Elevated Acetate Concentrations Research Articles

The geothermal gradient shapes microbial diversity and processes in natural-gas-bearing sedimentary aquifers

Abstract. Deep subsurface microorganisms constitute over 80 % of Earth's prokaryotic biomass and play an important role in global biogeochemical cycles. Geochemical processes driven by geothermal heating are key factors influencing their biomass and activities, yet their full breadth remains uncaptured. Here, we investigated the microbial community composition and metabolism in microbial-natural-gas-bearing aquifers at temperatures ranging from 38 to 81 °C, situated above nonmicrobial-gas- and oil-bearing sediments at temperatures exceeding 90 °C. Cultivation-based and molecular gene analyses, including radiotracer measurements, of formation water indicated variations in predominant methanogenic pathways across different temperature regimes of upper aquifers: high potential for hydrogenotrophic–methylotrophic, hydrogenotrophic, and acetoclastic methanogenesis at temperatures of 38, 51–65, and 73–81 °C, respectively. The potential for acetoclastic methanogenesis correlated with elevated acetate concentrations with increasing depth, possibly due to the decomposition of sedimentary organic matter. In addition to acetoclastic methanogenesis, in aquifers at temperatures as high as or higher than 65 °C, acetate is potentially utilized by microorganisms responsible for the dissimilatory reduction of sulfur compounds other than sulfate because of their high relative abundance at greater depths. The stable sulfur isotopic analysis of sulfur compounds in water and oil samples suggested that hydrogen sulfide, generated through the thermal decomposition of sulfur compounds in oil, migrates upward and is subsequently oxidized with iron oxides present in sediments, yielding elemental sulfur and thiosulfate. These compounds are consumed by sulfur-reducing microorganisms, possibly reflecting elevated microbial populations in aquifers at temperatures as high as or higher than 73 °C. These findings reveal previously overlooked geothermal-heat-driven geochemical and microbiological processes involved in carbon and sulfur cycling in the deep sedimentary biosphere.

Turning Carbon Dioxide into Sustainable Food and Chemicals: How Electrosynthesized Acetate Is Paving the Way for Fermentation Innovation.

ConspectusThe agricultural and chemical industries are major contributors to climate change. To address this issue, hybrid electrocatalytic-biocatalytic systems have emerged as a promising solution for reducing the environmental impact of these key sectors while providing economic onboarding for carbon capture technology. Recent advancements in the production of acetate via CO2/CO electrolysis as well as advances in precision fermentation technology have prompted electrochemical acetate to be explored as an alternative carbon source for synthetic biology. Tandem CO2 electrolysis coupled with improved reactor design has accelerated the commercial viability of electrosynthesized acetate in recent years. Simultaneously, innovations in metabolic engineering have helped leverage pathways that facilitate acetate upgrading to higher carbons for sustainable food and chemical production via precision fermentation. Current precision fermentation technology has received much criticism for reliance upon food crop-derived sugars and starches as feedstock which compete with the human food chain. A shift toward electrosynthesized acetate feedstocks could help preserve arable land for a rapidly growing population.Technoeconomic analysis shows that using electrochemical acetate instead of glucose as a fermentation feedstock reduces the production costs of food and chemicals by 16% and offers improved market price stability. Moreover, given the rapid decline in utility-scale renewable electricity prices, electro-synthesized acetate may become more affordable than conventional production methods at scale. This work provides an outlook on strategies to further advance and scale-up electrochemical acetate production. Additional perspective is offered to help ensure the successful integration of electrosynthesized acetate and precision fermentation technologies. In the electrocatalytic step, it is critical that relatively high purity acetate can be produced in low-concentration electrolyte to help ensure that minimal treatment of the electrosynthesized acetate stream is needed prior to fermentation. In the biocatalytic step, it is critical that microbes with increased tolerances to elevated acetate concentrations are engineered to help promote acetate uptake and accelerate product formation. Additionally, tighter regulation of acetate metabolism via strain engineering is essential to improving cellular efficiency. The implementation of these strategies would allow the coupling of electrosynthesized acetate with precision fermentation to offer a promising approach to sustainably produce chemicals and food. Reducing the environmental impact of the chemical and agricultural sectors is necessary to avoid climate catastrophe and preserve the habitability of the planet for future generations.

