Biodegradation of Isopropanol by a Solvent-Tolerant Paracoccus denitrificans Strain

The biodegradation of high concentration isopropanol (2-propanol, IPA) at 16 g/L was investigated by a solvent-tolerant strain of bacteria identified as Paracoccus denitrificans for the first time by 16S rDNA gene sequencing. The strain P. denitrificans GH3 was able to utilize the high concentration of IPA as the sole carbon source within a minimal salts medium with a cell density of 1.5 × 10⁸ cells/mL. The optimal conditions were found as follows: initial pH 7.0, incubation temperature 30°C, with IPA concentration 8 g/L. Under the optimal conditions, strain GH3 utilized 90.3% of IPA in 7 days. Acetone, the major intermediate of aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Both IPA and acetone were completely removed from the medium following 216 hr and 240 hr, respectively. The growth of strain GH3 on IPA as a sole carbon and energy source was well described by the Andrews model with a maximum growth rate (μ _max ) = 0.0277/hr, a saturation constant (K _S ) = 0.7333 g/L, and an inhibition concentration (Ki) = 8.9887 g/L. Paracoccus denitrificans GH3 is considered to be well used in degrading IPA in wastewater.     

Fed batch bioconversion of 2-propanol by a solvent tolerant strain of <Emphasis Type="Italic">Alcaligenes </Emphasis><Emphasis Type="Italic">faecalis</Emphasis> entrapped in Ca-alginate gel

A gram-negative, rod-shaped, aerobe, capable of converting 2-propanol (isopropanol, IPA) to acetone was isolated from an oil/sump, and identified by 16 S rDNA analysis as Alcaligenes faecalis. Investigations showed this strain to be extremely solvent-tolerant and it was subsequently named ST1. In this study, A. faecalis ST1 cells were immobilized by entrapment in Ca-alginate beads (3 mm in diameter), and used in the bioconversion of high concentration IPA. The biodegradation rates and the corresponding microbial growth inside the beads were measured at four different IPA concentration ranges from 2 to 15 g l(-1). The maximum cell concentration obtained was 9.59 g dry cell weight (DCW) l(-1) medium which equated to 66 g DCW l(-1) gel, at an initial IPA concentration of 15 g l(-1) after 216 h of incubation. A maximum biodegradation rate of 0.067 g IPA g cells(-1) h(-1) was achieved for 5 g l(-1) IPA where an increase in IPA concentration to 38 g l(-1) caused reduction in bead integrity. A modified growth medium was developed which allowed repeated use of the beads for more than 42 days without any loss of integrity and continued bioconversion activity.     

Biodegradation of high-concentration isopropanol by a solvent-tolerant thermophile,Bacillus pallidus

The aerobic biodegradation of high-concentration, to 24 g l(-1), 2-propanol (IPA) by a thermophilic isolate ST3, identified as Bacillus pallidus, was successfully carried out for the first time. This solvent-tolerant B. pallidus utilized IPA as the sole carbon source within a minimal salts medium. Cultivation was carried out in 100-ml shake flasks at 60 degrees C and compared with cultivation within a 1-l stirred tank reactor (STR). Specific growth rate (micro) was about 0.2 h(-1) for both systems, with a maximum cell density of 2.4 x 10(8) cells ml(-1) obtained with STR cultivation. During exponential growth and stationary phase, IPA biodegradation rates were found to be 0.14 and 0.02 g l(-1) h(-1), respectively, in shake-flask experiments, whereas corresponding values of 0.09 and 0.018 g l(-1) h(-1) were achievable in the STR. Generation of acetone, the major intermediate in aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Acetone levels reached a maximum of 2.2-2.3 g l(-1) after 72 and 58 h for 100-ml and 1-l systems, respectively. Both IPA and acetone were completely removed from the medium following 160 and 175 h, respectively, during STR growth, although this was not demonstrated within shake-flask reactions. Growth of B. pallidus on acetone or IPA alone demonstrated that the maximum growth rate ( micro ) obtainable was 0.247 h(-1) at 4 g l(-1) acetone and 0.202 h(-1) at 8 g l(-1) IPA within shake-flask cultivation. These results indicate the potential of the solvent-tolerant thermophile B. pallidus ST3 in the bioremediation of hot solvent-containing industrial waste streams.     

