Isoenzymes of Lactate Dehydrogenase in Swine Stability During Storage at Different Temperatures and By Heat Treatment

The isoenzymes of lactate dehydrogenase (LDH) in serum from normal pigs were studied after separation by agar gel electrophoresis with subsequent staining with a tetrazolium salt. Experiment 1. The stability of isoenzymes was investigated for 5 successive days after storage at room temperature (22°C), in the refrigerator (4°G), and once after storage for 32 days in the deep-freezer (—20°C). Greatest loss of activity was seen after storage in the refrigerator, where LDH2 and LDH3 lost most of its activity after 5 days. In LDH4 and LDH5 no loss had occurred at this time. Also at room temperature great losses were seen in LDH2 and LDH3. After storage in the deep-freezer an increase in LDH3 activity was recorded. Experiment 2. Serum samples were kept in water baths for 30 min. at 50, 53, and 56°C. A simultaneous and increasing loss in activity of LDH3, LDH4, and LDH5 was seen from 50° to 56°C. At 56° no activity was left in LDH3, LDH4, or LDH5, and only about 15 % of the original activity was present in LDH2. LDH1 showed no loss at 56°, but all activity was lost at 65°. A close correlation was found between total lactate dehydrogenase and α-hydroxybutyrate dehydrogenase activity in both experiments.     

Ecdysteroids and diapause in pupae of Heliothis punctiger

Most pupae of H. punctiger enter diapause when reared at 19°C, 12L:12D. When pharate pupae were treated for only 12 hr at 28°C about 50% developed at 19°C. The proportion of non-diapausing pupae increased as the temperature at which the pharate pupal stage was spent increased.The quantity of injected 20-hydroxyecdysone necessary to promote development in diapausing pupae varied from about 1 μg g^?1 soon after pupation to about 4 μg g^?1 after 50 days. It fell somewhat after 150 days.Removing brains from non-diapausing pupae showed that the brain secreted its hormone at the time of pupation (or just before). However, if the pupae were kept at 19°C development did not occur unless the brain remained in situ for at least 20 hr at 28°C. Implanting brains from non-diapausing pupae into diapausing ones had no measurable effect.These results may be explained by postulating that the prothoracic gland is ‘activated’ by exposure to high temperature, but that it reverts to inactivity over a period at 19°C. The ‘active’ gland must then be stimulated by brain hormone for a long period to trigger secretion of its hormone, which results in development. Diapause is thus the result of the failure of the prothoracic gland to secrete.     

Activation of guinea-pig and bovine neutrophil NADPH oxidase by N,N′-dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) is a potent stimulant of superoxide generation in guinea-pig peritoneal and bovine blood neutrophils. The dependence of DCCD-elicited respiratory burst on the compositon of the medium was investigated. At 37°C, the superoxide generation was short-lived and a rapid losses of enzymatic activity was observed; at 0°C, the activity could be preserved for hours. Superoxide generation by whole cells was accompanied by exocytic degranulation. Prolonged incubation with DCCD at 37°C resulted also in a progressive loss of cellular integrity evidenced by the release of a fraction of lactate dehydrogenase. K_m values of the particulate NADPH oxidase isolated from DCCD-triggered guinea-pig and bovine cells were 31.7 and 50.0 μM, respectively. Cells pre-equilibrated with the potential sensitive dye Di-S-C₃-(5) exhibited changes in the transmembrane potential upon stimulation. Stimulation with DCCD resulted also in the release of membrane-associated calcium, indicated by quenching of the fluorescence of chlortetracyclineloaded neutrophils. Both effects were observed also in human neutrophils which did not generate superoxide upon exposure to DCCD. The mechanism of DCCD-induced responses is discussed.     

