Effect of organic solvents on the yield of solvent-tolerant Pseudomonas putida S12.

Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h-1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.     

Improved stability of the continuous production of acetone-butanol by Clostridium acetobutylicum in a two-stage process

A stable continuous culture has been maintained for 30 days at a high 20 g/l solvent concentration. This substantial increase in the stability of the continuous culture ofClostridium acetobutylicum at the maximal solvent level was achieved by using a two-stage process with a dilution rate of 0.1 h^–1 in the first fermentor and 0.04 h^–1 in the second fermentor. The two-stage continuous fermentation allows an optimal growth of cells and induction of solvent metabolism in the first stage, and a maximal production yield of solvents in the second stage.     

Composition of Toluene-Degrading Microbial Communities from Soil at Different Concentrations of Toluene

Toluene-degrading bacteria were isolated from hydrocarbon-contaminated soil by incubating liquid enrichment cultures and agar plate cultures in desiccators in which the vapor pressure of toluene was controlled by dilution with vacuum pump oil. Incubation in desiccators equilibrated with either 100, 10, or 1% (wt/wt) toluene in vacuum pump oil and testing for genomic cross-hybridization resulted in four genomically distinct strains (standards) capable of growth on toluene (strains Cstd1, Cstd2, Cstd5, and Cstd7). The optimal toluene concentrations for growth of these standards on plating media differed considerably. Cstd1 grew best in an atmosphere equilibrated with 0.1% (wt/wt) toluene, but Cstd5 failed to grow in this atmosphere. Conversely, Cstd5 grew well in the presence of 10% (wt/wt) toluene, which inhibited growth of Cstd1. 16S ribosomal DNA sequencing and cross-hybridization analysis indicated that both Cstd1 and Cstd5 are members of the genus Pseudomonas. An analysis of the microbial communities in soil samples that were incubated with 10% (wt/wt) toluene with reverse sample genome probing indicated that Pseudomonas strain Cstd5 was the dominant community member. However, incubation of soil samples with 0.1% (wt/wt) toluene resulted in a community that was dominated by Pseudomonas strain Q7, a toluene degrader that has been described previously (Y. Shen, L. G. Stehmeier, and G. Voordouw, Appl. Environ. Microbiol. 64:637–645, 1998). Q7 was not able to grow by itself in an atmosphere equilibrated with 0.1% (wt/wt) toluene but grew efficiently in coculture with Cstd1, suggesting that toluene or metabolic derivatives of toluene were transferred from Cstd1 to Q7.     

The growth of Pseudomonas putida on m-toluic acid and on toluene in batch and in chemostat cultures

Summary The present study describes the growth of Pseudomonas putida cells (ATCC 33015) in batch and continuous cultures on two toxic substrates; toluene and m-toluic acid as sole carbon and energy sources. In fed-batch cultures on m-toluic acid up to 3.55 g cell dry weight/1 were achieved with a maximal specific growth rate (_max) of 0.1 h^-1. The average cellular yield was 1.42 g cell dry weight/g m-toluic acid utilized. When liquid toluene was added to shake-flask cultures in the presence of 0.7 g/1 m-toluic acid, the average cellular yield obtained was 1.3 g cell dry weight/g toluene utilized and the _max was 0.13 h^-1. Growth on toluene vapour in the presence of 0.7 g/l m-toluic acid in batch cultures resulted in a cellular yield of 1.28 g cell dry weight/g toluene utilized, with growth kinetics almost identical to those with liquid toluene (_max liquid=0.13 h^-1, _max vapour=0.12 h^-1). The maximal biomass concentration was 3.8 g cell dry weight/l, obtained in both cases after 100 h of incubation. Pseudomonas putida was grown in a chemostat initially on 0.7 g/l m-toluic acid and vapour toluene and then in the steady state on toluene as the sole source of carbon and energy. Toluene was added continuously to the culture as vapour with the inflowing airstream. Chemostat cultures could be maintained at steady state for several months on toluene. The maximal biomass concentration obtained in the chemostat culture was 3.2 g cell dry weight/l. The maximum specific growth rate was 0.13 h^-1, with a cellular yield of 1.05 g cell dry weight/g toluene utilized. Approximately 70% of the toluene consumed was converted into biomass, and the remainder was converted to CO₂ and unidentified byproducts.     