Potential Role of SLC5A8 Expression in the Etiology of Subacute Ruminal Acidosis.

Rumen fluid of cows with subacute ruminal acidosis (SARA) has high concentrations of short chain fatty acids (SCFA). However, the mechanism of SCFA accumulation is unknown. The solute-linked carrier 5a8 (SLC5A8) plays a key role in the transportation and absorption of SCFA in the intestinal epithelium. The objective of the current study was to investigate (1) SLC5A8 gene and protein expression in various parts of the bovine gastrointestinal tract, (2) the effect of SCFA on SLC5A8 expression in rumen epithelial cells, and (3) SLC5A8 gene and protein expression in SARA and healthy cows. A total of 10 dairy cows, 84 ± 26 days in milk and in their second to fourth parity were allocated to control (n = 5) and SARA groups (n = 5). Three cows from the control group and three calves (1-day-old, female, 45–50 kg, healthy, fasting) were chosen to collect a total of 10 sections of digestive tract, from rumen to rectum, and then bovine ruminal epithelial cells were isolated from the three calves. Gene and protein expression of SLC5A8 was detected in all tested regions of the gastrointestinal tract in calves and adult cows by Western blot and quantitative real-time PCR and were both highest in the rumen. Gene and protein expression of SLC5A8 was more than 50% lower in the rumen epithelium of SARA vs. control cows and was partly restored after therapy of SARA cows. Compared with SCFA concentrations typical for control cows (60 mM acetate, 30 mM propionate, and 20 mM butyrate), gene and protein expression of SLC5A8 in rumen epithelium was lower at elevated SCFA concentrations typical for SARA cows (90 mM acetate, 40 mM propionate, and 30 mM butyrate), specifically for elevated concentrations of propionate or butyrate in contrast to elevated concentrations of acetate increased gene and protein expression of SLC5A8 in rumen epithelium. In conclusion, the elevated concentrations of propionate and butyrate inhibit ruminal absorption of SCFA via downregulation of SLC5A8 in SARA cows; the expression of SLC5A8 plays an important role in the etiology of SARA.

Hemerythrins enhance aerobic respiration in Methylomicrobium alcaliphilum 20ZR, a methane-consuming bacterium.

Numerous hemerythrins, di-iron proteins, have been identified in prokaryote genomes, but in most cases their function remains elusive. Bacterial hemerythrin homologs (bacteriohemerythrins, Bhrs) may contribute to various cellular functions, including oxygen sensing, metal binding and antibiotic resistance. It has been proposed that methanotrophic Bhrs support methane oxidation by supplying oxygen to a core enzyme, particulate methane monooxygenase. In this study, the consequences of the overexpression or deletion of the Bhr gene (bhr) in Methylomicrobiam alcaliphillum 20ZR were investigated. We found that the bhrknockout (20ZRΔbhr) displays growth kinetics and methane consumption rates similar to wild type. However, the 20ZRΔbhr accumulates elevated concentrations of acetate at aerobic conditions, indicating slowed respiration. The methanotrophic strain overproducing Bhr shows increased oxygen consumption and reduced carbon-conversion efficiency, while its methane consumption rates remain unchanged. These results suggest that the methanotrophic Bhr proteins specifically contribute to oxygen-dependent respiration, while they have minimal, if any, input of oxygen for the methane oxidation machinery.

Unexpected competitiveness of Methanosaeta populations at elevated acetate concentrations in methanogenic treatment of animal wastewater.