Osmotic effects on the plasma membrane of thymocytes

Rat thymocytes were treated with media of different hypotonic osmolarity and the resulting damage of the plasma membrane was monitored by measuring the permeation of biochemical markers (K⁺, fluorescein, lactate dehydrogenase) and by observation of morphological changes in the plasma membrane using the freeze-etch technique. Release is dependent on the molecular weight of the markers; those with higher molecular weights permeate predominantly at a lower osmolarity of the medium and those with lower molecular weights permeate at a higher osmolarity. The marker release is temperature dependent: at 4°C it takes place at a lower osmolarity of the medium than at 37°C.With decreasing osmolarity of the medium at 4°C a decrease in the number of the intramembranous particles and increasing aggregation of the intramembranous particles occurs.     

Desulforhopalus vacuolatus gen. nov., sp. nov., a new moderately psychrophilic sulfate-reducing bacterium with gas vacuoles isolated from a temperate estuary

A new type of gas-vacuolated, sulfate-reducing bacterium was isolated at 10° C from reduced mud (E₀ < 0) obtained from a temperate estuary with thiosulfate and lactate as substrates. The strain was moderately psychrophilic with optimum growth at 18–19° C and a maximum growth temperature of 24° C. Propionate, lactate, and alcohols served as electron donors and carbon sources. The organism grew heterotrophically only with hydrogen as electron donor. Propionate and lactate were incompletely oxidized to acetate; traces of lactate were fermented to propionate, CO₂, and possibly acetate in the presence of sulfate. Pyruvate was utilized both with and without an electron acceptor present. The strain did not contain desulfoviridin. The G+C content was 48.4 mol%. The differences in the 16S rRNA sequence of the isolate compared with that of its closest phylogenetic neighbors, bacteria of the genus Desulfobulbus, support the assignment of the isolate to a new genus. The isolate is described as the type strain of the new species and genus, Desulforhopalus vacuolatus. Received: 4 March 1996 / Accepted: 17 June 1996     

Effect of Tetanus Toxin on Oxytocin and Vasopressin Release from Nerve Endings of the Neurohypophysis

The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 microgram/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100 degrees C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8-Bromo-cyclic GMP, which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.     

Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N,?N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1?M solute were heated for 10?min at temperatures between 35–75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1?M stabilizer solutions were frozen at ?30°C and freeze-dried for 24?h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

Lactate dehydrogenase from the tetraploid weatherfish <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> during temperature adaptation: Determination of structural differences in two forms of the enzyme by molecular modeling methods

A structural analysis of two lactate dehydrogenase M4 protein forms has been performed. These structures are the protein products of two lactate dehydrogenase gene (LDH-A) copies in the weatherfish Misgurnus fossilis genome after thermal adaptation (acclimation) to 5°C and 18°C. The localization of three earlier identified amino acid substitutions (Gly214Val, Leu304Ile, Asp312Glu) has been determined, and the molecular dynamics simulation and computer modeling of two forms of the enzyme from skeletal muscles LDH-M4 have been carried out. After molecular dynamics trajectory calculations carried out at 5, 18, and 25°C, the intersubunit distances for all structures used in calculations have been determined. It has been found that the Gly214Val substitution localized in the intersubunit region leads to a new intersubunit interaction, which plays a role in the stabilization of tetrameric enzyme structure after the adaptation to 18°C.     

Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between elongation growth and the incorporation of [³H]gibberellin A₁ ([³H]GA₁) into a 2,000g pelletable (2KP) fraction from lettuce (Lactuca sativa L., cv. Arctic) hypocotyl sections has been examined. Sections were loaded with incremental amounts of GA₁ under conditions where growth was arrested (5° C) or permitted (30° C) and, after 16 h, all were transferred to a GA-free medium at 30° C. Growth and 2KP radioactivity were measured at this point and after a further 24 h in the chase medium. Uptake was reduced by 80% at 5° C, as compared to 30° C, but 2KP labelling and protein synthesis were only reduced by half. The growth rate of the 5° C pretreated sections during the chase period was comparable to that observed during the pulse in the 30° C material but the dose/response relationship was flatter. Low temperature sections incorporated a much higher percentage of GA₁ uptake into the 2KP fraction (27% at maximum) but the absolute levels of labelling at this temperature were lower than those measured at 30° C. The data are interpreted as showing that 2KP labelling is not a consequence of growth. It must either precede response or be an unconnected concurrent process.     

