Development of a microtitre plate method for determination of phenol utilization,biofilm formation and respiratory activity by environmental bacterial isolates

Twenty-five aerobic phenol-degrading bacteria, isolated from different environmental samples on phenol agar after several subcultures in phenol broth, utilized phenol (0.2 g l⁻¹) within 24 h, but removal of phenol was more rapid when other carbon sources were also present. A microtitre plate method was developed to determine growth rate, biofilm formation and respiratory activity of the strains isolated. Pseudomonas putida strains C5 and D6 showed maximum growth (as O.D. at 600 nm), P. putida D6 and unidentified bacterial strain M1 were more stable at high concentrations of phenol (0.8 g l⁻¹), and P. putida C5 formed the greatest amount of biofilm in 0.5 g phenol l⁻¹ medium. Measurement of dehydrogenase activity as reduction of triphenyl tetrazolium chloride supported data on growth rate and biofilm formation. The microtitre plate method provided a selective method for detection of the best phenol degrading and biofilm-forming microorganisms, and was also a rapid, convenient means of studying the effect of phenol concentration on growth rate and biofilm formation.     

Characteristics of fed-batch cultures of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> containing human-like collagen cDNA at different specific growth rates

Fed-batch cultures of recombinant Escherichia coli BL21 for producing human-like collagen were performed at different specific growth rates (0.1~0.25 h⁻¹) before induction and at a constant value of 0.05 h⁻¹ after induction by the method of pseudo-exponential feeding. Although the final biomass (around 69 g l⁻¹) was almost the same in all fed-batch cultures, the highest product concentration (13.6 g l⁻¹) was achieved at the specific growth rate of 0.15 h⁻¹ and the lowest (9.6 g l⁻¹) at 0.25 h⁻¹. The mean productivity of human-like collagen was the highest at 0.15 h⁻¹ (0.57 g l⁻¹ h⁻¹) and the lowest at 0.1 h⁻¹ (0.35 g l⁻¹ h⁻¹). In the phase before induction, the cell yield coefficient (Y_X/S) decreased when the specific growth rate increased, while the formation of acetic acid increased upto 2.5 g l⁻¹ at 0.25 h⁻¹. The mean product yield coefficient (Y_P/S) also decreased with specific growth rate increasing. The respiration quotient (RQ) increased slightly with specific growth rate increasing before induction, and the mean value of RQ was around 72%. The optimum growth rate for human-like collagen production was 0.15~0.2 h⁻¹.     

Degradation of phenol by a defined mixed culture immobilized by adsorption on activated carbon and sintered glass

Summary A defined mixed culture of the yeast Cryptococcus elinovii H1 and the bacterium Pseudomonas putida P8 was immobilized by adsorption on activated carbon and sintered glass, respectively. Depending on its adsorption capacity for phenol the activated carbon system could completely degrade 17 g/l in batch culture, whereas the sintered glass system was able to degrade phenol up to 4 g/l. During semicontinuous degradation of phenol (1 g/l) both systems reached constant degradation times with the fourth batch that lasted 8 h when using the activated carbon system and 10 h in the sintered glass system. In the course of continuous degradation of phenol the activated carbon system reached a maximum degradation rate of 9.2 g l^–1 day^–1 compared to 6.4 g l^–1 day^–1degraded by the sintered glass system. 2-Hydroxymuconic acid semialdehyde could be identified and quantitatively determined as a metabolite of phenol degradation by P. putida P8. Increased membrane permeability under the influence of phenol was demonstrated by the examination of K⁺ efflux from P. putida P8. Offprint requests to: H.-J. Rehm     

The isolation and characterisation of a salicylate-hydroxylase-producing strain of Pseudomonas putida

Summary A salicylate-hydroxylase-producing strain of Pseudomonas putida with an unusual capability to grow at toxic levels of salicylate up to 10 g l^–1 has been isolated. It grew well under continuous culture conditions, with optimum growth at pH 6.5 and a temperature of 25° C. The use of an ammonium salt as a nitrogen source, instead of nitrate, resulted in a 30–40% increase in its biomass yield coefficient. Optimum growth under continuous culture conditions was achieved using 4 g l^–1 salicylate at 25° C, pH 6.5 and 0.2 h^–1 dilution rate. High salicylate hydroxylase enzyme activity [236 units (U) l^–1] and productivity (424.8 U h^–1) were obtained at a dilution rate of 0.45 h^–1 using a mineral medium containing 4 g l^–1 of salicylate. Operating under continuous culture conditions with oxygen limitation and a slight accumulation of residual salicylate (0.2 g l^–1) resulted in a decrease in culture performance and enzyme productivity. Correspondence to: R. Marchant     

