Sulphide production and corrosion in seawaters during exposure to FAME diesel

Experiments were designed to evaluate the corrosion-related consequences of storing/transporting fatty acid methyl ester (FAME) alternative diesel fuel in contact with natural seawater. Coastal Key West, FL (KW), and Persian Gulf (PG) seawaters, representing an oligotrophic and a more organic- and inorganic mineral-rich environment, respectively, were used in 60?day incubations with unprotected carbon steel. The original microflora of the two seawaters were similar with respect to major taxonomic groups but with markedly different species. After exposure to FAME diesel, the microflora of the waters changed substantially, with Clostridiales (Firmicutes) becoming dominant in both. Despite low numbers of sulphate-reducing bacteria in the original waters and after FAME diesel exposure, sulphide levels and corrosion increased markedly due to microbial sulphide production. Corrosion morphology was in the form of isolated pits surrounded by an intact, passive surface with the deepest pits associated with the fuel/seawater interface in the KW exposure. In the presence of FAME diesel, the highest corrosion rates measured by linear polarization occurred in the KW exposure correlating with significantly higher concentrations of sulphur and chlorine (presumed sulphide and chloride, respectively) in the corrosion products.     

Lipase-catalyzed biodiesel production from waste activated bleaching earth as raw material in a pilot plant

The production of fatty acid methyl esters (FAMEs) from waste activated bleaching earth (ABE) discarded by the crude oil refining industry using lipase from Candida cylindracea was investigated in a 50-L pilot plant. Diesel oil or kerosene was used as an organic solvent for the transesterification of triglycerides embedded in the waste ABE. When 1% (w/w) lipase was added to waste ABE, the FAME content reached 97% (w/w) after reaction for 12 h at 25 degrees C with an agitation rate of 30 rpm. The FAME production rate was strongly dependent upon the amount of enzyme added. Mixtures of FAME and diesel oil at ratios of 45:55 (BDF-45) and 35:65 (BDF-35) were assessed and compared with the European specifications for biodiesel as automotive diesel fuel, as defined by pr EN 14214. The biodiesel quality of BDF-45 met the EN 14214 standard. BDF-45 was used as generator fuel, and the exhaust emissions were compared with those of diesel oil. The CO and SO2 contents were reduced, but nitrogen oxide emission increased by 10%. This is the first report of a pilot plant study of lipase-catalyzed FAME production using waste ABE as a raw material. This result demonstrates a promising reutilization method for the production of FAME from industrial waste resources containing vegetable oils for use as a biodiesel fuel.     

Dynamics of corrosion rates associated with nitrite or nitrate mediated control of souring under biological conditions simulating an oil reservoir

Representative microbial cultures from an oil reservoir and electrochemical techniques including potentiodynamic scan and linear polarization were used to investigate the time dependent corrosion rate associated with control of biogenic sulphide production through addition of nitrite, nitrate and a combination of nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB) and nitrate. The addition of nitrate alone did not prevent the biogenic production of sulphide but the produced sulphide was eventually oxidized and removed from the system. The addition of nitrate and NR-SOB had a similar effect on oxidation and removal of sulphide present in the system. However, as the addition of nitrate and NR-SOB was performed towards the end of sulphide production phase, the assessment of immediate impact was not possible. The addition of nitrite inhibited the biogenic production of sulphide immediately and led to removal of sulphide through nitrite mediated chemical oxidation of sulphide. The real time corrosion rate measurement revealed that in all three cases an acceleration in the corrosion rate occurred during the oxidation and removal of sulphide. Amendments of nitrate and NR-SOB or nitrate alone both gave rise to localized corrosion in the form of pits, with the maximum observed corrosion rates of 0.72 and 1.4 mm year⁻¹, respectively. The addition of nitrite also accelerated the corrosion rate but the maximum corrosion rate observed following nitrite addition was 0.3 mm year⁻¹. Furthermore, in the presence of nitrite the extent of pitting was not as high as those observed with other control methods.     

Effects of oxygen on biodegradation of fuels in a corroding environment

The relationship between corrosion and biodegradation of bio- and petroleum-based fuels was evaluated using aerobic seawater, fuel and unprotected carbon steel coupons under stagnant conditions to simulate a potential fuel storage condition. Aerobic respiration and corrosion reactions consumed oxygen in the incubations in a short time. The transient oxygen influenced the microbial biodegradation of all fuels and resulted in a suite of characteristic metabolites, including catechols. The corrosion was believed to be the result of biogenic sulfide production and in all cases, the black corrosion products contained chlorine and sulfur (presumed chloride and sulfide) in addition to iron. There were few differences in electrochemically measured corrosion rates in incubations amended with any of the fuels or their blends. Clone library analysis demonstrated higher proportions of Firmicutes, Deltaproteobacteria (primarily sulfate-reducing bacteria), Chloroflexi, and Lentisphaerae in incubations exposed to fuels than the original seawater. Relative proportions of sequences affiliated with these bacterial groups varied with fuel. Methanogen sequences similar to those of Methanolobus were also found in multiple incubations. Despite the dominance of characteristically anaerobic taxa, sequences coding for an alkane monooxygenase from marine hydrocarbon-degrading genera and aerobically produced intermediates were observed, indicative that organisms with this metabolic potential were active at some point during the incubation. Aerobic oxidation of fuel components resulted in the formation of a series of intermediates that could be used by anaerobic seawater microbial communities to support metabolism, sulfide production, and carbon steel corrosion.     