温度对谷胱甘肽分批发酵的影响及动力学模型

研究了24～32℃范围内产朊假丝酵母生产谷胱甘肽的分批发酵过程,发现较高温度对细胞生长有促进作用,而较低温度则更有利于谷胱甘肽产量的提高。应用改进的Logistic和LuedekingPiret方程分别对细胞生长动力学和谷胱甘肽合成动力学进行了模拟,得到不同温度下各种动力学参数。在此基础上,进一步研究了温度同细胞生长动力学参数之间的内在联系,得到谷胱甘肽分批发酵过程中细胞浓度的变化同温度以及底物浓度之间的一般关系式：dX-dt＝［0.0224(T+1.7)］2X(1-X/X_max)1+S｛8.26×10.6×exp［-31477/R/(T+273)］｝。验证实验结果表明,该模型具有很好的适用性。     

Growth kinetics of Pseudomonas putida in cometabolism of phenol and 4-chlorophenol in the presence of a conventional carbon source

Growth kinetics of Pseudomonas putida (ATCC 49451) in cometabolism of phenol and 4-chlorophenol (4-cp) in the presence of sodium glutamate (SG) were studied. In the ternary substrate mixture, phenol and SG are growth substrates while 4-cp is a nongrowth substrate. Cell growth on phenol was found to follow Andrews kinetics and cells displayed substrate inhibition pattern on sodium glutamate in the range of 0-4 g L(-1) as well. A cell growth model for the ternary substrate system was established based on a simplified cell growth mechanism and subsequently modified by experimental results. Model analysis over a wide range of substrate concentrations shows that the inhibition of SG is much larger than phenol at low phenol concentrations (/=600 mg L(-1)). The nongrowth substrate, 4-cp, inhibits cell growth mainly through inactivation of cells (cell decay) and competitive inhibition to cell growth on phenol. In the absence of SG, 4-cp retards cell growth severely and cells cannot grow at 250 mg L(-1) 4-cp. Addition of sodium glutamate, however, greatly attenuates the toxicity of 4-cp and supports cell growth at 4-cp concentration higher than 250 mg L(-1). By using the proposed cell growth model, we were able to optimize the amount of SG needed to enhance cell growth rate and validate model predictions against experimental data.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

Growth characteristics of bakers' yeast in ethanol

The influence of temperature (15 degrees -40 degrees C) and pH (2.5-6.0) on the continuous growth of bakers' yeast (Saccharomyces cerevisiae) at steady state in 1% ethanol was investigated. Optimal temperature and pH were 30 degrees C and 4.5, respectively. The short-term effect of ethanol concentration (0.1-10.0%) on the yeast growth was assessed in batch culture. Up to 1% of ethanol, the yeast growth increased in function of the ethanol concentration in the medium. The biomass reached a maximum within the interval of 1-4% of ethanol (7.9 and 31.6 g/L, respectively) and decreased at higher concentrations. The residual ethanol concentration in the medium increased rapidly when the initial ethanol concentration exceeded 4%. The best-fit model obtained for growth inhibition as a function of ethanol concentrations was that of Tseng and Wayman: mu(m)S/)K + S( - i (S - S(theta)). With this model, the specific growth rate (mu) decreased linearly as the ethanol concentration increased between the threshold value (S(theta)) of 11.26 g/L to be fully inhibited at 70.00 g/L (S;) an inhibition constant (i) of 0.0048 g L(-1) h(-1), a maximum specific growth rate (mu(m)) of 0.284 h(-1), and a saturation constant (K) of 0.611 g/L were obtained.     

