Enhanced biodegradation of methylhydrazine and hydrazine contaminated NASA wastewater in fixed-film bioreactor

The aerobic biodegradation of National Aeronautics and Space Administration (NASA) wastewater that contains mixtures of highly concentrated methylhydrazine/hydrazine, citric acid and their reaction product was studied on a laboratory-scale fixed film trickle-bed reactor. The degrading organisms, Achromobacter sp., Rhodococcus B30 and Rhodococcus J10, were immobilized on coarse sand grains used as support-media in the columns. Under continuous flow operation, Rhodococcus sp. degraded the methylhydrazine content of the wastewater from a concentration of 10 to 2.5 mg/mL within 12 days and the hydrazine from 0.8 to 0.1 mg/mL in 7 days. The Achromobacter sp. was equally efficient in degrading the organics present in the wastewater, reducing the concentration of the methylhydrazine from 10 to 5 mg/mL within 12 days and that of the hydrazine from 0.8 to 0.2 mg/mL in 7 days. The pseudo first-order rate constants of 0.137 day^-1 and 0.232 day^-1 were obtained for the removal of methylhydrazine and hydrazine, respectively, in wastewater in the reactor column. In the batch cultures, rate constants for the degradation were 0.046 and 0.079 day^-1 for methylhydrazine and hydrazine respectively. These results demonstrate that the continuous flow bioreactor afford greater degradation efficiencies than those obtained when the wastewater was incubated with the microbes in growth-limited batch experiments. They also show that wastewater containing hydrazine is more amenable to microbial degradation than one that is predominant in methylhydrazine, in spite of the longer lag period observed for hydrazine containing wastewater. The influence of substrate concentration and recycle rate on the degradation efficiency is reported. The major advantages of the trickle-bed reactor over the batch system include very high substrate volumetric rate of turnover, higher rates of degradation and tolerance of the 100% concentrated NASA wastewater. The results of the present laboratory scale study will be of great importance in the design and operation of an industrial immobilized biofilm reactor for the treatment of methylhydrazine and hydrazine contaminated NASA wastewater.     

Degradation and O-methylation of chlorinated phenolic compounds by Rhodococcus and Mycobacterium strains

Three polychlorophenol-degrading Rhodococcus and Mycobacterium strains were isolated independently from soil contaminated with chlorophenol wood preservative and from sludge of a wastewater treatment facility of a kraft pulp bleaching plant. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, isolated from tetrachloroguaiacol enrichment, and Rhodococcus sp. strain CP-2, isolated from pentachlorophenol enrichment, mineralized pentachlorophenol and degraded several other polychlorinated phenols, guaiacols (2-methoxyphenols), and syringols (2,6-dimethoxyphenols) at micromolar concentrations and were sensitive to the toxic effects of pentachlorophenol. All three strains initiated degradation of the chlorophenols by para-hydroxylation, producing chlorinated para-hydroquinones, which were then further degraded. Parallel to degradation, strains CG-1, CG-2, and CP-2 also O-methylated nearly all chlorinated phenols, guaiacols, syringols, and hydroquinones. O-methylation of chlorophenols was a slow reaction compared with degradation. The preferred substrates of the O-methylating enzyme(s) were those with the hydroxyl group flanked by two chlorine substituents. O-methylation was constitutively expressed, whereas degradation of chlorinated phenolic compounds was inducible.     

Degradation and O-methylation of chlorinated phenolic compounds by Rhodococcus and Mycobacterium strains.

Three polychlorophenol-degrading Rhodococcus and Mycobacterium strains were isolated independently from soil contaminated with chlorophenol wood preservative and from sludge of a wastewater treatment facility of a kraft pulp bleaching plant. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, isolated from tetrachloroguaiacol enrichment, and Rhodococcus sp. strain CP-2, isolated from pentachlorophenol enrichment, mineralized pentachlorophenol and degraded several other polychlorinated phenols, guaiacols (2-methoxyphenols), and syringols (2,6-dimethoxyphenols) at micromolar concentrations and were sensitive to the toxic effects of pentachlorophenol. All three strains initiated degradation of the chlorophenols by para-hydroxylation, producing chlorinated para-hydroquinones, which were then further degraded. Parallel to degradation, strains CG-1, CG-2, and CP-2 also O-methylated nearly all chlorinated phenols, guaiacols, syringols, and hydroquinones. O-methylation of chlorophenols was a slow reaction compared with degradation. The preferred substrates of the O-methylating enzyme(s) were those with the hydroxyl group flanked by two chlorine substituents. O-methylation was constitutively expressed, whereas degradation of chlorinated phenolic compounds was inducible.     

