New application of single and mixed immobilized cells for furfural biodegradation

In this study, a new application of immobilized microbial cells for biodegradation of furfural in aqueous solution was investigated using spouted bed bioreactor. Pseudomonas sp., as a single type specie as well as activated sludge as mixed cultures were individually immobilized in 3 different bio-carrier matrices which were prepared by reinforcement of natural polysaccharides including sodium alginate, guar-gum and agar-agar with polyvinyl alcohol. The results demonstrated a complete removal (100%) of furfural from aqueous solutions using immobilized cells (IC) of Pseudomonas sp., and mixed cultures as well. Recycling of used IC for furfural removal in successive treatment cycles provided significant removal rates up to 96%. In general, results revealed that IC exhibited better performance compared to free cells in regard with the removal rate of furfural, duration of biodegradation process, as well as the ability for recycling and sustaining the high concentrations of furfural.     

Enrichment, isolation and characterization of dialkyl sulfosuccinate degrading bacteriaComamonas terrigena N3H andComamonas terrigena N1C

Microorganisms derived from soil, sludge, sewage or river water contaminated by detergents or mineral oils were enriched and tested for the capability of primary biodegradation of dialkyl sulfosuccinates (DASS). Adapted mixed cultures, containing predominantly Gram-negative bacteria, degraded di-n-hexyl sulfosuccinate (DHSS) more readily than di-n-octyl sulfosuccinate (DOSS). Bacterial isolates obtained from enriched mixed cultures that show the highest rates of primary biodegradation and assimilation of DASS belong to the genusComamonas. Under nongrowth conditions, the DHSS primary biodegradation by these strains follows the 1st order reaction kinetics. The rate constants of primary degradation of DHSS by resting cells ofC. terrigena N3H were found to be 30 times higher than when catalyzed byC. terrigena N1C. The capacity for accelerated removal of surface-active properties of DHSS byC. terrigena N3H was retained within the range of pH 4–10 and at temperatures up to 55°C, while DHSS degradation byC. terrigena N1C was completely inhibited above 48°C. Temperature and pH limits for DHSS primary degradation by both strains were to a great extent wider than those for the viability of the cultures. Biodegradation capacity and metabolic stability ofC. terrigena N3H make this strain interesting from both scientific and technological point of view.     

Biodegradation of Petroleum Tar by Pseudomonas Spp. From Oil Field of Assam,India

ABSTRACT

Petroleum tar produced during the processing of crude oil is one of the earth's major pollutants. The potential of certain soil bacteria in the biodegradation of petroleum tar was assessed to develop an active indigenous bacterial consortium for bioremediation of petroleum tar–polluted sites of Assam, India. In vitro enrichment cultures of five Pseudomonas spp. were found to metabolize petroleum tar. The Fourier transform infrared (FTIR) analyses of the enrichment cultures revealed the presence of the functional groups, viz., –OH, –CHO, C?O, and –COOH, which provided evidence for the biodegradation of petroleum tar. Further, gas chromatography–flame ionization detection (GC-FID) analyses revealed complete degradation of low-molecular-weight hydrocarbons, and the subsequent appearance of some additional peaks reflected the formation of intermediate metabolites during the degradation of petroleum tar. A mixed culture with 0.1% Tween 80 as a surfactant exhibited almost complete degradation in contrast to the degradation by the mixed culture without Tween 80. This confirmed the effect of a surfactant for acceleration of the biodegradation process of petroleum tar.     

Cometabolic degradation of chloroallyl alcohols in batch and continuous cultures

The biodegradation of chloroallyl alcohols by pure and mixed bacterial cultures was investigated. Only 2-chloroallyl alcohol and cis- and trans-3-chloroallyl alcohol served as growth substrate for pure cultures. The other chloroallyl alcohols could be cometabolically degraded during growth on 2-chloroallyl alcohol. Cometabolic degradation of trichloroallyl alcohol, which was the most recalcitrant congener, by a Pseudomonas strain isolated on 2-chloroallyl alcohol resulted in 60% dechlorination. Efficient degradation of a mixture of chloroallyl alcohols in continuous culture could only be achieved in the presence of a satellite population. The mixed culture degraded 99% of the total chloroallyl alcohols added with 71% chloride release. The culture contained strains with a new catabolic potential. The results indicate the importance of mixed cultures and genetic adaptation for efficient chloroallyl alcohol removal.     