Viral Lysis and Bacterivory during a Phytoplankton Bloom in a Coastal Water Microcosm

The relative importance of viral lysis and bacterivory as causes of bacterial mortality were estimated. A laboratory experiment was carried out to check the kind of control that viruses could exert over the bacterial assemblage in a non-steady-state situation. Virus-like particles (VLP) were determined by using three methods of counting (DAPI [4′,6-diamidino-2-phenylindole] staining, YOPRO staining, and transmission electron microscopy). Virus counts increased from the beginning until the end of the experiment. However, different methods produced significantly different results. DAPI-stained VLP yielded the lowest numbers, while YOPRO-stained VLP yielded the highest numbers. Bacteria reached the maximal abundance at 122 h (3 × 10⁷ bacteria ml⁻¹), after the peak of chlorophyll a (80 μg liter⁻¹). Phototrophic nanoflagellates followed the same pattern as for chlorophyll a. Heterotrophic nanoflagellates showed oscillations in abundance throughout the experiment. The specific bacterial growth rate increased until 168 h (2.6 day⁻¹). The bacterivory rate reached the maximal value at 96 hours (0.9 day⁻¹). Bacterial mortality due to viral infection was measured by using two approaches: measuring the percentage of visibly infected bacteria (%VIB) and measuring the viral decay rates (VDR), which were estimated with cyanide. The %VIB was always lower than 1% during the experiment. VDR were used to estimate viral production. Viral production increased 1 order of magnitude during the experiment (from 10⁶ to 10⁷ VLP ml⁻¹ h⁻¹). The percentage of heterotrophic bacterial production consumed by bacterivores was higher than 60% during the first 4 days of the experiment; afterwards, this percentage was lower than 10%. The percentage of heterotrophic bacterial production lysed by viruses as assessed by the VDR reached the highest values at the beginning (100%) and at the end (50%) of the experiment. Comparing both sources of mortality at each stage of the bloom, bacterivory was found to be higher than viral lysis at days 2 and 4, and viral lysis was higher than bacterivory at days 7 and 9. A balance between bacterial losses and bacterial production was calculated for each sampling interval. At intervals of 0 to 2 and 2 to 4 days, viral lysis and bacterivory accounted for all the bacterial losses. At intervals of 4 to 7 and 7 to 9 days, bacterial losses were not balanced by the sources of mortality measured. At these time points, bacterial abundance was about 20 times higher than the expected value if viral lysis and bacterivory had been the only factors causing bacterial mortality. In conclusion, mortality caused by viruses can be more important than bacterivory under non-steady-state conditions.     

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10⁵ cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10⁷ cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Isolation and Characterization of Toluene-Sensitive Mutants from the Toluene-Resistant Bacterium Pseudomonas putida GM73

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10⁻⁶). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.     

Predation of bacteria by the protozoa Tetrahymena pyriformis in toluene-degrading cultures

The influence of the toluene concentration on predation of toluene-degrading bacteria by the protozoa Tetrahymena pyriformis was investigated in suspended batch cultures continuously aerated with toluene-contaminated air. At gas phase concentrations of 0.035 to 0.74 g m–3, toluene did not significantly affected protozoan activity and the final bacteria concentration was reduced by growing protozoa by 98 to 99.9% compared to protozoa-free controls. As the toluene concentration was increased to 1.16–1.33 g m–3, the reduction of the bacteria cell concentration was 80%. At 3.35 g toluene m–3, growth of T. pyriformis was completely inhibited. Overall, the results presented herein demonstrate that protozoa grazing on bacteria play a major role in controlling bacterial cell concentration, but that the toxicity of the treated pollutants to the protozoa is an important factor that needs to be taken into account in biological treatment processes.     