The potential for establishment of axial temperature profiles during solid-state fermentation in rotating drum bioreactors

The mixing and heat transfer phenomena within rotating drum bioreactors (RDBs) used for solid-state fermentation processes are poorly studied. The potential for the establishment of axial temperature gradients within the substrate bed was explored using a heat transfer model. For growth of Aspergillus oryzae on wheat bran within a 24 L RDB with air at a superficial velocity of 0.0023 m s(-1) and 15% relative humidity, the model predicts an axial gradient between the air inlet and outlet of 2 degrees C during rapid growth, compared to experimental axial temperature gradients of between 1 and 4 degrees C. Undesirably high temperatures occur throughout the bed under these operating conditions, but the model predicts that good temperature control can be achieved using humid air (90% relative humidity) at superficial velocities of 1 m s(-1) for a 204 L RDB. For a 2200 L RDB, good temperature control is predicted with superficial velocities as low as 0.4 m s(-1) with the airflow being switched from 90% to 15% relative humidity whenever the temperature at the outlet end of the drum exceeds the optimal temperature for growth. This work suggests that significant axial temperature gradients can arise in those RDBs that lack provision for axial mixing. It is therefore advisable to use angled lifters within RDBs to promote axial mixing.     

High cell density cultivation of Pseudomonas oleovorans: growth and production of poly (3-hydroxyalkanoates) in two-liquid phase batch and fed-batch systems

Pseudomonas oleovorans is able to accumulate poly(3-hydroxyalkanoates) (PHAs) under conditions of excess n-alkanes, which serve as sole energy and carbon source, and limitation of an essential nutrient such as ammonium. In this study we aimed at an efficient production of these PHAs by growing P. oleovorans to high cell densities in fed-batch cultures.To examine the efficiency of our reactor system, P. oleovorans was first grown in batch cultures using n-octane as growth substrate and ammonia water for pH regulation to prevent ammonium limiting conditions. When cell growth ceased due to oxygen limiting conditions, a maximum cell density of 27 g .L(-1) dry weight was obtained. When the growth temperature was decreased from the optimal temperature of 30 degrees -18 degrees C, cell growth continued to a final cell density of 35 g . L(-1) due to a lower oxygen demand of the cells at this lower incubation temperature.To quantify mass transfer rates in our reactor system, the volumetric oxygen transfer coefficient (k(L)a) was determined during growth of P. oleovorans on n-octane. Since the stirrer speed and airflow were increased during growth of the organism, the k(L)a also increased, reaching a constant value of 0.49 s(-1) at maximum airflow and stirrer speed of 2 L . min(-1) and 2500 rpm, respectively. This k(L)a value suggests that oxygen transfer is very efficient in our stirred tank reactor.Using these conditions of high oxygen transfer rates, PHA production by P. oleovorans in fed-batch cultures was studied. The cells were first grown batchwise to a density of 6 g . L(-1), after which a nutrient feed, consisting of (NH(4))(2)SO(4) and MgSO(4), was started. The limiting nutrient ammonium was added at a constant rate of 0.23 g NH(4) (+) per hour, and when after 38 h the feed was stopped, a biomass concentration of 37.1 g . L(-1) was obtained. The Cellular PHA content was 33% (w/w), which is equal to a final PHA yield of 12.1 g . L(-1) and an overall PHA productivity of 0.25 g PHA produced per liter medium per hour. (c) 1993 John Wiley & Sons, Inc.     

Biokinetic evaluation and modeling of continuous thiocyanate biodegradation by Klebsiella sp