乳白耙齿菌F17对单一和复合多环芳烃的降解差异解析

[目的] 针对菲、蒽、荧蒽多环芳烃（PAHs）污染物,利用乳白耙齿菌F17,研究单一和复合PAHs污染物的生物降解规律。[方法] 采用气相色谱-质谱法（GC-MS）分析降解过程中PAHs的浓度,并采用准一级反应动力学模型对降解结果进行拟合。[结果] 对于单一PAHs,第15天时菲、蒽、荧蒽的降解率由高到低依次为菲（97.8%） > 蒽（89.3%） > 荧蒽（81.5%）。菲、蒽和荧蒽的降解过程具有准一级反应动力学特征,菲的生物降解速率最快,其次是蒽,荧蒽的降解速率最慢。与单一PAHs的降解相比,在复合PAHs的降解过程中,乳白耙齿菌F17的生长和锰过氧化物酶的合成均表现出不同的特征。此外,水溶性极可能是复合污染物降解的重要控制因子,三者水溶性为：菲 > 荧蒽 > 蒽。因此,在菲或荧蒽加入条件下,微生物能优先降解这些污染物,抑制了污染物蒽的降解;同时,蒽或菲的存在对荧蒽的降解也有抑制作用;然而外源加入水溶性较差的蒽和荧蒽,则对菲的生物降解无显著影响。[结论] 复合PAHs的生物降解主要表现为相互竞争的特点,通过GC-MS分析了PAHs的生物降解途径。     

Biodegradation of sodium sulfite liquor (NaSSL) and sodium lignosulfonate (NaLS) by a mixed culture of microorganisms

Summary In this work we discuss the aerobic biodegradation of sodium sulfite liquor of (NaSSL) and sodium lignosulfonate (NaLS) in a firwood sulfite waste liquor by a mixed culture of microorganisms consisting of two Trichosporon yeasts and bacteria in the Arthrobacter (two species), Pseudomonas and Chromabacterium genera. Under established process parameters, the NaSSL was biodegradated in one or two stages by mixed cultures. The kinetics in each stage was studied. The optimal ratio of NaLS and sugars in the substrate for the growth of mixed culture was determined. The growth of the monocultures of the bacteria on the NaLS and the growth of the yeasts as monocultures on the NaSSL substrate were examined. UV absorption and IR spectra were employed as analytical methods to follow the microbial degradation of NaLS. The aim of this research was to study the biodegradation process and kinetics and to remove by means of mixed culture the maximum amount of organic matter from NaSSL.     

Biodegradation of native feather keratin by Bacillus subtilis recombinant strains

A mixed culture containing two recombinant Bacillus subtilis strains; was used to hydrolyze 1% chicken feather; both were previously transformed with late-expressed and early expressed alkaline protease (aprE) carrying plasmids pS1 and p5.2, respectively. Proteolytic and keratinolytic activities of the mixed culture increased in parallel with those of the culture of B. subtilis DB100 (p5.2), and both were higher than that of B. subtilis (pS1) cultures. On the other hand, data indicated that degradation of feather by the recombinant strains B. subtilis DB100 (p5.2), was greatly enhanced when using a previously optimized medium. High levels of free amino groups as well as soluble proteins were also obtained. The concentration of amino acids was considerably increased during the fermentation process. It was found that, the amino acids Phe, Gly and Tyr were the major amino acids liberated in the cultures initiated by both strains. Results render these recombinant strains suitable for application in feather biodegradation large scale processes.     

Interspecific interactions in mixed microbial cultures in a biodegradation perspective

In recent works, microbial consortia consisting of various bacteria and fungi exhibited a biodegradation performance superior to single microbial strains. A highly efficient biodegradation of synthetic dyes, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and other organic pollutants can be achieved by mixed microbial cultures that combine degradative enzyme activities inherent to individual consortium members. This review summarizes biodegradation results obtained with defined microbial cocultures and real microbial consortia. The necessity of using a proper strategy for the microbial consortium development and optimization was clearly demonstrated. Molecular genetic and proteomic techniques have revolutionized the study of microbial communities, and techniques such as the denaturing gradient gel electrophoresis, rRNA sequencing, and metaproteomics have been used to identify consortium members and to study microbial population dynamics. These analyses could help to further enhance and optimize the natural activities of mixed microbial cultures.     