Efflux Pumps Involved in Toluene Tolerance in Pseudomonas putida DOT-T1E

The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant P. putida DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of P. putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to P. putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-′phoA mutagenesis of wild-type P. putida DOT-T1E cells. The mutant, called P. putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[¹⁴C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[¹⁴C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of ¹⁴C-labeled substrate in whole cells increased in competition assays in which toluene and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type.The sensitivity of microorganisms to toxic organic solvents is related to the logarithm of the partition coefficient of the solvent in a mixture of octanol and water (log P_ow). Aromatic hydrocarbons with a log P_ow of between 1.5 and 3.5 are extremely toxic to living organisms (47). These chemicals dissolve in the cytoplasmic membrane, disorganize it, and collapse the cell membrane potential; this, together with the induced loss of lipids and proteins, leads to irreversible damage resulting in the death of the cell (8, 47, 50).Independent laboratories have isolated Pseudomonas putida strains tolerant to different aromatic hydrocarbons such as toluene, styrene, and p-xylene (6, 15, 42, 48). All four isolated strains were able to grow in liquid culture medium to which a high concentration (1% [vol/vol]) of these aromatic hydrocarbons was added. Tolerance to organic solvents in these P. putida strains is achieved by a series of biochemical mechanisms that actively remove the organic solvent from cell membranes (16, 43) and by physical barriers that help the cell to become (to a certain degree) impermeable to the solvent (13, 37, 43, 48). The physical barriers involve the ordered organization of the cell surface lipopolysaccharides (37) together with modified phospholipids (4, 37, 43, 49). Modifications in phospholipids upon exposure to an organic solvent involve both a short-term response, in which the level of the trans isomers of unsaturated phospholipids increases, and a long-term response consisting of a modification of the polar head groups of phospholipids (4, 43, 49) and an increase in the total amount of phospholipids per dry weight (49). For P. putida DOT-T1, it was suggested that an energy-dependent exclusion system (such as an efflux pump) is critical for tolerance to solvents (43). This conclusion was based on the following findings: (i) P. putida DOT-T1 treated with the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone accumulated higher levels of 1,2,4-[¹⁴C]trichlorobenzene in cell membranes than did untreated cells, and (ii) P. putida DOT-T1 mutants which were sensitive to toluene, octanol, and other chemicals accumulated 5- to 20-fold-higher levels of 1,2,4-[¹⁴C]trichlorobenzene in cell membranes than did the wild-type strain. Similar observations have been reported for Pseudomonas sp. strain S12 (16).In this study, we report that P. putida DOT-T1 uses at least two efflux pumps for toluene exclusion, one that seems to be expressed constitutively and a second inducible one. A mini-Tn5′phoA-Km^r knocked out the constitutive efflux system of P. putida DOT-T1E. The mutant was shown to be hypersensitive to toluene but not to octanol. The Km^r marker of the mini-Tn5 and the 3′ adjacent chromosomal DNA were cloned, and the wild-type gene was rescued by colony screening hybridization and sequenced. Sequence analysis showed that the knocked-out gene in the mutant was a homolog of the mexB gene, which belongs to the efflux pump family of the resistant nodulator division type (34–36, 38–41).     

The Two Major Types of Plant Plasma Membrane H+-ATPases Show Different Enzymatic Properties and Confer Differential pH Sensitivity of Yeast Growth