Temperature responses of membrane-associated activities from spring and winter oats

Abstract Two-week old, glasshouse-grown seedlings of spring and winter oats (Avena sativa cv. Margam and Pennal respectively) were transferred to growth rooms where the daylength was 8 h and the temperature was either 20° or 5°C. Leaves from the 20°C treatment were harvested 1 week after transfer and those from the 5°C treatment after 3 weeks. Measurements were made, at temperatures in the range 5–20°C, of the rate of senescence of excised leaf sections; the rate of in vivo tetrazolium (TTC) reduction; the NADH-MTT tetrazolium diaphorase (NMD) activity of a membrane preparation isolated by sucrose gradient centri-fugation; and Hill activity of isolated chloroplasts. Data from these experiments, either untransformed or plotted in accordance with the Arrhenius equation, were analysed by the method of maximum likelihood for the occurrence of rate-temperature discontinuities. Distinct breakpoints at 14.2–16.3°C were detected for senescence, TTC reduction and NMD in winter oat leaves from the 20°C treatment, but spring oats grown at the same temperature gave a significant discontinuity only in the rate of senescent yellowing. After the hardening treatment, 3 weeks at 5°C, senescence and TTC reduction in winter oats exhibited breakpoints at 10.1–12.4°C and spring oats gave breaks at 11.8–17.3°C. No breakpoint was observed for Hill activity from either variety under either temperature treatment. The use of this approach in studies of growth at chilling temperatures and its potential as a screening method are discussed.     

The effects of injections of D,L-5-hydroxytryptophan on the efflux of endogenous serotonin and 5-hydroxyindoleacetic acid from a synaptosomal fraction.

Abstract— The effects of i.p. injections of SO mg/kg d,l-5-hydroxytryptophan (5-HTP) and saline alone on the in uitro release of endogenous serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were studied using preparations of axon terminals (P₂ isolated from the telencephalon of rats. The level of 5-HT was 2-fold greater and the level of 5-HIAA was 5-fold greater in the P₂ fraction isolated from rats given the d,l-5-HTP injection than from rats given saline injections. At 37°C the in vitro efflux of 5-HT and 5-HIAA from the P₂ fractions of animals injected with 5-HTP 30min before killing was approx 3 times higher than the saline control group. The amount of 5-HT and 5-HIAA released at 37°C was 3–5 times higher than the amount released at 0°C for both the 5-HTP and saline injected rats. Increasing the concentration of potassium ions in the media to 55 mm significantly increased the release of 5-HT but not 5-HIAA in both groups of animals. The amount of 5-HT released by 55mm-K⁺ was about 2-fold higher from the P₂ fraction isolated from rats given 5-HTP injections with respect to those given saline injections. The potassium stimulated release of 5-HT was calcium dependent. The data thus indicate that injection of 50 mg/kg d,l-5-HTP in rats can cause an increase in the level of 5-HT and 5-HIAA in a crude synaptosomal fraction and that as a result of this increase, there is a temperature dependent increased release of 5-HT and 5-HIAA under normal resting membrane conditions. There is also an increased release of 5-HT as a result of membrane depolarizing conditions induced by elevated potassium levels which is calcium dependent.     