Biokinetics for autotrophic degradation of thiocyanate using batch culture of Klebsiella sp. were evaluated both analytically and numerically. A sequential approach with an analytical method followed by a numerical approximation was used to evaluate and to ensure the accuracy of the parameter estimation. The nonlinear least-squares method with a 95% confidence interval was employed. The growth conditions were maintained at pH 7 and 38 degrees C for all experiments. With an automated incubation and turbidity reader, a total of 16 different initial thiocyanate concentrations, ranging from 10 to 300 mg L(-1), were used to develop a kinetic expression of specific growth rate as a function of substrate concentration. The biodegradation of thiocyanate with Klebsiella sp. followed a substrate inhibition pattern. Three identical automated bioreactors with working volumes of 1.5 L, equipped with sterilizable sampling ports, were also used for the numerical approximation of the biokinetic parameters in batch mode. A fourth order Runge-Kutta method was used to approximate the substrate inhibition kinetics of the Klebsiella sp. utilizing thiocyanate. Although the kinetic coefficients estimated by analytical and numerical methods were not statistically different at a 0.05 alpha level, model responses of numerical approximation generated a better prediction of changes in thiocyanate and cell mass concentrations. The hypothetical maximum growth rate, micro m, half saturation coefficient, Ks, microbial yield coefficient, Y, cell mass decay rate coefficient, kd, and substrate inhibition coefficient, Ksi, were evaluated as being 0.62 +/- 0.05 d(-1), 85 +/- 8 mg SCN- L(-1), 0.076 +/- 0.011 mg cell mass (mg SCN)(-1), 0.03 +/- 0.002 d(-1), and 131 +/- 22 mg SCN- L(-1), respectively. The calculated maximal substrate concentration, Sm, and apparent maximum specific growth rate, micro'm, were 105.5 +/- 8.7 mg SCN- L(-1) and 0.24 +/- 0.01 d(-1), respectively. Using these estimated parameters, the theoretical performance of the continuous operation was also illustrated, which depicts the residual thiocyanate and Klebsiella sp. concentrations in the non-steady and steady states at different hydraulic retention times (HRTs). Assuming the influent concentration of 250 mg SCN- L(-1), the expected treatment efficiency ranged from 94.9% to 69.4% between 20 and 5 days HRT, respectively. Klebsiella sp. was expected to be washed out at 4.8 days HRT, thus resulting in no treatment of thiocyanate.     

Influence of temperature on growth rate and competition between two psychrotolerant Antarctic bacteria: low temperature diminishes affinity for substrate uptake.

The growth kinetics of two psychrotolerant Antarctic bacteria, Hydrogenophaga pseudoflava CR3/2/10 (2/10) and Brevibacterium sp. strain CR3/1/15 (1/15), were examined over a range of temperatures in both batch culture and glycerol-limited chemostat cultures. The maximum specific growth rate (mu max) and Ks values for both bacteria were functions of temperature, although the cell yields were relatively constant with respect to temperature. The mu max values of both strains increased up to an optimum temperature, 24 degrees C for 2/10 and 20 degrees C for 1/15. Strain 1/15 might therefore be considered to be more psychrophilic than strain 2/10. For both bacteria, the specific affinity (mu max/Ks) for glycerol uptake was lower at 2 than at 16 degrees C, indicating a greater tendency to substrate limitation at low temperature. As the temperature increased from 2 to 16 degrees C, the specific affinity of 1/15 for glycerol increased more rapidly than it did for 2/10. Thus 1/15, on the basis of this criterion, was less psychrophilic than was 2/10. The steady-state growth kinetics of the two strains at 2 and 16 degrees C imply that 1/15 would be able to outgrow 2/10 only at relatively low substrate concentrations (< 0.32 g of glycerol.liter-1) and high temperatures (> 12 degrees C), which suggests that 1/15 has a less psychrotolerant survival strategy than does 2/10. Our data were compared with other data in the literature for bacteria growing at low temperatures. They also showed an increase of substrate-specific affinity with increasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic study and mathematical modeling of methanogenesis of acetate using pure cultures of methanogens

Kinetics of methanogenesis from acetate was studied using pure cultures of Methanosarcina barkeri and Methanosarcina mazei. Methane formation was found to be associated with cell growth. Nearly equimolar methane was produced from acetate during the methanogenic growth, and about 1.94 g of cells were formed from each mole of acetate consumed. Cell growth can be estimated from methane production. Significant substrate inhibition was found when acetate concentration was higher than 0.12 M. Among the three methanogenic strains studied, M. mazei strain S6 had the highest specific growth rate at all acetate concentrations studied and was least sensitive to environmental factors investigated (e.g., acetate concentration). The maximum specific growth rate found for strain S6 was 0.022 hr(-1) at acetic acid concentration around 7 g/L. The other two strains studied were M. barkeri strain 227 and strain MS. Growth of M. barkeri was completely inhibited at sodium acetate concentrations higher than 0.24 M. The maximum specific growth rate found for strains 227 and MS was 0.019 and 0.021 h(-1) at acetic acid concentrations of 3.6 and 6.8 g/L, respectively. A kinetic model with substrate inhibition was developed and can be used to simulate the methane formation from M. mazei strain S6 grown on acetate at 35 degrees C, pH 7.     