The proton-pumping ATPase (H⁺-ATPase) of the plant plasma membrane is encoded by two major gene subfamilies. To characterize individual H⁺-ATPases, PMA2, an H⁺-ATPase isoform of tobacco (Nicotiana plumbaginifolia), was expressed in Saccharomyces cerevisiae and found to functionally replace the yeast H⁺-ATPase if the external pH was kept above 5.0 (A. de Kerchove d'Exaerde, P. Supply, J.P. Dufour, P. Bogaerts, D. Thinès, A. Goffeau, M. Boutry [1995] J Biol Chem 270: 23828–23837). In the present study we replaced the yeast H⁺-ATPase with PMA4, an H⁺-ATPase isoform from the second subfamily. Yeast expressing PMA4 grew at a pH as low as 4.0. This was correlated with a higher acidification of the external medium and an approximately 50% increase of ATPase activity compared with PMA2. Although both PMA2 and PMA4 had a similar pH optimum (6.6–6.8), the profile was different on the alkaline side. At pH 7.2 PMA2 kept more than 80% of the maximal activity, whereas that of PMA4 decreased to less than 40%. Both enzymes were stimulated up to 3-fold by 100 μg/mL lysophosphatidylcholine, but this stimulation vanished at a higher concentration in PMA4. These data demonstrate functional differences between two plant H⁺-ATPases expressed in the same heterologous host. Characterization of two PMA4 mutants selected to allow yeast growth at pH 3.0 revealed that mutations within the carboxy-terminal region of PMA4 could still improve the enzyme, resulting in better growth of yeast cells.     

Formate Oxidation-Driven Calcium Carbonate Precipitation by Methylocystis parvus OBBP

Microbially induced carbonate precipitation (MICP) applied in the construction industry poses several disadvantages such as ammonia release to the air and nitric acid production. An alternative MICP from calcium formate by Methylocystis parvus OBBP is presented here to overcome these disadvantages. To induce calcium carbonate precipitation, M. parvus was incubated at different calcium formate concentrations and starting culture densities. Up to 91.4% ± 1.6% of the initial calcium was precipitated in the methane-amended cultures compared to 35.1% ± 11.9% when methane was not added. Because the bacteria could only utilize methane for growth, higher culture densities and subsequently calcium removals were exhibited in the cultures when methane was added. A higher calcium carbonate precipitate yield was obtained when higher culture densities were used but not necessarily when more calcium formate was added. This was mainly due to salt inhibition of the bacterial activity at a high calcium formate concentration. A maximum 0.67 ± 0.03 g of CaCO₃ g of Ca(CHOOH)₂⁻¹ calcium carbonate precipitate yield was obtained when a culture of 10⁹ cells ml⁻¹ and 5 g of calcium formate liter⁻¹ were used. Compared to the current strategy employing biogenic urea degradation as the basis for MICP, our approach presents significant improvements in the environmental sustainability of the application in the construction industry.     

Toluene Induction and Uptake Kinetics and Their Inclusion in the Specific-Affinity Relationship for Describing Rates of Hydrocarbon Metabolism

The kinetics of concentration-dependent toluene metabolism were examined by evaluating each term in the second-order rate equation. Marine and freshwater pseudomonads were used. Uptake for Pseudomonas sp. strain T2 was characterized by a completely saturatable system with small transport constant (K_t = 44 μg/liter) and large specific affinity. Kinetics for Pseudomonas putida PpF1 were similar. Induction had little effect on K_t, but it caused the specific affinity to increase from about 0.03 to 320 liters/g of cells per h. The level of induction depended on the time of exposure, the concentration of inducer, and the initial level of induction. If loss of the inducible system was not severe, toluene caused a linear increase in specific affinity with time, and the maximal value achieved at intermediate times (1 to 3 days) was hyperbolic with concentration when K_ind was 96 μg/liter (A. T. Law and D. K. Button, Appl. Environ. Microbiol. 51:469-476, 1986). As repression became complete, specific affinities were greatly reduced. Then induction required higher toluene concentrations and longer times, and the shape of the specific-affinity curve became sigmoidal with concentration. Cell yields (0.10 to 0.17 g of cells per g of toluene used) were low owing to liberation of organic products: 2-hydroxy-6-oxohepta-2,4-dienoic acid, toluene dihydrodiol, 3-methylcatechol, acetate, formate, and possibly pyruvate, which in turn caused lower rates of growth. Michaelis constants for the reaccumulation of products exceeded those for toluene, but specific affinities were lower and maximal velocities were higher, so that recycling was favored in cultures with high toluene concentration. Although these kinetics predict deviation from the linear relationship between uptake rate and biomass, we could detect none. Effects of saturation and induction were incorporated into the basic specific-affinity relationship. The result appears to be an improvement in the equation used for describing the kinetics of uptake and growth.     