Properties and primary structure of a thermostable l-malate dehydrogenase from Archaeoglobus fulgidus

A thermostable l-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of 70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidus l-malate dehydrogenase was stable for 5 h at 90° C, and the half-life at 101° C was 80 min. Thus, A. fulgidus l-malate dehydrogenase is the most thermostable l-malate dehydrogenase characterized to date. Addition of K₂HPO₄ (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to l-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to l-malate dehydrogenase from the archaeon Haloarcula marismortui and other l-lactate-dehydrogenase-like l-malate dehydrogenases. Received: 20 November 1997 / Accepted: 28 February 1997     

Regulation of Synthesis of Chlorophyll, Carotene, Ribulosel,5-diP Carboxylase and Phosphoribulokinase in a Temperature-sensitive Chlorophyll Mutant of Medicago sativa

The activities of several enzymes were studied in a temperature-sensitive chlorophyll mutant of alfalfa (Medicago saliva). In leaves grown at 10°C photosynthetic capacity was essentially nil with ribulose-1,5-diP carboxylase, chlorophyll, and carolene present in greatly limiting concentrations. The activity of phosphoribulokinase was 3.5 times lower at 10°C than at 27°C, but was still sufficiently high at 10°C to not limit the rate of CO₂ fixation. Activities of phosphoriboisomerase, phosphoenolpyruvate carboxylase, glucose-6-P dehydrogenase and malate dehydrogenase were not different at 10°C and 27°C. The low fraction I protein content (which also accounts for the ribulose-1,5-dip carboxylase activity in alfalfa) indicated that synthesis of the carboxylase was effectively blocked at 10°C. A large, comparable increase in carboxylase activity and in concentration of fraction I protein in alfalfa leaves grown at 27°C indicated that the carboxylase was synthesized de novo. The initial induction of the carboxylase, chlorophyll, and carotene may be related, but after induction the carboxylase was not linearly correlated with the other two and had a different temperature optima. Nevertheless, the synthesis of each appeared to be regulated by the temperature-sensitive gene of this mutant.     

Phytophthora cryptogea root rot of tomato in rock woo] nutrient culture. II. Effect of root zone temperature on infection, sporulation and symptom development

The effect of root-zone temperature on Phytophthora cryptogea root rot was studied in tomato cv. Counter grown under winter and summer conditions in rockwool culture. A nutrient temperature of 25°C resulted in increased root initiation and growth, higher in winter-grown than in summer-grown plants. Rhizosphere zoospore populations were greatly reduced at 25°C and above. Growth of P. cryptogea in vitro was optimal between 20°C and 25°C and completely suppressed at 30°C. Encystment was enhanced by increased temperatures above 20°C. Zoospore release in vitro occurred in cultures maintained at constant temperatures in the absence of the normal chilling stimulus. Optimal release was at 10°C; no zoospores were released at 30°C. Inoculated, winter-grown tomato plants maintained at 15°C developed acute aerial symptoms and died after 21 days. Comparable plants grown at a root-zone temperature of 25°C remained symptomless for the 3-months duration of the experiment. Summer-grown infected plants at the higher root temperature wilted but did not die. Enhanced temperature was ineffective as a curative treatment in summer-grown plants with established infection. Aerial symptoms of Phytophthora infection are seen as a function of the net amount of available healthy root. With high root zone temperatures this is determined by new root production and decreased inoculum and infection.     

Characterization of synaptosomes from the central nervous system of insects

Nerve terminals from the head ganglia of Locusta migratoria were isolated by means of a modified microscale flotation technique. Enzymatic, ultrastructural and chemical analysis revealed that the synaptosomal fraction was highly enriched in well-preserved nerve endings containing almost no free mitochondria. Cholinergic activities (choline acetyltransferase, acetylcholinesterase, acetylcholine receptors) were found to be concentrated in the synaptosomal fraction. The cholinergic nature and the functional integrity of nerve endings isolated from locusts were further supported by the existence of a high affinity choline uptake system, which is abolished by hemicholinium-3 as well as by low temperature, is essentially sodium dependent and inhibited by elevated potassium concentrations. After slight modifications of the gradient densities, synaptosomes could also be isolated from other insect species.     

