Hugo Emiliano de Jesus

Microbial diversity and hydrocarbon depletion in low and high diesel-polluted soil samples from Keller Peninsula, South Shetland Islands

Abstract The bioremediation of Antarctic soils is a challenge due to the harsh conditions found in this environment. To characterize better the effect of total petroleum hydrocarbon (TPH) concentrations on bacterial, archaeal and microeukaryotic communities in low (LC) and high (HC) hydrocarbon-contaminated soil samples from the Maritime Antarctic clone libraries (small-subunit rRNA genes) were constructed. The results showed that a high concentration of hydrocarbons resulted in a decrease in bacterial and eukaryotic diversity; however, no effect of the TPH concentration was observed for the archaeal community. The HC soil samples demonstrated a high relative abundance of bacterial operational taxonomic units (OTUs) affiliated with unclassified group TM7 and eukaryotic OTUs affiliated with unclassified fungi from Pezizomycotina subphyla. Chemical analyses of the LC and HC soil samples revealed the presence of negligible amounts of nitrogen, thereby justifying the use of biostimulation to remediate these Antarctic soils. Microcosm experiments showed that the application of fertilizers led to an increase of up to 27.8% in the TPH degradation values. The data presented here constitute the first step towards developing the best method to deploy bioremediation in Antarctic soils and provide information to indicate an appropriate action plan for immediate use in the case of new accidents.

Comparison of different protocols for the extraction of microbial DNA from reef corals

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Bioremediation in Antarctic Soils

With the increase of human activities in cold environments, the risk of oil spill has become higher due the necessity of energy generation using oils. Several accidents were related in Arctic and Antarctic involving greatcontaminated areas and chronically levels of contamination. Despite that, Antarctic continent present peculiar characteristics and need to be analyzed separately due to geographic, physic and topologic properties, beyond different proposes of human activities in Antarctic continent. In Antarctic, the main occupations are permanent scientific and military stations and most of them are in activity during whole year. Several studies evaluating the potential of biodegradation were performed using Antarctic soils and the results show promising, however, there is no works about bioremediation process in soils from the core of the continent, only from the shore regions. Antarctic continent present a diverse microbial community able to degrade oils even in extreme conditions, being the most found strains belonging to genera Rhodococcus, Acinetobacter, Sphingomonas, Pseudomonas, Stenotrophomonas, Pedobacter and Brevundimonas. In this regard, bioremediation treatments are indicated to promote a sustainable low cost efficient recovery, that need to be performed as soon as possible after the spill to improve this efficiency. Moreover, in Antarctic environment is necessary to take in account the physical characteristics of the soil, environmental conditions as temperature, water availability, nutrients, season of the year and freeze-thaw cycles. In this sense, activities of maintenance like refueling, cleanup of oil tanks and transport of oils could be made in the winter or near the winter period, as snow can serve as a physic barer containing the contaminant. Besides, bioremediation application could be made preferentially in the summer, due the higher temperature, but after the thaw period, once the unfrozen ice can spread the oil to more sensitive regions and also the nutrients added. This paper proposes an unprecedented review about bioremediation process exclusive to Antarctic soils, and provide necessary knowledge to consolidation of the bioremediation in Antarctic environment, besides suggesting applying strategies. The need to previously study the area to be treated, considering as many abiotic factors as possible, to indicate the best treatment strategy, is even more necessary in Antarctic environments.

Bacterial and Archaeal Communities Variability Associated with Upwelling and Anthropogenic Pressures in the Protection Area of Arraial do Cabo (Cabo Frio region - RJ)

Upwelling systems contain a high diversity of pelagic microorganisms and their composition and activity are defined by factors like temperature and nutrient concentration. Denaturing gradient gel electrophoresis (DGGE) technique was used to verify the spatial and temporal genetic variability of Bacteria and Archaea in two stations of the Arraial do Cabo coastal region, one under upwelling pressure and another under anthropogenic pressure. In addition, biotic and abiotic variables were measured in surface and deep waters from three other stations between these stations. Six samplings were done during a year and adequately represented the degrees of upwelling and anthropogenic pressures to the system. Principal Component Analysis (PCA) showed negative correlations between the concentrations of ammonia and phosphorous with prokaryotic secondary production and the total heterotrophic bacteria. PCA also showed negative correlation between temperature and the abundance of prokaryotic cells. Bacterial and archaeal compositions were changeable as were the oceanographic conditions, and upwelling had a regional pressure while anthropogenic pressure was punctual. We suggest that the measurement of prokaryotic secondary production was associated with both Bacteria and Archaea activities, and that substrate availability and temperature determine nutrients cycling.

Distribution of Anaerobic Hydrocarbon-Degrading Bacteria in Soils from King George Island, Maritime Antarctica

Anaerobic diesel fuel Arctic (DFA) degradation has already been demonstrated in Antarctic soils. However, studies comparing the distribution of anaerobic bacterial groups and of anaerobic hydrocarbon-degrading bacteria in Antarctic soils containing different concentrations of DFA are scarce. In this study, functional genes were used to study the diversity and distribution of anaerobic hydrocarbon-degrading bacteria (bamA, assA, and bssA) and of sulfate-reducing bacteria (SRB—apsR) in highly, intermediate, and non-DFA-contaminated soils collected during the summers of 2009, 2010, and 2011 from King George Island, Antarctica. Signatures of bamA genes were detected in all soils analyzed, whereas bssA and assA were found in only 4 of 10 soils. The concentration of DFA was the main factor influencing the distribution of bamA-containing bacteria and of SRB in the analyzed soils, as shown by PCR-DGGE results. bamA sequences related to genes previously described in Desulfuromonas, Lautropia, Magnetospirillum, Sulfuritalea, Rhodovolum, Rhodomicrobium, Azoarcus, Geobacter, Ramlibacter, and Gemmatimonas genera were dominant in King George Island soils. Although DFA modulated the distribution of bamA-hosting bacteria, DFA concentration was not related to bamA abundance in the soils studied here. This result suggests that King George Island soils show functional redundancy for aromatic hydrocarbon degradation. The results obtained in this study support the hypothesis that specialized anaerobic hydrocarbon-degrading bacteria have been selected by hydrocarbon concentrations present in King George Island soils.