Effects of pH and acetic acid on homoacetic fermentation of lactate by Clostridium formicoaceticum

Clostridium formicoaceticum homofermentatively converts lactate to acetate at 37 degrees C and pH 6.6-9.6. However, this fermentation is strongly inhibited by acetic acid at acidic pH. The specific growth rate of this organism decreased from a maximum at pH 7.6 to zero at pH 6.6. This inhibition effect was found to be attributed to both H(+) and undissociated acetic acid. At pH values below 7.6, the H(+) inhibited the fermentation following non-competitive inhibition kinetics. The acetic acid inhibition was found to be stronger at a lower medium pH. At pH 6.45-6.8, cell growth was found to be primarily limited by a maximum undissociated acetic acid concentration of 0.358 g/L (6mM). This indicates that the undissociated acid, not the dissociated acid, is the major acid inhibitor. At pH 7.6 or higher, this organism could tolerate acetate concentrations of higher than 0.8M, but salt (Na(+)) became a strong inhibitor at concentrations of higher than 0.4M. Acetic acid inhibition also can be represented by noncompetitive inhibition kinetics. A mathematical model for this homoacetic fermentation was also developed. This model can be used to simulate batch fermentation at any pH between 6.9 and 7.6.     

SARS冠状病毒主蛋白酶抑制剂的筛选及抑制动力学研究

对SARS冠状病毒主蛋白酶(SARS-CoV M^pro)进行异源重组表达与提纯,并以其为靶点,利用基于荧光共振能量转移(FRET)技术的体外药物筛选模型,对蛋白酶抑制剂聚焦库96种化合物进行了体外抑制活性的评价,并从动力学的角度探讨筛选出的阳性化合物对SARS-CoV M^pro的抑制能力与机制。结果表明:通过筛选获得抑制率>80%、淬灭率<20%的化合物5种,为P-1-08、P-1-19、P-2-24、P-2-28、P-2-54,其半数有效抑制浓度(IC₅₀)分别为:0.69±0.05μmol/L、1.19±0.41μmol/L、0.14±0.01μmol/L、1.36±0.07μmol/L、0.36±0.03μmol/L。其中化合物P-1-08、P-1-19、P-2-24、P-2-54对SARS冠状病毒主蛋白酶的抑制作用为不可逆抑制,化合物P-2-28的抑制作用为可逆抑制。根据Lineweaver-Burk图和Dixon图的研究,发现化合物P-2-28对SARS冠状病毒主蛋白酶呈竞争性抑制,抑制常数Ki为0.81μmol/L。通过对底物浓度,IC₅₀值及Ki值关系的研究,进一步验证了P-2-28的抑制作用为竞争性抑制。该抑制剂的发现为SARS冠状病毒主蛋白酶抑制剂的研究打下基础,为抗SARS病毒药物开发提供了先导化合物。     

Mild heat treatment of lettuce enhances growth of Listeria monocytogenes during subsequent storage at 5 degrees C or 15 degrees C