Use of trout serum to prepare primary attached monolayer cultures of hepatocytes from rainbow trout (Salmo gairdneri)

Summary The influence of trout serum on the attachment and spreading of isolated trout hepatocytes maintained in primary culture at different temperatures was evaluated. Hepatocytes were obtained from young rainbow trout (Salmo gairdneri) by collagenase dissociation and maintained in modified Leibowitz L15 medium at 10° or 27° C for 24 h in plastic dishes previously coated with type I bovine collagen. In the absence of serum, fewer than 10% of hepatocytes attached and none of them spread on the collagen substrate. Trout serum at concentrations as low as 1.25% in the medium resulted in a pronounced concentration-dependent increase in hepatocyte attachment, as determined by direct counts by phase contrast microscopy, or by percentage of lactate dehydrogenase activity attached to the dishes after washing away unattached cells. Attachment rates were greater at the lower temperature (10° C). Trout serum also substantially increased the proportion of attached hepatocytes that spread as monolayers on the collagen substrate, especially at 10° C. By comparison, fetal bovine serum had little influence on the attachment or spreading of trout hepatocytes. These studies demonstrate a simple inexpensive method for preparing attached monolayer trout hepatocyte cultures. This procedure may be useful in toxicologic or functional studies in which fish hepatocyte attachment is an operational requirement.     

Characterization of a thermally stable and organic solvent-adaptative NAD+ -dependent formate dehydrogenase from Bacillus sp. F1

Aims: To characterize a robust NAD⁺‐dependent formate dehydrogenase firstly obtained from a nonmethylotroph, Bacillus sp. F1. Methods and Results: The Bacillus sp. F1 NAD⁺‐dependent formate dehydrogenase (BacFDH) gene was cloned by TAIL‐PCR and heterologous expressed in Escherichia coli. BacFDH was stable at temperatures below 55°C, and the half‐life at 60°C was determined as 52·9 min. This enzyme also showed a broad pH stability and retained more than 80% of the activities after incubating in buffers with different pH ranging from 4·5 to 10·5 for 1 h. The activity of BacFDH was significantly enhanced by some metal ions. Moreover, BacFDH exhibited high tolerance to 20% dimethyl sulfoxide, 60% acetone, 10% methanol, 20% ethanol, 60% isopropanol and 20% n‐hexane. Like other FDHs, BacFDH displayed strict substrate specificity for formate. Conclusion: We isolated a robust formate dehydrogenase, designated as BacFDH, which showed excellent thermal stability, organic solvent stability and a broad pH stability. Significance and Impact of the Study: The multi‐aspect stability makes BacFDH a competitive candidate for coenzyme regeneration in practical applications of chiral chemicals and pharmaceuticals synthesis with a relatively low cost, especially for the catalysis performed in extreme pH conditions and organic solvents.     

Microcosm evaluation of bioaugmentation after field-scale thermal treatment of a TCE-contaminated aquifer

This paper investigates effects of combining thermal and biological remediation, based on laboratory studies of trichloroethene (TCE) degradation. Aquifer material was collected 6 months after terminating a full-scale Electrical Resistance Heating (ERH), when the site had cooled from approximately 100°C to 40°C. The aquifer material was used to construct bioaugmented microcosms amended with the mixed anaerobic dechlorinating culture, KB-1^TM, and an electron donor (5 mM lactate). Microcosms were bioaugmented during cooling at 40, 30, 20, and 10°C, as temperatures continually decreased during laboratory incubation. Redox conditions were generally methanogenic, and electron donors were present to support dechlorination. For microcosms bioaugmented at 10°C and 20°C, dechlorination stalled at cis-dichloroethene (cDCE) and vinyl chloride (VC) 150 days after bioaugmentation. However, within 300 days of incubation ethene was produced in the majority of these microcosms. In contrast, dechlorination was rapid and complete in microcosms bioaugmented at 30°C. Microcosms bioaugmented at 40°C also showed rapid dechlorination, but stalled at cDCE with partial VC and ethene production, even after 150 days of incubation when the temperature had decreased to 10°C. These results suggest that sequential bioremediation of TCE is possible in field-scale thermal treatments after donor addition and bioaugmentation and that the optimal bioaugmentation temperature is approximately 30°C. When biological and thermal remediations are to be applied at the same location, three bioremediation approaches could be considered: (a) treating TCE in perimeter areas outside the source zone at temperatures of approximately 30°C; (b) polishing TCE concentrations in the original source zone during cooling from approximately 30°C to ambient groundwater temperatures; and (c) using bioremediation in downgradient areas taking advantages of the higher temperature and potential release of organic matter.     

