Marc Viñas

Bacterial Community Dynamics and Polycyclic Aromatic Hydrocarbon Degradation during Bioremediation of Heavily Creosote-Contaminated Soil

ABSTRACT Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the α-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the γ-Proteobacteria group (genus Xanthomonas), the α-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the γ-Proteobacteria group (genus Xathomonas), the β-Proteobacteria group (genera Alcaligenes and Achromobacter), and the α-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.

Bacterial communities from shoreline environments (Costa da Morte, Northwestern Spain) affected by the Prestige oil spill

ABSTRACT The bacterial communities in two different shoreline matrices, rocks and sand, from the Costa da Morte, northwestern Spain, were investigated 12 months after being affected by the Prestige oil spill. Culture-based and culture-independent approaches were used to compare the bacterial diversity present in these environments with that at a nonoiled site. A long-term effect of fuel on the microbial communities in the oiled sand and rock was suggested by the higher proportion of alkane and polyaromatic hydrocarbon (PAH) degraders and the differences in denaturing gradient gel electrophoresis patterns compared with those of the reference site. Members of the classes Alphaproteobacteria and Actinobacteria were the prevailing groups of bacteria detected in both matrices, although the sand bacterial community exhibited higher species richness than the rock bacterial community did. Culture-dependent and -independent approaches suggested that the genus Rhodococcus could play a key role in the in situ degradation of the alkane fraction of the Prestige fuel together with other members of the suborder Corynebacterineae. Moreover, other members of this suborder, such as Mycobacterium spp., together with Sphingomonadaceae bacteria (mainly Lutibacterium anuloederans), were related as well to the degradation of the aromatic fraction of the Prestige fuel. The multiapproach methodology applied in the present study allowed us to assess the complexity of autochthonous microbial communities related to the degradation of heavy fuel from the Prestige and to isolate some of their components for a further physiological study. Since several Corynebacterineae members related to the degradation of alkanes and PAHs were frequently detected in this and other supralittoral environments affected by the Prestige oil spill along the northwestern Spanish coast, the addition of mycolic acids to bioremediation amendments is proposed to favor the presence of these degraders in long-term fuel pollution-affected areas with similar characteristics.

The Prestige oil spill: bacterial community dynamics during a field biostimulation assay.

A field bioremediation assay using the oleophilic fertilizer S200 was carried out 12xa0months after the Prestige heavy fuel-oil spill on a beach on the Cantabrian coast (north Spain). This assay showed that S200-enhanced oil degradation, particularly of high-molecular-weight n-alkanes and alkylated PAHs, suggesting an increase in the microbial bioavailability of these compounds. The bacterial community structure was determined by cultivation-independent analysis of polymerase chain reaction-amplified 16S rDNA by denaturing gradient gel electrophoresis. Bacterial community was mainly composed of α-Proteobacteria (Rhodobacteriaceae and Sphingomonadaceae). Representatives of γ-Proteobacteria (Chromatiales, Moraxellaceae, and Halomonadaceae), Bacteroidetes (Flavobacteriaceae), and Actinobacteria group (Nocardiaceae and Corynebacteriaceae) were also found. The addition of the fertilizer led to the appearance of the bacterium Mesonia algae in the early stages, with a narrow range of growth substrates, which has been associated with the common alga Achrosiphonia sonderi. The presence of Mesonia algae may be attributable to the response of the microbial community to the addition of N and P rather than indicating a role in the biodegradation process. The Rhodococcus group appeared in both assay plots, especially at the end of the experiment. It was also found at another site on the Galician coast that had been affected by the same spill. This genus has been associated with the degradation of n-alkanes up to C36. Taking into account the high content of heavy alkanes in the Prestige fuel, these microorganisms could play a significant role in the degradation of such fuel. A similar bacterial community structure was observed at another site that showed a similar degree of fuel weathering.