AIMS: The objective of this study was to determine the influence of mild heat treatment, storage temperature and storage time on the survival and growth of Listeria monocytogenes inoculated onto cut iceberg lettuce leaves. METHODS AND RESULTS: Before or after inoculation with L. monocytogenes, cut iceberg lettuce leaves were dipped in water (20 or 50 degrees C) containing or not 20 mg l(-1) chlorine, for 90 s, then stored at 5 degrees C for up to 18 days or 15 degrees C for up to 7 days. The presence of 20 mg l(-1) chlorine in the treatment water did not significantly (alpha=0.05) affect populations of the pathogen, regardless of other test parameters. The population of L. monocytogenes on lettuce treated at 50 degrees C steadily increased throughout storage at 5 degrees C for up to 18 days. At day 10 and thereafter, populations were 1.7-2.3 log10 cfu g(-1) higher on lettuce treated at 50 degrees C after inoculation compared with untreated lettuce or lettuce treated at 20 degrees C, regardless of chlorine treatment. The population of L. monocytogenes increased rapidly on lettuce stored at 15 degrees C. At 2 and 4 days, significantly higher populations were detected on lettuce that had been treated at 50 degrees C, compared with respective samples that had been treated at 20 degrees C, regardless of inoculation before or after treatment, or the presence of 20 mg l(-1) chlorine in the treatment water. CONCLUSIONS: The results clearly demonstrated that mild heat treatment of cut lettuce leaves enhances the growth of L. monocytogenes during subsequent storage at 5 or 15 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Mild heat treatment of cut lettuce may result in a prolonged shelf life as a result of delaying the development of brown discoloration. However, heat treatment also facilitates the growth of L. monocytogenes during storage at refrigeration temperature, thereby increasing the potential risk of causing listeriosis.     

Multiple substrate growth kinetics of Leptothrix discophora SP-6

The growth parameters of Leptothrix discophora SP-6 were quantified on the basis of the steady-state concentrations and utilization rates of pyruvate, dissolved oxygen, and concentration of microorganisms in a chemostat operated at 25 degrees C, pH 7.2, and an agitation rate of 350 rpm. The results showed that the microbial growth was limited by both pyruvate and dissolved oxygen. A combined growth kinetics model using Monod growth kinetics for pyruvate and Tessier growth kinetics for oxygen showed the best correlation with the experimental data when analyzed using an interactive multiple substrate model. The growth kinetics parameters and the respective confidence limits, estimated using the Monte Carlo simulation, were mu(max) = 0.576 +/- 0.021 h(-1), K(sMp) = 38.81 +/- 4.24 mg L(-1), K(sTo) = 0.39 +/- 0.04 mg L(-1), Y(X/p) = 0.150 (mg microorganism mg(-1) pyruvate), Y(X/o) = 1.24 (mg microorganism mg(-1) oxygen), the maintenance factors for pyruvate and oxygen were m(p) = 0.129 (mg pyruvate consumed mg(-1) microorganism h(-1)) and m(o) = 0.076 (mg oxygen consumed mg(-1) microorganism h(-1)), respectively.     

The influence of temperature upon the hydrolysis of cellobiose by beta-1,4-glucosidases from Aspergillus niger

We have made experimental studies into the enzymatic hydrolysis of cellobiose within the temperature range of 40 degrees C to 70 degrees C at pH 4.9, by using beta-1,4-glucosidase from Aspergillus niger. At 70 degrees C there was significant enzyme deactivation, which could be fitted to a potential deactivation model with values of n equal to 1.09 and k(d) to 0.1564 (g/l)(-0.09) min(-1), whereas the rate of hydrolysis could be fitted to the Michaelis-Menten equation. Between 40 degrees C and 60 degrees C we noted a substrate inhibition and that the CEC compound formed contributed to glucose production. The apparent activation energies had values of 4.66, 8.45, 4.82, and 3.99 kJ/mol for the kinetic constants k(a) and k(a2) the Michaelis constant and the substrate inhibition constant, respectively.     

Influence of some physicochemical parameters on bacterial activity of biofilm: Ferrous iron oxidation by Thiobacillus ferrooxidans

The influence of temperature, pH, and substrate and product concentrations on the oxidation rate of ferrous iron by biofilm of Thiobacillus ferrooxidans was determined. The experiments were performed in an inverse fluidized-bed biofilm reactor in which the biofilm thickness was kept constant at 80 mum. Oxygen concentration and diffusion through the biofilm did not limit the oxidation rate. The oxidation rate was almost unaffected by temperature between 13 and 38 degrees C, pH between 1.3 and 2.2, ferric iron concentration up to 14 g/L, or ferrous iron concentration from 4 to 13 g/L. The kinetics of the process was described by the Monod equation with respect to the mass of the biofilm and with ferrous ions as the limiting substrate.