Change in Size of Chromatium minus Cells in Relation to Growth Rate, Sulfur Content, and Photosynthetic Activity: A Comparison of Pure Cultures and Field Populations

The size frequency distribution of planktonic cells of purple sulfur phototrophic bacteria was measured at several depths in a bacterial layer of Lake Cisó (Spain). The bacterioplankton was dominated by Chromatium minus (87 to 94% of the total biomass). The largest cells of C. minus were found in the top part of the bacterial layer. In addition, the in situ and potential specific photosynthetic activity (CO₂ fixation and acetate uptake) and specific pigment content were measured in relation to several key environmental parameters that determine the activity of cells. Potential growth rates were estimated from production rates and biomass. A maximal specific growth rate of 0.074 h⁻¹ was found for the top part of the bacterial layer. Photosynthesis versus light and versus sulfide curves among field samples indicated that light was the main limiting factor controlling the activity of C. minus in Lake Cisó. The specific bacteriochlorophyll a content was very high in all samples (0.27 to 0.36 μg μg of C⁻¹). Results of laboratory experiments performed with pure cultures indicated that the average cell volume changes from 5.9 to 20.0 μm³ and that differences in growth rate, breakdown, or synthesis of sulfur and glycogen and degradation of the photosynthetic apparatus are the main factors accounting for the observed changes in cell volume across the bacterial layer.     

Continuous acetone-butanol fermentation: influence of vitamins on the metabolic activity of Clostridium acetobutylicum

Summary A detailed investigation was undertaken to examine the influence of biotin and paminobenzoic acid (PABA) in chemostat cultures of Clostridium acetobutylicum ATCC 824. Initiation of chemostat cultures with a basic synthetic medium (biotin 0.01 mg l^–1; PABA 1.0 mg l^–1) have resulted in a low biomass together with a low specific rate of solvent production. A different picture emerged on elevating the concentration of both vitamins 8-fold: biomass and specific rates (solvent production, glucose consumption) were increased and a solvent productivity of 2.54 g l^–1 h^–1 at the solvent concentration of 13.1 g l^–1 was achieved. It has also been shown that PABA was the only limiting factor for the metabolism of Clostridium acetobutylicum in the basic synthetic medium and that the optimised concentration was 8 mg l^–1 in the chemostat cultures with the growth conditions employed.     

Combined Effects of pH and Sugar on Growth Rate of Zygosaccharomyces rouxii, a Bakery Product Spoilage Yeast

The effects of citric acid-modified pH (pH 2.5, 2.75, 3, 3.5, 4, 4.5, 5, and 5.5) and a 30% glucose–70% sucrose mixture (300, 400, 500, 600, 700, 800, 875, and 900 g/liter) on an osmophilic yeast, Zygosaccharomyces rouxii, were determined by using synthetic medium. One hundred experiments were carried out; 50-ml culture flasks were inoculated with 10³ CFU ml⁻¹ by using a collection strain and a wild-type strain cocktail. The biomass was measured by counting cell colonies, and growth curves were fitted by using a Baranyi equation. The growth rate decreased linearly with sugar concentration, while the effect of pH was nonlinear. Indeed, the optimal pH range was found to be pH 3.5 to 5, and pH 2.5 resulted in a 30% reduction in the growth rate. Finally, we evaluated the performance of two nonlinear predictive models developed previously to describe bacterial contamination. Equations derived from the Rosso and Ratkowsky models gave similar results; however, the model that included dimensionless terms based on the Ratkowsky equation was preferred because it contained fewer estimated parameters and also because biological interpretation of the results was easier.     