Operational, design and microbial aspects related to power production with microbial fuel cells implemented in constructed wetlands

This work aimed at determining the amount of energy that can be harvested by implementing microbial fuel cells (MFC) in horizontal subsurface constructed wetlands (HSSF CWs) during the treatment of real domestic wastewater. To this aim, MFC were implemented in a pilot plant based on two HSSF CW, one fed with primary settled wastewater (Settler line) and the other fed with the effluent of a hydrolytic up-flow sludge blanket reactor (HUSB line). The eubacterial and archaeal community was profiled on wetland gravel, MFC electrodes and primary treated wastewater by means of 16S rRNA gene-based 454-pyrosequencing and qPCR of 16S rRNA and mcrA genes. Maximum current (219xa0mA/m(2)) and power (36xa0mW/m(2)) densities were obtained for the HUSB line. Power production pattern correlated well with water level fluctuations within the wetlands, whereas the type of primary treatment implemented had a significant impact on the diversity and relative abundance of eubacteria communities colonizing MFC. It is worth noticing the high predominance (13-16% of relative abundance) of one OTU belonging to Geobacter on active MFC of the HUSB line that was absent for the settler line MFC. Hence, MFC show promise for power production in constructed wetlands receiving the effluent of a HUSB reactor.

Fungal/bacterial interactions during the biodegradation of TEX hydrocarbons (toluene, ethylbenzene and p-xylene) in gas biofilters operated under xerophilic conditions

The treatment of air contaminated with toluene, ethylbenzene, and p-xylene was assayed in three laboratory-scale biofilters, each consisting of two modules connected in series, packed with a pelletized organic fertilizer and inoculated with a toluene-degrading liquid enrichment culture. Biofilters were operated in parallel for 185xa0days in which the volumetric organic loading rate was progressively increased. The operation regime was subjected to drying out, so that packing humidity generally remained below 40%. Significant process failure occurred with ethylbenzene and p-xylene, but the toluene biofilter comparatively sustained a significant elimination capacity. Microbial community characterization by quantitative PCR and denaturing gradient gel electrophoresis showed substantial fungal enrichment in the toluene biofilter. Ribotypes identical to the well-known toluene-degrading black yeast Exophiala oligosperma (Chaetotyriales) were found among the dominant species. The microbial community structure was similar in the biofilters loaded with toluene and ethylbenzene but with p-xylene was quite specific and encompassed other chaetothyrialean fungi. Several species of Actinomycetales were found in the packing while the inoculum was dominated by representatives of the Burkholderiales and Xanthomonadales. One single fungal ribotype homologous to Acremonium kiliense was detected in the inoculum. The implications of xerophilic biofilter operation on process biosafety and efficiency are discussed.

Polyphasic approach for assessing changes in an autochthonous marine bacterial community in the presence of Prestige fuel oil and its biodegradation potential

A laboratory experiment was conducted to identify key hydrocarbon degraders from a marine oil spill sample (Prestige fuel oil), to ascertain their role in the degradation of different hydrocarbons, and to assess their biodegradation potential for this complex heavy oil. After a 17-month enrichment in weathered fuel, the bacterial community, initially consisting mainly of Methylophaga species, underwent a major selective pressure in favor of obligate hydrocarbonoclastic microorganisms, such as Alcanivorax and Marinobacter spp. and other hydrocarbon-degrading taxa (Thalassospira and Alcaligenes), and showed strong biodegradation potential. This ranged from >99% for all low- and medium-molecular-weight alkanes (C15–C27) and polycyclic aromatic hydrocarbons (C0- to C2- naphthalene, anthracene, phenanthrene, dibenzothiophene, and carbazole), to 75–98% for higher molecular-weight alkanes (C28–C40) and to 55–80% for the C3 derivatives of tricyclic and tetracyclic polycyclic aromatic hydrocarbons (PAHs) (e.g., C3-chrysenes), in 60xa0days. The numbers of total heterotrophs and of n-alkane-, aliphatic-, and PAH degraders, as well as the structures of these populations, were monitored throughout the biodegradation process. The salinity of the counting medium affects the counts of PAH degraders, while the carbon source (n-hexadecane vs. a mixture of aliphatic hydrocarbons) is a key factor when counting aliphatic degraders. These limitations notwithstanding, some bacterial genera associated with hydrocarbon degradation (mainly belonging to α- and γ-Proteobacteria, including the hydrocarbonoclastic Alcanivorax and Marinobacter) were identified. We conclude that Thalassospira and Roseobacter contribute to the degradation of aliphatic hydrocarbons, whereas Mesorhizobium and Muricauda participate in the degradation of PAHs.

Aerobic nonylphenol degradation and nitro-nonylphenol formation by microbial cultures from sediments.