Detection of proteases from Sporosarcina aquimarina and Algoriphagus antarcticus isolated from Antarctic soil

Two psychrophilic bacterial samples were isolated from King George Island soil, in Antarctica. The phylogenetic analysis based on the 16S rRNA (rrs) gene led to the correlation with the closest related isolates as Sporosarcina aquimarina (99%) and Algoriphagus antarcticus (99%), with query coverage of 99% and 98%, respectively. The spent culture media from both isolates displayed proteolytic activities detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis containing gelatin as protein substrate. Under the employed conditions, S. aquimarina showed a 55 kDa protease with the best activity detected at pH 7.0 and at 27°C. A. antarcticus also showed a single extracellular protease, however its molecular mass was around 90kDa and its best activity was detected at pH 9.0 and at 37°C. The proteases from both isolates were inhibited by 1,10-phenanthroline and EDTA, two metalloprotease inhibitors. This is the first record of protease detection in both species, and our results may contribute to broaden the basic knowledge of proteases from the Antarctica environment and may help prospecting future biotechnological applications of these enzymes.

Topical Effect of a Medically Prescribed Pediatric Antibiotic on Dental Biofilm: A Cross-Over, In Situ Study

Objective This study aimed to investigate the possible topical effect of a broad-spectrum antibiotic on dental biofilm formed in situ in the absence or presence of sucrose. Methods A crossover study was conducted in three phases of 14 days each, during which 11 volunteers wore palatal devices containing 6 enamel blocks covered with meshes to allow biofilm formation. Dental blocks were extraorally submitted to a 20% sucrose solution at three different frequencies of exposure (0, 3 and 8 times/day), and to a suspension of amoxicillin/clavulanate potassium (A/CP) or a placebo (P) suspension at an 8-hour time interval application regimen. On the 14th day of each phase, biofilms were collected for microbiological (conventional culture) and molecular (Denaturing Gradient Gel Electrophoresis – DGGE) analyses. Results In the absence of sucrose exposure (SE) and at the 3-time daily frequency, dental biofilms treated with A/CP showed lower total biofilm weight and lower counts of total microbiota than the ones treated with P (p>0.05). A/CP presented higher counts of Candida spp. when compared with P in the presence of SE, especially at the 8-time daily frequency (p<0.05). Considering the DGGE analysis, the mean number of bands was higher for P (p>0.05), regardless of SE. However, DGGE profiles demonstrated large interindividual variability. Conclusion Both conventional culture and DGGE have demonstrated some differences on total microbiota of dental biofilms when exposed to the A/CP or P suspensions, mainly in the absence of sucrose, which suggests a possible topical effect of the sugar-free A/CP suspension on dental biofilm.

Bioremediation, hydrocarbon depletion and microbial genetic diversity of Antarctic oil-polluted soil

Natural environments have been affected by oil spills around the world for decades. In some cases, the attempt to cleanup can be made using physical and chemical methods. However, for the Antarctic environments this is not so simple. Displacement of the machinery necessary for the application of physical methods would be very expensive whereas the application of chemical methods would be dangerous considering the risks of additional environmental impacts. Oil contamination of soils of EACF was caused by a tank rupture in the mid eighties in addition to little spills and intense use of motor vehicles. In some sites the presence of oil can be visually detected, which leads us to believe that a monitored natural attenuation is not feasible. Bioremediation techniques are relatively more cost-effective and benign. These techniques are based on the ability of some microorganisms (especially some bacteria) to use the petroleum hydrocarbons as energy source. However, before any implementation of bioremediation action, it is important to perform studies for the chemical and biological characterization of the contaminated soil. We are performing physical-chemical and microbiological studies of soil samples of Brazilian Antarctic Station contaminated with diesel. The results show an absence of Nitrogen in soil, the presence of high content of petroleum hydrocarbons and a depletion effect of the microbial diversity in polluted soil.

Bioremediation of the Diesel-Contaminated Soil of the Brazilian Antarctic Station

Antarctic soils are under constant risks of oil contamination due the presence of scienti c stations. Bioremediation is the best choice for their recovery. However, before taking the initiative, it is important to test their e ect on hydrocarbon depletion and microorganisms. Furthermore, it is important to search for hydrocarbon degraders and bioindicators for monitoring. Our studies showed that the low concentration of N may be causing the recalcitrance of the hydrocarbons in the soil. e microbial characterization revealed alteration of structure and low diversity of the microbial communities in the diesel-polluted soils. e results of an ex situ microcosm experiment revealed depletion of the hydrocarbons content due the aeration and the application of N fertilizer, as well as e ects under the microbial communities. An in situ microcosm experiment with the application of N fertilizer and oil-degrader bacterial species previously isolated con rmed the changes under the microbial community. However, it is important to point out that the impact of the fertilizer under microbial community is lower than the oil impact. e present data provides information that allows us to propose the appropriate methodology that can be applied in the area of the Brazilian Antarctic Station for the bioremediation process. In addition, to provide information that allows us to propose an appropriate action plan using better recommended materials (e.g. type and dose of fertilizer; stock of consortia of degraders strains) that will be available for immediate use in the case of new contaminations due to fuel spills in the new Brazilian Antarctic Station.