Metabolism of anthracene by a Rhodococcus species

A Rhodococcus sp. isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons. The Rhodococcus sp. was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene. In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways. One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria. The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product. This represents a novel metabolic pathway only identified in Gram-positive bacteria.     

Production of citric acid using its extraction wastewater treated by anaerobic digestion and ion exchange in an integrated citric acid-methane fermentation process

In order to solve the problem of extraction wastewater pollution in citric acid industry, an integrated citric acid-methane fermentation process is proposed in this study. Extraction wastewater was treated by mesophilic anaerobic digestion and then used to make mash for the next batch of citric acid fermentation. The recycling process was done for seven batches. Citric acid production (82.4 g/L on average) decreased by 34.1?% in the recycling batches (2nd–7th) compared with the first batch. And the residual reducing sugar exceeded 40 g/L on average in the recycling batches. Pigment substances, acetic acid, ammonium, and metal ions in anaerobic digestion effluent (ADE) were considered to be the inhibitors, and their effects on the fermentation were studied. Results indicated that ammonium, Na⁺ and K⁺ in the ADE significantly inhibited citric acid fermentation. Therefore, the ADE was treated by acidic cation exchange resin prior to reuse to make mash for citric acid fermentation. The recycling process was performed for ten batches, and citric acid productions in the recycling batches were 126.6 g/L on average, increasing by 1.7 % compared with the first batch. This process could eliminate extraction wastewater discharge and reduce water resource consumption.     

Alkane utilization by Rhodococcus strain NTU-1 alone and in its natural association with Bacillus fusiformis L-1 and Ochrobactrum sp

Linear (n-hexadecane) and branched (pristane) alkanes were degraded by a mixed culture isolated from an oil-contaminated field. The degradation was accompanied by formation of biofloccules. The culture was composed of Rhodococcus strain NTU-1, Bacillus fusiformis L-1, and Ochrobactrum sp. Rhodococcus strain NTU-1 carried out the degradation of the alkane via a hydroxylase. Bacillus fusiformis L-1 and Ochrobactrum sp. did not degrade the alkanes but aided the flocculation by forming more rigid bacterial aggregates that enhanced the trapping of alkanes. In batch cultures, transformation and removal of the linear and branched alkanes was achieved within 66 h with more than 95% efficiency.     

Microbial biodegradation of 4-chlorobiphenyl, a model compound of chlorinated biphenyls

The biodegradation products of 4-chlorobiphenyl were analyzed in an Achromobacter sp. strain and a Bacillus brevis strain. Both strains generated the same metabolites, with 4-chlorobenzoic acid as the major metabolic product. Our results corroborate previous observations whereby most bacterial strains degrade the chlorobiphenyls via a major pathway which proceeds by an hydroxylation in position 2,3 and a meta-1,2 fission. However, we also detected several metabolites whose structure suggests the existence of other routes for the degradation of chlorinated biphenyls.     

Microbial biodegradation of 4-chlorobiphenyl, a model compound of chlorinated biphenyls.

The biodegradation products of 4-chlorobiphenyl were analyzed in an Achromobacter sp. strain and a Bacillus brevis strain. Both strains generated the same metabolites, with 4-chlorobenzoic acid as the major metabolic product. Our results corroborate previous observations whereby most bacterial strains degrade the chlorobiphenyls via a major pathway which proceeds by an hydroxylation in position 2,3 and a meta-1,2 fission. However, we also detected several metabolites whose structure suggests the existence of other routes for the degradation of chlorinated biphenyls.     

Removal of volatile fatty acids with immobilized Rhodococcus sp. B261

The removal of aqueous volatile fatty acids (VFA) in wastewater and spoiled waste-foods by immobilized Rhodococcus sp. B261 was investigated. The n-valeric acid (0.5%) was completely removed within 25 h under the following conditions; solution pH, 8.0; air flow rate, 0.2 l/min; superficial velocity, 0.96 h(-1); temperature, 37 degrees C. Under the optimized conditions, the acetic (8525 ppm), propionic (7310 ppm) and n-butyric (4360 ppm) except n-valeric (2572 ppm) acids from the wastewater were completely removed by immobilized Rhodococcus sp. B261 in 24 h. The acetic (7810 ppm), propionic (8942 ppm) and butyric (5730 ppm) acids from the solution of spoiled waste-foods were effectively removed by immobilized Rhodococcus sp. B261 from 48 h within 60 h but n-valeric acid (3625 ppm) took 72 h.     