Nonylphenol (NP) is an estrogenic pollutant which is widely present in the aquatic environment. Biodegradation of NP can reduce the toxicological risk. In this study, aerobic biodegradation of NP in river sediment was investigated. The sediment used for the microcosm experiments was aged polluted with NP. The biodegradation of NP in the sediment occurred within 8xa0days with a lag phase of 2xa0days at 30°C. During the biodegradation, nitro-nonylphenol metabolites were formed, which were further degraded to unknown compounds. The attached nitro-group originated from the ammonium in the medium. Five subsequent transfers were performed from original sediment and yielded a final stable population. In this NP-degrading culture, the microorganisms possibly involved in the biotransformation of NP to nitro-nonylphenol were related to ammonium-oxidizing bacteria. Besides the degradation of NP via nitro-nonylphenol, bacteria related to phenol-degrading species, which degrade phenol via ring cleavage, are abundantly present.

Microbial populations related to PAH biodegradation in an aged biostimulated creosote-contaminated soil

A previous bioremediation survey on a creosote-contaminated soil showed that aeration and optimal humidity promoted depletion of three-ringed polycyclic aromatic hydrocarbons (PAHs), but residual concentrations of four-ringed benzo(a)anthracene (B(a)A) and chrysene (Chry) remained. In order to explain the lack of further degradation of heavier PAHs such as four-ringed PAHs and to analyze the microbial population responsible for PAH biodegradation, a chemical and microbial molecular approach was used. Using a slurry incubation strategy, soil in liquid mineral medium with and without additional B(a)A and Chry was found to contain a powerful PAH-degrading microbial community that eliminated 89% and 53% of the added B(a)A and Chry, respectively. It is hypothesized that the lack of PAH bioavailability hampered their further biodegradation in the unspiked soil. According to the results of the culture-dependent and independent techniques Mycobacterium parmense, Pseudomonas mexicana, and Sphingobacterials group could control B(a)A and Chry degradation in combination with several microorganisms with secondary metabolic activity.

Nitrogen recovery from pig slurry in a two-chambered bioelectrochemical system.

Abiotic batch experiments showed that ammonia migration from anode to cathode was favored by an increase in voltage, from 39.9% to 44.6%, using synthetic media. A slight increase in ammonia migration was observed when using pig slurry, reaching a maximum of 49.9%. In a continuously MFC fed with pig slurry with a stripping/absorption unit coupled to the cathode chamber, the highest nitrogen flux (7.2 g N d(-1) m(-2)) was achieved using buffer as catholyte. Nitrogen flux increased to 10.3 g N d(-1) m(-2) when shifting to MEC mode. A clear improvement in nitrogen flux (25.5 g N d(-1) m(-2)) was observed when using NaCl as catholyte. Besides, ammonia stripping was favored, reaching a nitrogen recovery of 94.3% in the absorption column, due to the high pH reached in the cathode. The microbial community analysis revealed an enrichment of certain taxonomic Eubacterial and Archaeal groups when the system shifted from MFC to MEC mode.

Degradation of 4-n-nonylphenol under nitrate reducing conditions.

Nonylphenol (NP) is an endocrine disruptor present as a pollutant in river sediment. Biodegradation of NP can reduce its toxicological risk. As sediments are mainly anaerobic, degradation of linear (4-n-NP) and branched nonylphenol (tNP) was studied under methanogenic, sulphate reducing and denitrifying conditions in NP polluted river sediment. Anaerobic bioconversion was observed only for linear NP under denitrifying conditions. The microbial population involved herein was further studied by enrichment and molecular characterization. The largest change in diversity was observed between the enrichments of the third and fourth generation, and further enrichment did not affect the diversity. This implies that different microorganisms are involved in the degradation of 4-n-NP in the sediment. The major degrading bacteria were most closely related to denitrifying hexadecane degraders and linear alkyl benzene sulphonate (LAS) degraders. The molecular structures of alkanes and LAS are similar to the linear chain of 4-n-NP, this might indicate that the biodegradation of linear NP under denitrifying conditions starts at the nonyl chain. Initiation of anaerobic NP degradation was further tested using phenol as a structure analogue. Phenol was chosen instead of an aliphatic analogue, because phenol is the common structure present in all NP isomers while the structure of the aliphatic chain differs per isomer. Phenol was degraded in all cases, but did not affect the linear NP degradation under denitrifying conditions and did not initiate the degradation of tNP and linear NP under the other tested conditions.