S3}}{{Area}_{S4}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAnalyses were performed in triplicate, and average values were used to estimate FC. Proteins with FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5 or \u0026lt;\u0026thinsp;0.66 and p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (reactor-level) or \u0026lt;\u0026thinsp;0.1 (taxon-level) were considered significant. Areas were further normalized to the sum of all peptide areas in each sample to study taxon-level changes.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Madrid\u0026rsquo;s Community through the project BIVALIA-CM [TEC-2024/BIO-177] and by the Project VALPIG4FOOD (TED2021-129595B-I00), funded by the Spanish Ministry of Science, Innovation and Universities and the European Union\u0026rsquo;s NextGenerationEU. The work has received funding support by the European Project Purple4Life (Grant Agreement No. 101212806), funded by the Circular Bio-based Europe Joint Undertaking (CBE JU) under the EU\u0026rsquo;s Horizon Europe programme. This work was supported by research funding from the Research Foundation\u0026ndash;Flanders (FWO) and the Fonds National de la Recherche Scientifique (FNRS) under the Weave inter-federal research programme, Project Redoxome [G096623N]. L.D.A. is supported by a Junior Postdoctoral Fellowship from the Research Foundation \u0026ndash; Flanders (FWO-Vlaanderen) [12A2925N].\u0026nbsp;This work is based upon work from COST Action PurpleGain (CA21146), supported by COST (European Cooperation in Science and Technology).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.D.A. designed the study, performed the experiments, curated the data, carried out formal analysis, and wrote the manuscript. M.V. contributed to the investigation and formal analysis. S.E.V. supervised the work and contributed to manuscript review. J.A.M. contributed to the conceptualization, reviewed the manuscript, supervised the research, and secured funding. B.L. contributed to the conceptualization, developed methodology, performed formal analysis, and reviewed the manuscript. D.P. contributed to the conceptualization, supervised the project, acquired funding, and performed the final review of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest Statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials \u0026amp; Correspondence:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDaniel Puyol - [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCapson-Tojo, G., et al.: Purple phototrophic bacteria for resource recovery: Challenges and opportunities. Biotechnol. Adv. \u003cb\u003e43\u003c/b\u003e, 107567 (2020)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAllegue, L.D., Puyol, D., Melero, J.A.: Novel approach for the treatment of the organic fraction of municipal solid waste: Coupling thermal hydrolysis with anaerobic digestion and photo-fermentation. Sci. Total Environ. \u003cb\u003e714\u003c/b\u003e, 136845 (2020)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartin-Gamboa, M., Allegue, L.D., Puyol, D., Melero, J.A., Dufour, J.: Environmental life cycle assessment of polyhydroxyalkanoates production by purple phototrophic bacteria mixed cultures. J. Clean. Prod. \u003cb\u003e428\u003c/b\u003e, 139421 (2023)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlloul, A., et al.: Dehazing redox homeostasis to foster purple bacteria biotechnology. Trends Biotechnol. \u003cb\u003e41\u003c/b\u003e, 106\u0026ndash;119 (2023)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eH\u0026auml;dicke, O., Grammel, H., Klamt, S.: Metabolic network modeling of redox balancing and biohydrogen production in purple nonsulfur bacteria. BMC Syst. Biol. \u003cb\u003e5\u003c/b\u003e, 150 (2011)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAllegue, L.D., Ventura, M., Melero, J.A., Puyol, D.: Unraveling PHA production from urban organic waste with purple phototrophic bacteria via organic overload. Renew. Sustain. Energy Rev. \u003cb\u003e166\u003c/b\u003e, 112687 (2022)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcKinlay, J.B., Harwood, C.S.: Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 107, 11669\u0026ndash;11675 (2010)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGordon, G.C., McKinlay, J.B.: Calvin Cycle Mutants of Photoheterotrophic Purple Nonsulfur Bacteria Fail To Grow Due to an Electron Imbalance Rather than Toxic Metabolite Accumulation. J. Bacteriol. \u003cb\u003e196\u003c/b\u003e, 1231\u0026ndash;1237 (2014)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMadigan, M., Cox, S.S., Stegeman, R.A.: Nitrogen fixation and nitrogenase activities in members of the family Rhodospirillaceae. J. Bacteriol. \u003cb\u003e157\u003c/b\u003e, 73\u0026ndash;78 (1984)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChowdhury, N.B., Alsiyabi, A., Saha, R.: Characterizing the Interplay of Rubisco and Nitrogenase Enzymes in Anaerobic-Photoheterotrophically Grown Rhodopseudomonas palustris CGA009 through a Genome-Scale Metabolic and Expression Model. \u003cem\u003eMicrobiol Spectr\u003c/em\u003e 10 (2022)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarius, L., H\u0026auml;dicke, O., Grammel, H.: Stepwise reduction of the culture redox potential allows the analysis of microaerobic metabolism and photosynthetic membrane synthesis in \u003cem\u003eRhodospirillum rubrum\u003c/em\u003e. Biotechnol. Bioeng. \u003cb\u003e110\u003c/b\u003e, 573\u0026ndash;585 (2013)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBayon-Vicente, G., et al.: Analysis of the Involvement of the Isoleucine Biosynthesis Pathway in Photoheterotrophic Metabolism of Rhodospirillum rubrum. Front. Microbiol. \u003cb\u003e12\u003c/b\u003e (2021)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim, S., Cheong, S., Gonzalez, R.: Engineering Escherichia coli for the synthesis of short- and medium-chain α,β-unsaturated