Purification, Stability, and Mineralization of 3-Hydroxy-2- Formylbenzothiophene, a Metabolite of Dibenzothiophene

3-Hydroxy-2-formylbenzothiophene (HFBT) is a metabolite found in many bacterial cultures that degrade dibenzothiophene (DBT) via the Kodama pathway. The fate of HFBT in cultures and in the environment is unknown. In this study, HFBT was produced by a DBT-degrading bacterium and purified by sublimation. When stored in organic solvent or as a crystal, the HFBT slowly decomposed, yielding colored products. Two of these were identified as thioindigo and cis-thioindigo. The supernatant of the DBT-degrading culture contained thioindigo, which has not been reported previously as a product of DBT biodegradation. In mineral salts medium, HFBT was sufficiently stable to allow biodegradation studies with a mixed microbial culture over a 3- to 4-week period. High-performance liquid chromatography analyses showed that HFBT was removed from the medium. 2-Mercaptophenylglyoxalate, detected as benzothiophene-2,3-dione, was found in an HFBT-degrading mixed culture, and the former appears to be a metabolite of HFBT. This mixed culture also mineralized HFBT to CO₂.     

Biodegradation kinetics of 2,4-dichlorophenol by acclimated mixed cultures

The biodegradation kinetics of 2,4-dichlorophenol (2,4-DCP) by culture (Culture M) acclimated to mixture of 4-chlorophenol (4-CP) and 2,4-DCP and the culture (Culture 4) acclimated to 4-CP only were investigated in aerobic batch reactors. Also, pure strains isolated from mixed cultures were searched for their ability towards the biodegradation of 2,4-DCP. Culture 4 was able to completely degrade 2,4-DCP up to 80 mg/L within 30 h and removal efficiency dropped to 21% upon increasing initial concentration to 108.8 mg/L. When the Culture M was used, complete degradation of 2,4-DCP in the range of 12.5-104.4 mg/L was attained. A linear relationship between time required for complete degradation and initial 2,4-DCP concentrations was observed for both mixed cultures. It was observed that the Haldane equation can be used to predict specific degradation rate (SDR) (R(2)>0.99) as a function of initial 2,4-DCP concentrations and it adequately describes 2,4-DCP concentration profiles. Both of the mixed cultures settled well, which is important to maintain good removal efficiency for longer periods of time for real full-scale applications. Although the pure strains isolated from mixed cultures were found to have higher SDR of 2,4-DCP compared to mixed cultures, they did not settle well under quiescent conditions.     

Microbial degradation of formaldehyde in white mineral dispersions preserved with formaldehyde-releasing biocides

Two different formaldehyde-degrading microorganisms, Pseudomonas putida and Methylobacterium extorquens, were isolated from calcium carbonate slurry containing the formaldehyde-releasing biocide (ethylenedioxy) dimethanol. Their relative formaldehyde biodegradation and formic acid production kinetics were studied in broth and in calcium carbonate slurry for each microorganism individually, as well as in mixed cultures. Furthermore, the minimal inhibition concentration (MIC) was determined. The results indicated that in slurry, M. extorquens is more tolerant of formaldehyde than P. putida. In slurry, microbial-induced oxidation of formaldehyde caused a temporary accumulation of formic acid, which is presumed to be responsible for pH drop and destabilisation of the calcium carbonate slurry suspension systems. In addition, the residual formaldehyde concentration was observed to drive dominance and recovery of individual formaldehyde-resistant microorganisms in the slurry. Overall, this investigation indicated that biodegradation of formaldehyde in calcium carbonate slurry is brought about by alternating dominance of bacterial genera of mixed formaldehyde-resistant microbial populations.     

Aerobic MTBE biodegradation in the presence of BTEX by two consortia under batch and semi-batch conditions

This study explores the effect of microbial consortium composition and reactor configuration on methyl tert-butyl ether (MTBE) biodegradation in the presence of benzene, toluene, ethylbenzene and p-xylenes(BTEX). MTBE biodegradation was monitored in the presence and absence of BTEX in duplicate batch reactors inoculated with distinct enrichment cultures: MTBE only (MO—originally enriched on MTBE) and/or MTBE BTEX (MB—originally enriched on MTBE and BTEX). The MO culture was also applied in a semi-batch reactor which received both MTBE and BTEX periodically in fresh medium after allowing cells to settle. The composition of the microbial consortia was explored using a combination of 16S rRNA gene cloning and quantitative polymerase chain reaction targeting the known MTBE-degrading strain PM1^T. MTBE biodegradation was completely inhibited by BTEX in the batch reactors inoculated with the MB culture, and severely retarded in those inoculated with the MO culture (0.18 ± 0.04 mg/L-day). In the semi-batch reactor, however, the MTBE biodegradation rate in the presence of BTEX was almost three times as high as in the batch reactors (0.48 ± 0.2 mg/L-day), but still slower than MTBE biodegradation in the absence of BTEX in the MO-inoculated batch reactors (1.47 ± 0.47 mg/L-day). A long lag phase in MTBE biodegradation was observed in batch reactors inoculated with the MB culture (20 days), but the ultimate rate was comparable to the MO culture (0.95 ± 0.44 mg/L-day). Analysis of the cultures revealed that strain PM1^T concentrations were lower in cultures that successfully biodegraded MTBE in the presence of BTEX. Also, other MTBE degraders, such as Leptothrix sp. and Hydrogenophaga sp. were found in these cultures. These results demonstrate that MTBE bioremediation in the presence of BTEX is feasible, and that culture composition and reactor configuration are key factors.     