Acetoclastic methanogenesis is a key metabolic process in anaerobic digestion, a technology with broad applications in biogas production and waste treatment. Acetoclastic methanogenesis is known to be performed by two archaeal genera, Methanosaeta and Methanosarcina. The conventional model posits that Methanosaeta populations are more competitive at low acetate levels (<1mM) than Methanosarcina and vice versa at higher acetate concentrations. While this model is supported by an extensive body of studies, reports of inconsistency have grown that Methanosaeta were observed to outnumber Methanosarcina at elevated acetate levels. In this study, monitoring of anaerobic digesters treating animal wastewater unexpectedly identified Methanosaeta as the dominant acetoclastic methanogen population at both low and high acetate levels during organic overloading. The surprising competitiveness of Methanosaeta at elevated acetate was further supported by the enrichment of Methanosaeta with high concentrations of acetate (20mM). The dominance of Methanosaeta in the methanogen community could be reproduced in anaerobic digesters with the direct addition of acetate to above 20mM, again supporting the competitiveness of Methanosaeta over Methanosarcina at elevated acetate levels. This study for the first time systematically demonstrated that the dominance of Methanosaeta populations in anaerobic digestion could be linked to the competitiveness of Methanosaeta at elevated acetate concentrations. Given the importance of acetoclastic methanogenesis in biological methane production, findings from this study could have major implications for developing strategies for more effective control of methanogenic treatment processes.

Effects of acetate on Kluyveromyces marxianus DSM 5422 growth and metabolism.

Metabolically active cells produce a wide array of metabolites that can inhibit their growth. Acetate is a widely known preservative, and it is also produced by yeast cells during their growth. Kluyveromyces marxianus DSM 5422 is a promising yeast strain that could be employed in biotechnological processes, but the knowledge of its stress physiology is scarce. Here, we investigate the effects of acetate on growth and changes in cell population structure during adaptation to elevated concentrations of acetate in K. marxianus DSM 5422. Our results indicate that acetate inhibits growth in a pH-dependent manner and has pronounced effects if yeast is grown on lactose or galactose. When challenged with acetate, culture extends lag phase, during which cells adapt to elevated acetate concentrations, and growth reoccurs, albeit at a slower rate, when majority of the population is acetate resistant. Acetate resistance is maintained only if acetate is present in the media or if the culture has reached end of active growth phase. This study shows possible caveats in lactose fermentation with K. marxianus and gives a further perspective in non-conventional yeast applications in biotechnology.

Effects of adding protein, condensed tannins, and polyethylene glycol to diets of sheep and goats fed one-seed juniper and low quality roughage

The biochemical mechanisms that limit voluntary intake of one-seed juniper by browsing ruminants are not well understood. Twelve Rambouillet ewes (78±2.3kgBW) and 12 Boer-Spanish does (54±1.4kgBW) were used in a split-plot sequence design to investigate the effects of adding protein, quebracho tannins (QTs), and polyethylene glycol (PEG) on one-seed juniper intake and preference and on ruminal VFA and plasma AA concentrations. Animals received sudangrass hay and isoenergetic basal diets (4 does and 4 ewes/diet) with either rumen degradable (RDP) or rumen undegradable (RUP) protein (12.5% CP) or no additional protein (control, 5% CP) during four 15-d periods. The control, RDP, and RUP diets were considered the main plot, whereas the four supplement treatments were analyzed as the split-plot effects. Period 1 allowed for adaptation to basal diets and served as a baseline phase. In periods 2–4, animals were offered juniper leaves and twigs (period 2), juniper plus QT (10% of basal diet; period 3), and juniper plus QT plus PEG (50g/animal; period 4). Juniper intake by sheep and goats was not affected (P>0.88) by RDP or RUP when animals were first exposed to juniper, but marginally increased (P<0.10) in period 4 (QT+PEG) regardless of the basal diet. Prior exposure to juniper did not affect (P=0.61) the preference ratio for juniper, but goats had higher preference ratio for juniper (P<0.01) when receiving PEG (period 4). Concentrations of total VFA tended to increase in sheep (P=0.10) and goats (P=0.14) fed protein supplements and molar proportions shifted toward acetate for goats fed RDP and RUP (P=0.07) and to butyrate for sheep fed RDP (P=0.01). Initial juniper exposure (period 2) elevated concentrations of acetate, propionate, and butyrate (P<0.01), but the effect was extinguished in periods 3 and 4 with addition of QT (P<0.05). Supplementation with PEG transiently mitigated the depressor effect of QT on acetate, propionate, and butyrate concentration at 12h post juniper feeding (P<0.01). RDP in goats and to a greater extent RUP in sheep increased plasma concentrations of various AA, especially the branched chained Val, Ile, and Leu (P<0.05). Plasma concentrations of several AA, including Met, Cyst, Glu, Gly, Gln, Asn, Thr, Ser, and Phe, decreased with ingestion of juniper (period 2) and juniper plus QT (period 3). Concentrations of some AA that were depressed by ingestion of juniper and QT were partially restored with supplemental PEG (P<0.05). Protein, terpenes, tannins, and PEG interacted to influence rumen VFA and plasma AA, which were related to intake of juniper and basal diets.