carboxylic acids. Metab. Eng. \u003cb\u003e36\u003c/b\u003e, 90\u0026ndash;98 (2016)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchada, L., von Borzyskowski, et al.: Replacing the Ethylmalonyl-CoA Pathway with the Glyoxylate Shunt Provides Metabolic Flexibility in the Central Carbon Metabolism of \u003cem\u003eMethylobacterium extorquens\u003c/em\u003e AM1. ACS Synth. Biol. \u003cb\u003e7\u003c/b\u003e, 86\u0026ndash;97 (2018)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCabecas Segura, P., et al.: Preferential photoassimilation of volatile fatty acids by purple non-sulfur bacteria: Experimental kinetics and dynamic modelling. Biochem. Eng. J. \u003cb\u003e186\u003c/b\u003e, 108547 (2022)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFradinho, J.C., Oehmen, A., Reis, M.A.M.: Photosynthetic mixed culture polyhydroxyalkanoate (PHA) production from individual and mixed volatile fatty acids (VFAs): Substrate preferences and co-substrate uptake. J. Biotechnol. \u003cb\u003e185\u003c/b\u003e, 19\u0026ndash;27 (2014)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCabecas Segura, P., et al.: Effects of Mixing Volatile Fatty Acids as Carbon Sources on Rhodospirillum rubrum Carbon Metabolism and Redox Balance Mechanisms. \u003cem\u003eMicroorganisms\u003c/em\u003e 9, (2021). (1996)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAllegue, L.D., Ventura, M., Melero, J.A., Puyol, D.: Integrated sustainable process for polyhydroxyalkanoates production from lignocellulosic waste by purple phototrophic bacteria. GCB Bioenergy. \u003cb\u003e13\u003c/b\u003e, 862\u0026ndash;875 (2021)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBayon-Vicente, G., Wattiez, R., Leroy, B.: Global Proteomic Analysis Reveals High Light Intensity Adaptation Strategies and Polyhydroxyalkanoate Production in Rhodospirillum rubrum Cultivated With Acetate as Carbon Source. Front. Microbiol. \u003cb\u003e11\u003c/b\u003e (2020)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFuller, R.C.: Polyesters and Photosynthetic Bacteria. In: Anoxygenic Photosynthetic Bacteria. Advances in Photosynthesis and Respiration, pp. 1245\u0026ndash;1256. Kluwer Academic (1995)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHirakata, Y., et al.: Identification and cultivation of anaerobic bacterial scavengers of dead cells. ISME J. \u003cb\u003e17\u003c/b\u003e, 2279\u0026ndash;2289 (2023)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHallenbeck, P.C.: Fundamentals of the fermentative production of hydrogen. Water Sci. Technol. \u003cb\u003e52\u003c/b\u003e, 21\u0026ndash;29 (2005)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026iacute;az-Rullo, S., Edreira, et al.: Elucidating metabolic tuning of mixed purple phototrophic bacteria biofilms in photoheterotrophic conditions through microbial photo-electrosynthesis. Commun. Biol. \u003cb\u003e7\u003c/b\u003e, 1526 (2024)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePuyol, D., Barry, E.M., H\u0026uuml;lsen, T., Batstone, D.J.: A mechanistic model for anaerobic phototrophs in domestic wastewater applications: Photo-anaerobic model (PAnM). Water Res. \u003cb\u003e116\u003c/b\u003e, 241\u0026ndash;253 (2017)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7242062/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7242062/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePurple phototrophic bacteria (PPB) are renowned for versatile metabolic strategies to maintain redox balance, including CO₂ fixation, H₂ production, and polymer storage. Here we report a previously unknow redox-balancing mechanism in PPB: the extracellular release of crotonate as a metabolic overflow. In batch photoheterotrophic cultures of enriched PPB, crotonate accumulated transiently under conditions of carbon excess. Crotonate excretion coincided with the depletion of other electron sinks (polyhydroxybutyrate and H₂) and was reversed when an alternate electron acceptor (DMSO) was provided, indicating a regulated overflow rather than irreversible fermentation. Using metaproteomics, we found that dominant Rhodopseudomonas species redirect metabolism toward crotonyl-CoA production when conventional acetyl-CoA assimilation routes are limited. Key enzymes of the glyoxylate cycle were suppressed, while enzymes converting acetyl-CoA to crotonyl-CoA were up-regulated, leading to crotonyl-CoA accumulation. Notably, a CoA-transferase enzyme was identified as a candidate responsible for converting crotonyl-CoA to free crotonate, enabling excretion. These results reveal crotonate as an \u0026ldquo;escape valve\u0026rdquo; metabolite that PPB naturally deploy to dissipate excess reductant. The discovery of crotonate overflow expands our understanding of microbial redox homeostasis and highlights a novel facet of PPB metabolism with potential implications for optimizing biotechnological processes such as bioplastic production\u003c/p\u003e","manuscriptTitle":"Purple phototrophic bacteria release crotonate as metabolic overflow pathway to complement other redox-balancing routes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-02 14:02:33","doi":"10.21203/rs.3.rs-7242062/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bfb1e852-b60e-4ba5-b16c-d2e94c8313c8","owner":[],"postedDate":"September 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53587740,"name":"Biological sciences/Biotechnology/Environmental biotechnology"},{"id":53587741,"name":"Biological sciences/Microbiology/Applied microbiology"},{"id":53587742,"name":"Biological sciences/Molecular biology/Proteomics"}],"tags":[],"updatedAt":"2026-05-11T12:29:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-02 14:02:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7242062","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7242062","identity":"rs-7242062","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}