Design of bacterial defined mixed cultures for biodegradation of specific crude oil fractions, using population dynamics analysis by DGGE

Hydrocarbon-degrading bacteria isolated from oil-polluted soils, were used to design three defined mixed cultures (DMC) for biodegradation of Maya crude oil fractions. The first degrading culture, DMC A was made up with 10 strains. Design of DMC B (six strains) and DMC C (three strains) was based on DGGE profiles obtained throughout biodegradation assays of different petroleum fractions. Biodegradation of the aliphatic fraction (10 000 mg l⁻¹) and an aromatic–polar mixture (5000 mg l⁻¹) was evaluated for the DMC B. Biodegradation of total hydrocarbons (10 000 mg l⁻¹) and its fractions was evaluated for DMC B and DMC C. During biodegradation assays, O₂ consumption and CO₂ production were assessed by respirometry, while population dynamics of predominant strains was based on PCR-DGGE profiles of partial 16S rDNA. Aliphatic fraction was completely biodegraded by DMC B, while degradation of the aromatic–polar mixture was 12.5% and for total hydrocarbons 40.5%. DMC B was able to degrade the aromatic fraction (31%) and even the polar fraction (19.6%) present in total hydrocarbons. DMC C degraded the aromatic and polar fractions (5.6% and 2%, respectively) present in total hydrocarbons. DGGE profiles of the DMCs indicated that Pseudomonas sp., Gordonia rubripertincta and a non-identified strain were predominant and probably responsible of the hydrocarbons biodegradation. The use of DGGE-fingerprinting to track microbial populations, allowed selecting strains to design efficient oil-degrading defined mixed cultures.     

Biodegradation of phenol and 4-chlorophenol by the yeast <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Biodegradation of phenol and 4-chlorophenol (4-cp) using a pure culture of Candida tropicalis was studied. The results showed that C. tropicalis could degrade 2,000 mg l⁻¹ phenol alone and 350 mg l⁻¹ 4-cp alone within 66 and 55 h, respectively. The capacity of the strain to degrade phenol was obviously higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Phenol beyond 800 mg l⁻¹ could not be degraded in the presence of 350 mg l⁻¹ 4-cp. Comparatively, low-concentration phenol from 100 to 600 mg l⁻¹ supplied a sole carbon and energy source for C. tropicalis in the initial phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in the fact that 4-cp biodegradation velocity was higher than that without phenol. And the capacity of C. tropicalis to degrade 4-cp was increased up to 420 mg l⁻¹ with the presence of 100–160 mg l⁻¹ phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and mixed substrates in batch cultures. The results illustrated that the models proposed adequately described the dynamic behaviors of biodegradation by C. tropicalis.     

Anaerobic biodegradation of ethylthionocarbamate by the mixed bacteria under various electron acceptor conditions

Biodegradation behavior and kinetics of ethylthionocarbamate under nitrate, sulfate and ferric reducing conditions by mixed cultures enriched from the anaerobic digester sludge was investigated. The results showed that ethylthionocarbamate could be degraded independently by the mixed cultures coupled to nitrate, sulfate, and ferric reduction, and meanwhile, nitrite, sulfide, and ferrous were accumulated as a result of nitrate, sulfate and ferric reduction, respectively. Ferric was a more favorable terminal electron acceptor compared to nitrate and sulfate. The order of the electron acceptors with decreasing biodegradation rates of the ethylthionocarbamate was: ferric>nitrate>sulfate, and the corresponding maximum biodegradation rate was 7.240, 6.267, and 4.602 mg/(L·d), respectively. The anaerobic biodegradation of ethylthionocarbamate under various electron acceptor conditions can be accurately described by first order exponential decay kinetics.     