Elucidating acetate tolerance in E. coli using a genome-wide approach

Engineering organisms for improved performance using lignocellulose feedstocks is an important step towards a sustainable fuel and chemical industry. Cellulosic feedstocks contain carbon and energy in the form of cellulosic and hemicellulosic sugars that are not metabolized by most industrial microorganisms. Pretreatment processes that hydrolyze these polysaccharides often also result in the accumulation of growth inhibitory compounds, such as acetate and furfural among others. Here, we have applied a recently reported strategy for engineering tolerance towards the goal of increasing Escherichia coli growth in the presence of elevated acetate concentrations (Lynch et al., 2007). We performed growth selections upon an E. coli genome library developed using a moderate selection pressure to identify genomic regions implicated in acetate toxicity and tolerance. These studies identified a range of high-fitness genes that are normally involved in membrane and extracellular processes, are key regulated steps in pathways, and are involved in pathways that yield specific amino acids and nucleotides. Supplementation of the products and metabolically related metabolites of these pathways significantly increased growth rate (a 130% increase in specific growth) at inhibitory acetate concentrations. Our results suggest that acetate tolerance will not involve engineering of a single pathway; rather we observe a range of potential mechanisms for overcoming acetate based inhibition.

Rapid strain improvement through optimized evolution in the cytostat

Acetate is present in lignocellulosic hydrolysates at growth inhibiting concentrations. Industrial processes based on such feedstock require strains that are tolerant of this and other inhibitors present. We investigated the effect of acetate on Saccharomyces cerevisiae and show that elevated acetate concentrations result in a decreased specific growth rate, an accumulation of cells in the G1 phase of the cell cycle, and an increased cell size. With the cytostat cultivation technology under previously derived optimal operating conditions, several acetate resistant mutants were enriched and isolated in the shortest possible time. In each case, the isolation time was less than 5 days. The independently isolated mutant strains have increased specific growth rates under conditions of high acetate concentrations, high ethanol concentrations, and high temperature. In the presence of high acetate concentrations, the isolated mutants produce ethanol at higher rates and titers than the parental strain and a commercial ethanol producing strain that has been analyzed for comparison. Whole genome microarray analysis revealed gene amplifications in each mutant. In one case, the LPP1 gene, coding for lipid phosphate phosphatase, was amplified. Two mutants contained amplified ENA1, ENA2, and ENA5 genes, which code for P-type ATPase sodium pumps. LPP1 was overexpressed on a plasmid, and the growth data at elevated acetate concentrations suggest that LPP1 likely contributes to the phenotype of acetate tolerance. A diploid cross of the two mutants with the amplified ENA genes grew faster than either individual haploid parent strain when 20 g/L acetate was supplemented to the medium, which suggests that these genes contribute to acetate tolerance in a gene dosage dependent manner.

Metabolic characterisation of E. coli citrate synthase and phosphoenolpyruvate carboxylase mutants in aerobic cultures

E. coli is still one of the most commonly used hosts for protein production. However, when it is grown with excess glucose, acetate accumulation occurs. Elevated acetate concentrations have an inhibitory effect on growth rate and recombinant protein yield, and thus elimination of acetate formation is an important aim towards industrial production of recombinant proteins. Here we examine if over-expression of citrate synthase (gltA) or phosphoenolpyruvate carboxylase (ppc) can eliminate acetate production. Knock-out as well as over-expression mutants were constructed and characterized. Knocking out ppc or gltA decreased the maximum cell density by 14% and increased the acetate excretion by 7%, respectively decreased it by 10%. Over-expression of ppc or gltA increased the maximum cell dry weight by 91% and 23%, respectively. No acetate excretion was detected at these increased cell densities (35 and 23 g/l, respectively).