Effects of circadian rhythms of fluctuating temperature on growth and biochemical composition of <Emphasis Type="Italic">Ulva pertusa</Emphasis>

The marcoalga Ulva pertusa was cultured under (20 ± 2)°C, (20 ± 4)°C, (20 ± 6)°C, (20 ± 8)°C and (20 ± 10)°C circadian rhythms of fluctuating temperature conditions, and constant temperature of 20°C was used as the control. The growth rate of macroalga at (20 ± 2)°C, (20 ± 4)°C and (20 ± 6)°C were significantly higher than that at constant temperature of 20°C, while growth rate at (20 ± 8)°C and (20 ± 10)°C were significantly lower than that at constant temperature of 20°C. The growth rate of macroalga was a quadratic function of the thermal amplitude. Such a growth model can be described by G = β ₀ + β ₁(TA) + β ₂(TA)², where G represents the relative growth rate, TA is thermal amplitude in degree Celsius, β ₀ is the intercept on the G axis, and β ₁ and β ₂ are the regression coefficients. The optimal thermal amplitude for the growth of thallus at mean temperature of 20°C was estimated to be ± 3.69°C. Analysis of biochemical composition at the final stages of thaulls growth revealed that diel fluctuating temperature caused various influences (P < 0.05). The content of chlorophyll, protein and total solute carbohydrate at (20 ± 2)°C and (20 ± 4)°C were slightly higher than those at constant temperature of 20°C, however no statistically significant differences were found among them (P > 0.05). While osmolytes (total solute carbohydrate and free proline) at (20 ± 10)°C were significantly higher than that at 20°C (P < 0.05). Therefore, more chlorophyll and carbohydrate production might account for the enhancement in the growth of macroalga at the diel fluctuating temperatures in the present study. Handling editor: S. M. Thomaz     

Biodegradation of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether by cold-adapted mixed and pure bacterial cultures

An aerobic mixed bacterial culture (CL-EMC-1) capable of utilizing methyl tert-butyl ether (MTBE) as the sole source of carbon and energy with a growth temperature range of 3 to 30°C and optimum of 18 to 22°C was enriched from activated sludge. Transient accumulation of tert-butanol (TBA) occurred during utilization of MTBE at temperatures from 3°C to 14°C, but TBA did not accumulate above 18°C. The culture utilized MTBE at a concentration of up to 1.5 g l⁻¹ and TBA of up to 7 g l⁻¹. The culture grew on MTBE at a pH range of 5 to 9, with an optimum pH of 6.5 to 7.1. The specific growth rate of the CL-EMC-1 culture on 0.1 g l⁻¹ of MTBE at 22°C and pH 7.1 was 0.012 h⁻¹, and the growth yield was 0.64 g (dry weight) g⁻¹. A new MTBE-utilizing bacterium, Variovorax paradoxus strain CL-8, isolated from the mixed culture utilized MTBE, TBA, 2-hydroxy isobutyrate, lactate, methacrylate, and acetate as sole sources of carbon and energy but not 2-propanol, acetone, methanol, formaldehyde, or formate. Two other isolates, Hyphomicrobium facilis strain CL-2 and Methylobacterium extorquens strain CL-4, isolated from the mixed culture were able to grow on C₁ compounds. The combined consortium could thus utilize all of the carbon of MTBE.