Characterization of the Polyurethanolytic Activity of Two Alicycliphilus sp. Strains Able To Degrade Polyurethane and N-Methylpyrrolidone

Two bacterial strains (BQ1 and BQ8) were isolated from decomposed soft foam. These were selected for their capacity to grow in a minimal medium (MM) supplemented with a commercial surface-coating polyurethane (PU) (Hydroform) as the carbon source (MM-PUh). Both bacterial strains were identified as Alicycliphilus sp. by comparative 16S rRNA gene sequence analysis. Growth in MM-PUh showed hyperbolic behavior, with BQ1 producing higher maximum growth (17.8 ± 0.6 mg·ml⁻¹) than BQ8 (14.0 ± 0.6 mg·ml⁻¹) after 100 h of culture. Nuclear magnetic resonance, Fourier transform infrared (IR) spectroscopy, and gas chromatography-mass spectrometry analyses of Hydroform showed that it was a polyester PU type which also contained N-methylpyrrolidone (NMP) as an additive. Alicycliphilus sp. utilizes NMP during the first stage of growth and was able to use it as the sole carbon and nitrogen source, with calculated K_s values of about 8 mg·ml⁻¹. Enzymatic activities related to PU degradation (esterase, protease, and urease activities) were tested by using differential media and activity assays in cell-free supernatants of bacterial cultures in MM-PUh. Induction of esterase activity in inoculated MM-PUh, but not that of protease or urease activities, was observed at 12 h of culture. Esterase activity reached its maximum at 18 h and was maintained at 50% of its maximal activity until the end of the analysis (120 h). The capacity of Alicycliphilus sp. to degrade PU was demonstrated by changes in the PU IR spectrum and by the numerous holes produced in solid PU observed by scanning electron microscopy after bacterial culture. Changes in the PU IR spectra indicate that an esterase activity is involved in PU degradation.     

Two-stage continuous fermentation of Clostridium acetobutylicum: effects of pH and dilution rate

Summary The kinetics of a two-stage continuous fermentation of Clostridium acetobutylicum have been studied. The pH and the dilution rate have been shown to be two essential factors for process optimization. An increase in pH or dilution rate in the first stage decreased solvent production in the second fermentor. To achieve optimal solvent production, the pH had to be maintained at 4.5 in the first stage and between 4.5 and 5.0 in the second stage. Dilution rates of 0.08 h^–1 and 0.04 h^–1,respectively, in the first and second fermentors allowed a high solvent concentration. When the pH was maintained at 4.5 in each stage and when the dilution rates were 0.08 h^–1 and 0.04 h^–1 in the first and second fermentors respectively, 21 g/l solvent concentration was achieved. A conversion yield of 0.36 g solvents/g glucose consumed was obtained with total consumption of glucose. Biomass was only produced in the first stage together with 40% of the solvents, indicating that solvent production had to be induced in the first fermentor. Offprint requests to: J. M. Engasser     

Anaerobic Benzene Biodegradation Linked to Nitrate Reduction

Benzene oxidation to carbon dioxide linked to nitrate reduction was observed in enrichment cultures developed from soil and groundwater microcosms. Benzene biodegradation occurred concurrently with nitrate reduction at a constant ratio of 10 mol of nitrate consumed per mol of benzene degraded. Benzene biodegradation linked to nitrate reduction was associated with cell growth; however, the yield, 8.8 g (dry weight) of cells per mol of benzene, was less than 15% of the predicted yield for benzene biodegradation linked to nitrate reduction. In experiments performed with [¹⁴C]benzene, approximately 92 to 95% of the label was recovered in ¹⁴CO₂, while the remaining 5 to 8% was incorporated into the nonvolatile fraction (presumably biomass), which is consistent with the low measured yield. In benzene-degrading cultures, nitrite accumulated stoichiometrically as nitrate was reduced and then was slowly reduced to nitrogen gas. When nitrate was depleted and only nitrite remained, the rate of benzene degradation decreased to almost zero. Based on electron balances, benzene biodegradation appears to be coupled more tightly to nitrate reduction to nitrite than to further reduction of nitrite to nitrogen gas.