Enhanced yield of medium-chain-length polyhydroxyalkanoates from nonanoic acid by co-feeding glucose in carbon-limited, fed-batch culture

Medium-chain-length polyhydroxyalkanoates (MCL-PHAs) were produced in carbon-limited, single-stage, fed-batch fermentations of Pseudomonas putida KT2440 by co-feeding nonanoic acid (NA) and glucose (G) to enhance the yield of PHA from NA. An exponential (μ = 0.25 h⁻¹) followed by a linear feeding strategy at a NA:G ratio of 1:1 (w/w) achieved 71 g l⁻¹ biomass containing 56% PHA. Although the same overall PHA productivity (1.44 g l⁻¹ h⁻¹) was obtained when NA alone was fed at the same specific growth rate, the overall yield of PHA from NA increased by 25% (0.66 g PHA g NA⁻¹ versus 0.53 g g⁻¹) with glucose co-feeding. Further increasing glucose in the feed (NA:G = 1:1.5) resulted in a slightly higher yield (0.69 g PHA g NA⁻¹) but lower PHA content (48%) and productivity (1.16 g l⁻¹ h⁻¹). There was very little change in the PHA composition.     

Characterization of two quaternary ammonium chloride-resistant bacteria isolated from papermaking processing water and the biocidal effect on their biofilm formation

Biofilm formation and growth on equipment surfaces is detrimental to papermaking processes. However, a fundamental understanding leading to an optimal control strategy is yet to be found. Quaternary ammonium compounds (QAC) are being increasingly applied in the papermaking processes. Among them, the most frequently applied, N-alkyl-benzyl-dimethyl ammonium chloride, was employed in this study. To foster fundamental understanding of QAC efficacy towards biofilm control, two of the highest QAC-resistant strains of bacteria were isolated from the papermaking processing water and employed as model organisms. By the 16S rRNA gene sequencing technique, two Gram-negative rods with QAC resistance were identified as Morganella morganii (HB22) and the biofilm-forming Pseudomonas putida (HB45). The minimal inhibition concentration (MIC) values were 8 mg L⁻¹ for HB22 and 16 mg L⁻¹ for HB45, respectively, against QAC in basal medium (BM). However, both strains could grow under more than 150 mg L⁻¹ QAC in basal medium at neutral pH. As observed by crystal violet assay and fluorescent confocal microscopy, HB45 formed biofilm more slowly on stainless steel coupon which is the prime material of papermachine than on the surface of polystyrene, the most common material for food packaging and semi-finished/finished products. HB45 formed biofilm more slowly on stainless steel coupons than on polystyrene Petri dish surfaces, as observed by crystal violet assay and fluorescent confocal microscopy. For HB45, there was a marginal increase of inhibition of biofilm formation by increasing QAC concentration from 50 to 75 mg L⁻¹. By comparison of inhibition concentration in liquid state and in biofilm formation, the results implicate that the current practice in papermaking processes of adding biocide to qualitatively control planktonic bacterial communities does not ensure control of biofilm formation.     

Intensification of phenol biodegradation by humic substances

Phenol biodegradation was carried out in a batch system by the bacterial strain Cupriavidus metallidurans in the presence of potassium humate that was prepared by alkaline extraction from oxyhumolite. The experiments were focused on the assessment of the humate effect on biodegradation activity of the tested bacterial strain. The achieved results demonstrated that the humate has a positive influence on the biodegradation of phenol and reduces the incubation time necessary for phenol removal. Higher biodegradation rate and more intensive growth were observed during the cultivation in presence of humate in comparison to the cultivation without its addition. Adsorption of the humate on bacterial biomass was observed as well. Subsequently, a phenol biodegradation testing in a continuous-flow system using a biofilm reactor was also carried out. Although the reactor was inoculated by C. metallidurans only, the microbial composition under an aerobic non-aseptic condition during this long-term cultivation changed. The phenol removal efficiency obtained in the biofilm reactor was higher than 92% when phenol concentration in a treated medium was 1200 mg l⁻¹.     

High-level production of arachidonic acid by fed-batch culture of <Emphasis Type="Italic"> Mortierella alpina</Emphasis> using NH<Subscript>4</Subscript>OH as a nitrogen source and pH control

Mortierella alpina was grown in a fed-batch culture using a 12-l jar fermenter with an initial 8-l working volume containing 20 g glucose l⁻¹ and 10 g corn-steep powder l⁻¹. Glucose was intermittently fed to give 32 g l⁻¹ at each time. The pH of culture was maintained using 14% (v/v) NH₄OH, which also acted as a nitrogen source. A final cell density of 72.5 g l⁻¹ was reached after 12.5 days with a content of arachidonic acid (ARA) at 18.8 g l⁻¹. These values were 4 and 1.8 times higher than the respective values in batch culture. Our results suggest that the combined feeding of glucose and NH₄⁺ to the growth of M. alpina could be applied for the industrial scale production of ARA.     

Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis

Aims: The aim of this work was to investigate the possible effect of human cathelicidin antimicrobial peptide LL37 on biofilm formation of Staphylococcus epidermidis, a major causative agent of indwelling device‐related infections. Methods and Results: We performed initial attachment assay and biofilm formation solid surface assay in microtitre plates, as well as growth experiment in liquid medium using laboratory strain Staph. epidermidis ATCC35984. We found that already a low concentration of the peptide LL37 (1 mg l^?1) significantly decreased both the attachment of bacteria to the surface and also the biofilm mass. No growth inhibition was observed even at 16 mg l^?1 concentration of LL37, indicating a direct effect of the peptide on biofilm production. Conclusions: As biofilm protects bacteria during infections in humans and allows their survival in a hostile environment, inhibition of biofilm formation by LL37 may have a key role to prevent bacterial colonization on indwelling devices. Significance and Impact of the Study: Our findings suggest that this host defence factor can be a potential candidate in prevention and treatment strategies of Staph. epidermidis infections in humans.     

Degradation of caffeine by immobilized cells of Pseudomonas putida strain C 3024

Summary A caffeine-resistant strain of Pseudomonas putida was isolated from soil and was grown with caffeine as the sole source of carbon, energy and nitrogen. Cells were immobilized in agar gel particles which were continuously supplied with a caffeine solution (0.52 g · l^–1, D=1.0 h^–1) in a homogeneously mixed aerated reaction vessel. In the presence of the ATPase inhibitor arsenate the caffeine was removed by the immobilized cells at an average rate of 0.25 mg caffeine · h^–1 · (mg cell carbon)^–1 during 6 days. Thereafter a rapid decline of activity was observed. From a similar system without arsenate supplied with a growth medium containing a limiting amount of caffeine (0.13 g · l^–1) the caffeine was almost completely oxidized by the immobilized cells. The concentration of the remaining caffeine was 1.4 mg · l^–1, which is much lower than the substrate constant for caffeine (9.7 mg · l^–1) observed with freshly harvested suspended resting cells.     

Conversion of xylose to ethanol by a novel phenol-tolerant strain of Enterobacteriaceae isolated from olive mill wastewater

A xylose-fermenting bacterium of the family Enterobacteriaceae was isolated from olive mill wastewater. It converted xylose to ethanol with a yield of 0.19 g ethanol g^–1 xylose. Although phenolic compounds normally inhibit pentose-utilizing microorganisms, this isolate was tolerant to phenol. Both the yield and the productivity of xylose fermentation decreased by 30% when phenol was added at a final concentration of 0.8 g phenol l^–1. Xylose (23 g l^–1) was totally fermented to ethanol (4.3 g l^–1) within 48 h in the absence of phenol; however, in the presence of 0.8 g phenol l^–1, only 3.3 g ethanol l^–1 was obtained from the same starting concentration of xylose after 70 h.     

Biofilms isolated from washing machines from three continents and their tolerance to a standard detergent

The goal of this comparative study was to investigate biofilm forming microorganisms living in washing machines (WMs). Biofilms were sampled from 11 washing machines from four countries and three continents. Among the 94 isolated strains, 30% were potential human pathogens. Representative strains were selected and biofilm formation was evaluated with the crystal violet (CV) assay. The majority of the WM isolates formed more biofilm than their reference strains. Biofilms of P. putida WM (the largest biofilm producer) were exposed to different concentrations (0.0007–7 g l^?1) of the standard detergent IEC-A* at 30°C for 30 min and observed with confocal laser scanning microscopy. Using quantitative CVA, P. putida WM biofilm removal required higher detergent concentrations than the type strain. However, for both strains the recommended detergent concentration (7 g l^?1) was insufficient to completely clean surfaces from cell debris and exopolymeric substances.     