Biodegradation of di-n-butyl phthalate by Rhodococcus sp. JDC-11 and molecular detection of 3, 4-phthalate dioxygenase gene

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.     

High efficiency degradation of tetrahydrofuran (THF) using a membrane bioreactor: identification of THF-degrading cultures of<Emphasis Type="Italic"> Pseudonocardia</Emphasis> sp. strain M1 and<Emphasis Type="Italic"> Rhodococcus ruber</Emphasis> isolate M2

A mixed microbial culture capable of growing aerobically on tetrahydrofuran (THF) as a sole carbon and energy source was used as the inoculum in a 10 l working volume membrane bioreactor. Following start-up, the reactor was operated in batch mode for 24 h and then switched to continuous feed with 100% biomass recycle. On average, greater than 96% of THF fed to the reactor was removed during the 8-month study. THF loading rates ranged from 0.62 to 9.07 g l^–1 day^–1 with a hydraulic retention time of 24 h. THF concentrations as high as 800 mg/l were tolerated by the culture. Biomass production averaged 0.28 kg total suspended solids/kg chemical oxygen demand removed, i.e., comparable to a conventional wastewater treatment process. Periodic batch wasting resulted in a solids retention time of 7–14 days. Reactor biomass typically ranged from 4 to 10 g/l volatile suspended solids and the effluent contained no solids. Pure THF-degrading cultures were isolated from the mixed culture based on morphological characteristics, Gram-staining and THF degradation. Based on 16S rDNA analysis the isolates were identified as Pseudonocardia sp. M1 and Rhodococcus ruber M2.     

Comparison of PCR-DGGE and selective plating methods for monitoring the dynamics of a mixed culture population in synthetic brewery wastewater

Enrichment of an activated sludge inoculum in synthetic brewery wastewater, which included glucose, maltose, and ethanol, was conducted in batch experiments to identify the dominant microbes present, to determine methodologies capable of monitoring the mixed culture population dynamics, and to determine the consortium's substrate degradation behavior. These results and methodologies were subsequently used in the determination of the population dynamics of suspended and attached microorganisms in a sequencing batch system in the second part of this research work. The three-membered microbial community comprised two bacterial and one fungal species that were identified as Acinetobacter sp., Enterobacter sp., and Candida sp. PCR-DGGE and plating on selective media were used to track the population dynamics of the consortium during the degradation of different substrates in synthetic wastewater containing glucose, maltose, and ethanol. Enterobacter sp. could degrade glucose and maltose but not ethanol, whereas Acinetobacter and Candida could degrade all three carbon sources. In buffered batch mixed culture experiments, Enterobacter was the predominant bacterium until the sugar concentrations decreased to levels that enabled Acinetobacter and Candida to degrade ethanol. PCR-DGGE was effective for detecting the dominant species, but culture-based methods were more accurate for monitoring the population dynamics of these microorganisms during growth in the wastewater medium.     

Biotransformation of D-methionine into L-methionine in the cascade of four enzymes

D-Methionine was converted to L-methionine in a reaction system where four enzymes were used. D-amino acid oxidase (D-AAO) from Arthrobacter protophormiae was used for the complete conversion of D-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was added to prevent 2-oxo-4-methylthiobutyric acid decarboxylation. In the second reaction step, L-phenylalanine dehydrogenase (L-PheDH) from Rhodococcus sp. was used to convert 2- oxo-4-methylthiobutyric acid to L-methionine, and formate dehydrogenase (FDH) from Candida boidinii was added for NADH regeneration. Enzyme kinetics of all enzymes was analyzed in detail. Mathematical models for separate reactions steps, as well as for the complete system were developed and validated in the batch reactor experiments. Complete conversion of D-methionine to L-methionine was achieved. Considering that both enzymes act on different substrates, such a system could be easily employed for the synthesis of other amino acids from D-isomer, as well as from the racemate of a certain amino acid (DL-amino acid).     

Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1.

The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful.     