Purification, stability, and mineralization of 3-hydroxy-2- formylbenzothiophene, a metabolite of dibenzothiophene

3-Hydroxy-2-formylbenzothiophene (HFBT) is a metabolite found in many bacterial cultures that degrade dibenzothiophene (DBT) via the Kodama pathway. The fate of HFBT in cultures and in the environment is unknown. In this study, HFBT was produced by a DBT-degrading bacterium and purified by sublimation. When stored in organic solvent or as a crystal, the HFBT slowly decomposed, yielding colored products. Two of these were identified as thioindigo and cis-thioindigo. The supernatant of the DBT-degrading culture contained thioindigo, which has not been reported previously as a product of DBT biodegradation. In mineral salts medium, HFBT was sufficiently stable to allow biodegradation studies with a mixed microbial culture over a 3- to 4-week period. High-performance liquid chromatography analyses showed that HFBT was removed from the medium. 2-Mercaptophenylglyoxalate, detected as benzothiophene-2,3-dione, was found in an HFBT-degrading mixed culture, and the former appears to be a metabolite of HFBT. This mixed culture also mineralized HFBT to CO2.     

Growth and survival ofAzospirillum brasilense andArthrobacter giacomelloi in binary continuous culture

Summary Azospirillum brasilense andArthrobacter giacomelloi were grown in single and mixed succinate-limited continuous cultures at a partial oxygen pressure of 0.01atm. Growth, viability and survival during nutrient starvation were examined at various dilution rates. At D=0.05 h^–1, K_s values for succinate consumed were calculated.Arthrobacter giacomelloi viability was inversely related to dilution rate whereasAzo. brasilense was directly related. Slightly lower values of viability were obtained in mixed culture, but the ratio between the microorganisms was constant. The survival ofArth. giacomelloi in single culture decreased with increasing growth rate while survival ofAzo. brasilense was directly related to dilution rate. Acetylene reduction activity was generally very low in both single and mixed cultures. Respiration rate was also determined and the mixed culture showed an oxygen uptake rate higher than that of single cultures.Research work supported by CNR, Italy. Special grant I.P.R.A. Sub-project 1. Paper N. 317.     

Decolourization of olive mill waste-waters by the white-rot fungus Phanerochaete chrysosporium: involvement of the lignin-degrading system

Summary The decolourization of olive mill waste-waters (OMW) by Phanerochaete chrysosporium was investigated. OMW decolourization occurred during the primary phase of growth when glycerol was used as the carbon source, and during secondary metabolism in nitrogen-limited cultures. The decolourization was found to be extensive (74% of colour removal, 80% of chemical oxygen demand removal) when the cultures were supplement d with veratryl alcohol and flushed with O₂. The biodegradation system was repressed with glutamate as a nitrogen source. These results suggest that all or part of the lignin-degrading system of P. chrysosporium played a role in biodegradation of OMW. The decolourization of OMW corresponds to depolymerization of high-molecular-mass aromatics combined with mineralization of a wide range of monoaromatic compounds. Correspondence to: S. Sayadi     

Microbial community shifts as a response to efficient degradation of chlorobenzene under hypoxic conditions

Limitations in the availability of oxygen restrict aerobic biodegradation of chloroaromatic compounds in groundwater ecosystems. In this context the activity of ring-cleaving chlorocatechol dioxygenases (CC12O) is crucial for effective mineralization. Previously we demonstrated that oxygen-related enzyme characteristics of CC12O can vary widely among the Proteobacteria (Balcke et al. submitted). Here, we investigated how strains with different ability to transform intermediary 3-chlorocatechol integrate into biodegradation of chlorobenzene (CB) under low or high oxygen availability. Pseudomonas veronii UFZ B549 and Acidovorax facilis UFZ B530, which had differing oxygen affinities for CC12O, were mixed together at different proportions (20:80; 80:20), and compared for degradation of chlorobenzene under oxic (215 μM O2) and hypoxic (11 μM O2) conditions. Changes in community composition in binary mixed cultures were determined and compared with an indigenous groundwater community, cultivated under comparable conditions. Community shifts were determined by FISH (fluorescent in situ hybridization) in our model system and SSCP (single stranded conformation polymorphism) fingerprinting in the groundwater community, as well as by analysis of respiratory quinones of taxonomic value. Hypoxia led to enrichment of Acidovoracae in the groundwater and binary cultures. Under hypoxic conditions cis,cis-2-chloromuconate released to the medium by A. facilis allowed for concomitant growth of P. veronii, although its low-affinity type CC12O would not imply growth on CB. Vice versa, increasing abundance of P. veronii induced intermediary 3-chlorocatechol accumulation, which was reduced by growth of A. facilis. Thus, reduced oxygen availability caused syntrophic rather than competitive interactions.