Production of xanthan by in-flow cultures of Xanthomonas campestris

The kinetics of xanthan formation in Xanthomonas campestris continuous and fed-batch fermentations was studied along with metabolic changes due to growth rate variation. A maximum growth rate within the range 0.11–0.12 h^–1 was obtained from the continuous culture data in defined medium, producing xanthan at rates up to 0.36 g l^–1 h^–1 corresponding to a maximum 67% glucose conversion at a dilution rate (D) of 0.05 h^–1. Comparatively, fed-batch cultivation was more efficient, producing maximum xanthan at 0.75 g l^–1 h^–1 and 63% glucose conversion at 0.1 h^–1. When reaching D=0.062 h^–1 in continuous cultures, a change was observed and the values of the specific rate of substrate consumption shifted, initiating an uncoupled growth region expressing a lack of balance of the catabolic and anabolic reactions. The deviation was not accompanied by a change in specific xanthan production indicating that xanthan metabolism was not affected by D. For fed-batch-grown X. campestris cells within the range D=0.03–0.1 h^–1, both metabolic parameters changed linearly with the growth rate showing a wide region coupled to growth. Outside that range, glucose accumulated and the specific xanthan production dropped, suggesting substrate inhibition. Correspondence to: J. C. Roseiro     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

Utilization of <Emphasis Type="Italic">fadA</Emphasis> knockout mutant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> for overproduction of medium chain-length-polyhydroxyalkanoate

The fadBA operon in the fatty acid β-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l⁻¹ stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l⁻¹ with a cell dry weight of 10.3 g l⁻¹.     

Biotransformation of oleic acid into 10-hydroxy-8E-octadecenoic acid by Pseudomonas sp. 42A2

Pseudomonas sp. 42A2 when incubated for 36 h with oleic acid (20 g l^–1) in a stirred bioreactor, accumulated 10-hydroxy-8E-octadecenoic acid. Production in a 2 l bioreactor with 1.4 l of working volume, was increased from 0.65 g l^–1 to 7.4 g l^–1 with K _L a values ranging between 15 and 200 h^–1. A linear relationship was found between volumetric productivity and oxygen transfer rates and an exponential relation between the specific rate of product formation and specific growth rate.     

A novel <Emphasis Type="Italic">Candida glycerinogenes</Emphasis> mutant with high glycerol productivity in high phosphate concentration medium

A novel Candida glycerinogenes mutant, which possesses high glycerol productivity in a high phosphate concentration medium, was obtained by mutagenesis of an industrial glycerol producer. The mutant accumulated a total biomass of 11.5 g l⁻¹, which is less than the 15 g l⁻¹of the wild-type strain, but it consumed glucose faster than the wild-type strain did. The mutant reached its maximal glycerol concentration of 129 g l⁻¹ in 84 h compared to 96 h for the wild-type strain. High cytoplasmic glycerol-3-phosphate dehydrogenase activity of the mutant in the early glycerol formation phase, leading to a rapid glycerol synthesis and accumulation, may be the main reason for the short fermentation process.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

The effect of ampicillin plus streptomycin on growth and photosynthesis of two halotolerant chlorophyte algae

Streptomycin and ampicillin are antibiotics commonly used to eliminate prokaryotes from the cultures of eukaryotic algae. We studied the effects of 25 mg l⁻¹ streptomycin plus 50 mg l⁻¹ ampicillin on the growth and photosynthesis of two broadly halotolerant algae, Picochlorum oklahomensis and Dunaliella sp. (Chlorophyceae). We measured growth rate, oxygen evolution, chlorophyll fluorescence kinetics, and pigment content in low (150 μmol photons m⁻² s⁻¹) and high (600 μmol photons m⁻² s⁻¹) light grown batch cultures. Our results show only a minor effect of the antibiotics on P. oklahomensis, and none on Dunaliella sp., so this combination of antibiotics is suitable for maintenance of stock cultures for physiological experiments. We also show that these antibiotics can be used in turbidostat cultures of P. oklahomensis, which otherwise tend to succumb to bacteria.     

Anaerobic fermentation of Salmonella typhimurium with and without pyruvate carboxylase

A plasmid that expressed pyruvate carboxylase (PYC) from Rhizobium etli was introduced into Salmonella typhimurium LT2. Anaerobic fermentations of S. typhimurium with and without PYC were compared with glucose as a carbon source. The presence of PYC increased the succinate yield from glucose from 0.044 g g^–1 to 0.22 g g^–1, while the lactate yield decreased from 0.31 g g^–1 to 0.16 g g^–1. Metabolic flux calculations during the early growth phase indicate that under these growth conditions in the presence of PYC more carbon flows to oxaloacetate via pyruvate carboxylase than via phosphoenolpyruvate carboxylase. Also, under these growth and induction conditions, the presence of PYC diminished the cell growth rate from 0.34 h^–1 to 0.28 h^–1, the specific rate of ATP formation from 45 mmol l^–1 h^–1 to 27 mmol l^–1 h^–1, and the specific rate of glucose consumption from 17 mmol l^–1 h^–1 to 10 mmol l^–1 h^–1.