Sulfidogenic volatile fatty acid degradation in a baffled reactor

The effect of staging the sludge bed on volatile fatty acid degradation by sulfidogenic reactors was evaluated in a baffled reactor. In a 5.4 l baffled reactor, containing three equal compartments, a volatile fatty acid (VFA) mixture (acetate:propionate:butyrate ratio 1:2:2 on COD basis; pH 8) was treated under mesophilic (30 degrees C) and sulfidogenic (COD:SO4(2-) ratio: 0.5) conditions for 38 days. At a specific sludge loading rate of 0.50 g COD.gVSS(-1) x d(-1), a COD and sulfate removal of 85% and 30%, respectively, was obtained. In the baffled reactor, staging of the sulfidogenic VFA degradation occurred. Propionate and butyrate were mainly degraded in the first compartment. Their degradation was incomplete, resulting in elevated acetate concentrations in compartment I. In the second and third compartment of the baffled reactor, a net degradation of acetate took place. Acetate was the sole substrate present in compartment III and residual acetate concentrations of about 200 mg/l were present in the effluent at a specific sludge loading of 0.50 g COD x gVSS(-1) x d(-1). Sludges with different maximum specific VFA and acetate degrading activities developed in the first and second compartment. These maximal specific activities were almost equal for sludge present in compartment II and III.

Effect of nitrate on acetate degradation in a sulfidogenic staged reactor

The optimisation of acetate removal in sulfidogenic reactors by adding nitrate to the last compartment of a baffled reactor was evaluated. In a 5.4 l baffled reactor, containing three equal compartments, a VFA mixture (acetate:propionate:butyrate ratio 1:2:2 on COD basis; pH 8) was treated under mesophilic (30°C) and sulfidogenic (COD:SO 2− 4 ratio: 0.5) conditions. At a specific sludge loading rate of 1.1 g COD gVSS −1 d −1, a COD and sulfate removal of 80 and 35%, respectively, was obtained. In the baffled reactor, staging of the sulfidogenic VFA degradation occurred. Propionate and butyrate were mainly degraded in the first compartment. Their degradation was incomplete, resulting in elevated acetate concentrations in Compartment I. In the second and third compartment of the baffled reactor, a net degradation of acetate took place. Acetate was the sole substrate present in Compartment III and residual acetate concentrations of about 600 mg/l were present in the effluent at the highest specific sludge loading applied. In the three compartments, development of sludges with different maximum specific acetate degrading activities was observed. The maximal specific activity with acetate was three times higher in sludge growing in Compartment III as compared to sludge growing in Compartment I. Batch experiments showed that the addition of about 500 mg/l nitrate (COD/nitrate ratio of 2.5) increased the specific acetate degradation rate by 300%. Nitrate was denitrified (pH optimum of 7.0–8.0) with a temporary accumulation of the intermediate nitrite. When 2.3 g/l nitrate (COD/nitrate ratio of 0.5) was dosed in compartment III of the baffled reactor, the effluent acetate concentration could be decreased from 600 to 100 mg/l.

The co‐disposal of a model brewery wastewater with domestic refuse

By use of refuse columns, it was shown that, at appropriate organic loading rates, the co-disposal of a synthetic brewery wastewater with refuse stimulated methane production and did not reduce leachate quality, in terms of pH or volatile fatty acid concentrations. When the application rate was doubled, from imposed dilution rate (D) 0·025 to 0·056 h−1, there was no breakthrough of volatile fatty acids and methane production was promoted. However, when the same total organic load was achieved by doubling the organic strength of the wastewater, but maintaining D = 0·025 h−1, leachate quality was temporarily reduced, with elevated concentrations of acetate and propionate.