Effect of propionic acid on citric acid fermentation in an integrated citric acid–methane fermentation process

In this study, an integrated citric acid-methane fermentation process was established to solve the problem of wastewater treatment in citric acid production. Citric acid wastewater was treated through anaerobic digestion and then the anaerobic digestion effluent (ADE) was further treated and recycled for the next batch citric acid fermentation. This process could eliminate wastewater discharge and reduce water resource consumption. Propionic acid was found in the ADE and its concentration continually increased in recycling. Effect of propionic acid on citric acid fermentation was investigated, and results indicated that influence of propionic acid on citric acid fermentation was contributed to the undissociated form. Citric acid fermentation was inhibited when the concentration of propionic acid was above 2, 4, and 6 mM in initial pH 4.0, 4.5 and, 5.0, respectively. However, low concentration of propionic acid could promote isomaltase activity which converted more isomaltose to available sugar, thereby increasing citric acid production. High concentration of propionic acid could influence the vitality of cell and prolong the lag phase, causing large amount of glucose still remaining in medium at the end of fermentation and decreasing citric acid production.     

Fed-batch production of citric acid by Candida lipolytica grown on n-paraffins

This study reports on the effects of fermentor agitation and fed-batch mode of operation on citric acid production from Candida lipolytica using n-paraffin as the carbon source. An optimum range of agitation speeds in the 800-1000 rpm range corresponding to Reynolds numbers of 50000-63000 (based on initial batch conditions) seemed to give the best balance between substrate utilization for biomass growth and citric acid production. Application of multiple fed-batch feedings can be used to extend the batch fermentation and increase final citric acid concentrations and product yield. The three-cycle fed-batch system increased overall citric acid yields to 0.8-1.0 g citricacid/g n-paraffin, approximately a 100% improvement in product yield from those observed in the single cycle fed-batch system and a 200% improvement over normal batch operation. The three-cycle fed-batch mode of operation also increased the final citric acid concentration to 42 g/l from about 12 and 6g/l for single fed-batch cycle and normal batch modes of operation, respectively. Increased citric acid concentrations in three-cycle fed-batch mode was achieved at longer fermentation times.     

一株乙腈降解菌的鉴定及其生物学特性

【目的】明确乙腈降解菌BX2的分类地位及生物学特性,评价其处理含乙腈废水的可行性。【方法】通过形态特征、生理生化特性以及系统发育分析对菌株BX2进行鉴定。考察温度、初始pH及接种量等因素对菌株生长的影响,确定菌株的最佳生长条件及在该条件下的乙腈降解能力。测定菌株BX2对NaCl的耐受能力。【结果】乙腈降解菌BX2的形态特征及生理生化特性与紫红红球菌(Rhodococcus rhodochrous)最相近。其16S rRNA、gyrB、secA1基因序列与紫红红球菌的相似性分别为99.37%、99.29%、97.87%。最佳生长条件为35℃,初始pH 7.5,接种量1%。此条件下,菌株BX2在16 h内对浓度为800 mg/L乙腈的降解率为95.87%。菌株BX2在NaCl含量高于6%的培养基中无法生长。【结论】菌株BX2被鉴定为紫红红球菌。该菌株有较强的环境适应能力,可降解高浓度乙腈,在含氰废水的生物修复中有很好的应用前景。     

Citric acid production by a novel Aspergillus niger isolate: I. Mutagenesis and cost reduction studies

Ultraviolet-irradiation (UV), ethyl methane sulfonate (EMS) and acridine orange (AO) were used to induce citric acid overproduction mutations in Aspergillus niger UMIP 2564. Among 15, eight of the mutant derivatives, were improved with respect to citric acid production from sucrose in batch cultures. Maximum product yield (60.25%) was recorded by W5, a stable UV mutant, with approximately 3.2-fold increase when compared to the parental wild type strain. In terms of the kinetic parameters for batch fermentation processes, the mutation doubled the specific substrate uptake rate and achieved 4.5- and 7.5-fold improvements in citric acid productivity and specific productivity, respectively. For reduction of the fermentation medium cost, corn steep liquor and calcium phosphate pre-treated beet molasses were successfully used as substituents of nitrogen and carbon sources in the growth medium, respectively. These medium substitutions resulted in a W5 citric acid fermentation culture with a product yield of 74.56%.     

mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.     

The use of neural networks to aid in microorganism identification: a case study ofHaemophilus species identification

The adipamidase of a mutant strainBrevibacterium sp. R312 involved in the degradation of adiponitrile to adipic acid was purified. Its N-terminal amino acid sequence was shown to be identical toBrevibacterium sp. R312 enantio-selective amidase andRhodococcus sp. N-774 amidase.