Factors that control the stable carbon isotopic composition of methane produced in an anoxic marine sediment

The carbon isotopic composition of methane produced in anoxic marine sediment is controlled by four factors: (1) the pathway of methane formation, (2) the isotopic composition of the methanogenic precursors, (3) the isotope fractionation factors for methane production, and (4) the isotope fractionation associated with methane oxidation. The importance of each factor was evaluated by monitoring stable carbon isotope ratios in methane produced by a sediment microcosm. Methane did not accumulate during the initial 42-day period when sediment contained sulfate, indicating little methane production from 'noncompetitive' substrates. Following sulfate depletion, methane accumulation proceeded in three distinct phases. First, CO2 reduction was the dominant methanogenic pathway and the isotopic composition of the methane produced ranged from -80 to -94 per thousand. The acetate concentration increased during this phase, suggesting that acetoclastic methanogenic bacteria were unable to keep pace with acetate production. Second, acetate fermentation became the dominant methanogenic pathway as bacteria responded to elevated acetate concentrations. The methane produced during this phase was progressively enriched in C-13, reaching a maximum delta(C-13) value of -42 per thousand. Third, the acetate pool experienced a precipitous decline from greater than 5 mM to less than 20 micro-M and methane production was again dominated by CO2 reduction. The delta(C-13) of methane produced during this final phase ranged from -46 to -58 per thousand. Methane oxidation concurrent with methane production was detected throughout the period of methane accumulation, at rates equivalent to 1 to 8 percent of the gross methane production rate. Thus methane oxidation was too slow to have significantly modified the isotopic signature of methane. A comparison of microcosm and field data suggests that similar microbial interactions may control seasonal variability in the isotopic composition of methane emitted from undisturbed Cape Lookout Bight sediment.

Factors that control the stable carbon isotopic composition of methane produced in an anoxic marine sediment

The carbon isotopic composition of methane produced in anoxic marine sediment is controlled by four factors: (1) the pathway of methane formation, (2) the isotopic composition of the methanogenic precursors, (3) the isotope fractionation factors for methane production, and (4) the isotope fractionation associated with methane oxidation. The importance of each factor was evaluated by monitoring stable carbon isotope ratios in methane produced by a sediment microcosm. Methane did not accumulate during the initial 42‐day period when sediment contained sulfate, indicating little methane production from “noncompetitive” substrates. Following sulfate depletion, methane accumulation proceeded in three distinct phases. First, CO2 reduction was the dominant methanogenic pathway and the isotopic composition of the methane produced ranged from −80 to −94‰. The acetate concentration increased during this phase, suggesting that acetoclastic methanogenic bacteria were unable to keep pace with acetate production. Second, acetate fermentation became the dominant methanogenic pathway as bacteria responded to elevated acetate concentrations. The methane produced during this phase was progressively enriched in 13C, reaching a maximum δ13C value of −42‰. Third, the acetate pool experienced a precipitous decline from &gt;5 mM to &lt;20 μM and methane production was again dominated by CO2 reduction. The δ13C of methane produced during this final phase ranged from −46 to −58‰. Methane oxidation concurrent with methane production was detected throughout the period of methane accumulation, at rates equivalent to 1 to 8% of the gross methane production rate. Thus methane oxidation was too slow to have significantly modified the isotopic signature of methane. A comparison of microcosm and field data suggests that similar microbial interactions may control seasonal variability in the isotopic composition of methane emitted from undisturbed Cape Lookout Bight sediment.

Acetothermus paucivorans, gen. nov., sp. nov., a Strictly Anaerobic, Thermophilic Bacterium from Sewage Sludge, Fermenting Hexoses to Acetate, CO 2 and H 2

Summary A new, Gram-negative, strictly anaerobic, non-sporeforming, thermophilic, non-motile rod-shaped bacterium, strain TN, was enriched and isolated from sewage sludge, digesting at 60°C. The optimum temperature for growth was 58°C., the optimum pH was between 7 and 8. Yeast extract and peptone (approximately 0.2% of each), as well as 1 μg/l of vitamin B12 were required for optimal growth. In the absence of vitamin B12 cells formed long filaments. From a variety of different substrates only glucose and fructose were utilized. Glucose was fermented via the Embden-Meyerhof-Parnas pathway, yielding 2 mol acetate, 2 mol CO2 and 4 mol H2. Increasing H2 concentrations in the gas phase inhibited growth of strain TN, while elevated acetate concentrations did not affect growth. Growth was also inhibited by kanamycin, streptomycin and cycloserin, while nisin, chloramphenicol, phosphomycin and penicillin were ineffective. The G+C content of the DNA was 62 mol%. DNA-DNA-hybridization with Acetomicrobium flavidum did not indicate a close relation between both strains (24%, spectrophotometric method). Thus, although both organisms had a similar metabolism, strain TN was considered a member of a new genus and was named Acetothermus paucivorans gen. nov., sp. nov.

The induction of acetone and butanol production in cultures ofClostridium acetobutylicumby elevated concentrations of acetate and butyrate

The induction of acetone and butanol production in cultures of<i>Clostridium acetobutylicum</i>by elevated concentrations of acetate and butyrate

The induction of acetone and butanol production in cultures of Clostridium acetobutylicum by elevated concentrations of acetate and butyrate

The induction of acetone and butanol production in cultures of Clostridium acetobutylicum by elevated concentrations of acetate and butyrate

Regulation of Chlorophyll Synthesis in the Green Alga Golenkinia

Chlorophyll synthesis in Golenkinia is inhibited 10-fold by growth in darkness on acetate or by growth on elevated concentrations of acetate in the light, particularly if the growth medium contains low levels of nitrogen. Glucose has no such inhibitory effect. delta-Aminolevulinic acid, with a maximal effect at 0.01 m, but not its precursors, overrides the inhibitory effect of acetate and darkness, restoring chlorophyll synthesis. Glycine, succinate, and alpha-ketoglutarate, the precursors tested, all enter the cell. Cells forming chlorophyll produce significantly more aminolevulinic acid than do cells becoming bleached, further indicating the important regulatory role of this compound. Cyclic AMP has no effect on chlorophyll synthesis. These results are compared with those obtained studying other algae, and a mechanism relating light and acetate to chlorophyll formation is proposed.

Studies of the Acetyl Coenzyme A Synthetase Reaction: IV. THE REQUIREMENT FOR MONOVALENT CATIONS

Abstract The monovalent cation requirement for a highly purified acetyl coenzyme A synthetase derived from bovine heart mitochondria was investigated. When removal of stimulatory cations from the reaction components was accomplished, no catalytic activity was detected. Adding chloride salts of rubidium, ammonium, potassium, tris(hydroxymethyl)aminomethane, sodium, or lithium to a final concentration of 5 to 10 mm stimulated the over-all reaction maximally. Stimulating monovalent cations acted catalytically and all of their apparent Km values fell into a range of 1 to 3 mm. Activating monovalent cations could be separated into two groups on the basis of their kinetic behavior in the over-all reaction. Cations in the first group (Rb+, NH4+, K+) gave optimal rates of acetyl coenzyme A formation at concentrations of 10 to 200 mm, showed atypical kinetics at elevated concentrations of acetate (10 to 40 mm), but failed to show altered Km values in the presence of increasing magnesium ion concentrations (3 to 27 mm). In contrast, cations in the second group (Na+ and Li+) inhibited the reaction at concentrations greater than 10 mm, had this inhibition partially reversed by monovalent cations in the first group, did not have their kinetics effected by acetate, but did show decreasing Km values as the magnesium ion concentration was increased from 3 to 27 mm. Two different activating cations, each at a concentration of 60% of its apparent Km, produced an appreciably greater reaction rate than a single cation at twice this concentration. Yet the maximal velocity obtainable with a single species was not exceeded by various combinations of activating alkali metal ions. Activating monovalent cations were also shown to be necessary for formation of enzyme-bound acetyladenylate, either from acetate and adenosine triphosphate or from acetyl coenzyme A and adenosine monophosphate. Substrate quantities of enzyme were used to demonstrate this product of both partial reactions under equilibrium conditions. Optimal yields of acetyladenylate were obtained with 5 mm rubidium or sodium chloride. Sodium chloride at 10 mm decreased this yield, whereas rubidium ions did not. These observations suggest that activating monovalent cations participate in the catalytic mechanism of acetyl coenzyme A synthetase.