Impacts of diesel fuel exposure on the microphytobenthic community of an intertidal sand flat

This study employed simulated spills of weathered diesel fuel and measured the initial effects on the intertidal sand flat microphytobenthic (MPB) communities. The goals were to examine the impacts of short-term (hours) and longer-term (days) exposure to petroleum on the native sand flat MPB in coastal North Carolina and to assess recovery of the community following the exposure. We assessed changes in biomass (chlorophyll a), primary productivity (¹⁴C bicarbonate incorporation), photophysiology (P vs. I curves) and species composition (microscopy) and compared diesel exposed samples to unamended controls. We found that short-term impacts of diesel fuel pollution were confined to primary productivity and photophysiology of sand flat MPB. Short-term effects were only detected at relatively high concentrations that are not common outside of a major spill event. In the longer term, diesel fuel was again found to have effects on primary productivity, but at higher concentrations than would be likely to occur in industrialized coastal areas. However, negative impacts on photophysiology were detected at diesel fuel concentrations slightly above typical ambient conditions in coastal waters in industrialized areas. Biomass as measured by chlorophyll a was not affected by any concentration in the longer-term exposure to diesel fuel. Cell counts in the longer-term experiments found cyanobacteria had larger negative impacts from diesel fuel exposure than did diatoms. The recovery portion of this study showed the sand flat MPB communities were fairly resilient following both additions of diesel fuel. However, photophysiology and cell counts did not return to conditions equivalent to the control. Data from this study indicate that the effects of petroleum pollution on the MPB community of tidal sand flats should be considered alongside effects on other coastal microalgae in ecological and damage assessments.     

Spatial distribution of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.     

Effect of Different Seawaters on the Development of Biochemically Deficient Mutants of Serratia marinorubra

The effect of different seawaters on survival and growth of biotin-, isoleucine-, and uracil-requiring mutants of the marine bacterium, Serratia marinorubra, has been investigated. Samples of seawater were collected from coastal waters, the California Current, and central North Pacific waters at depths of 1, 25, 50, 100, 250, and 500 m. The growth or survival of the test bacterium in basal medium prepared in these seawater samples was determined. The control water was synthetic or charcoal-treated natural seawater. In several experiments, the metabolite required by the bacterium was added to the basal medium 24 hr after inoculation, and the growth response was determined. Depending on the source, the seawater samples were both stimulating and inhibitory. Surface waters were more inhibitory than those taken at depth, where, in some cases, bacterial growth occurred. Seawater inhibition was related more to station depth than to the location of the station. The most toxic effects were found against the uracil-requiring mutant; the least, against the isoleucine-requiring mutant. The results of these studies and some laboratory experiments indicate that seawater toxicity is not primarily associated with the physical and biological properties of a particular water mass and that the same factor(s) may be responsible for the rapid death of bacteria in all waters.     

Spatial Distribution of Marine Crenarchaeota Group I in the Vicinity of Deep-Sea Hydrothermal Systems

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.     

Respiratory pumping in Aplysia fasciata as part of an integrated defensive response to increases and decreases in seawater concentration

Much of the neural circuitry controlling respiratory pumping in Aplysia has been well characterized, but the function of this movement is incompletely understood. To gain insight into possible functions of respiratory pumping, responses were examined for a 40 min exposure to two stimuli that modulate the movement: 1) increase and 2) decrease in seawater concentration. Thresholds were present for both stimuli to affect respiratory pumping. Above threshold, there were graded increases in the number of pumps elicited. There were decrements in respiratory pump frequency as a function of time exposed to the stimulus. Increased respiratory pumping did not contribute to volume regulation in response to exposure to altered seawaters, but was associated with increased defensive responses, such as escape locomotion (swimming) and inking. In addition, head shock, a well-established noxious stimulus, elicited temporal patterns of respiratory pumping similar to those elicited by altered seawaters. The data indicate that in our experimental conditions, respiratory pumping is elicited as part of an integrated defensive response to noxious seawaters.     

Enhanced biodegradation of diesel fuel through the addition of particulate organic carbon and inorganic nutrients in coastal marine waters

Diesel fuel pollution in coastal waters, resulting from recreational boating and commercial shipping operations, is common and can adversely affect marine biota. The purpose of this study was to examine the effect of additions of particulate organic carbon (POC) in the form of naturally-occurring marsh grass (Spartina alterniflora), inorganic nutrients (nitrogen and phosphorus), inert particles, and dissolved organic carbon (DOC) on diesel fuel biodegradation and to attempt to formulate an effective bioremedial treatment for small diesel fuel spills in marine waters. Various combinations of treatments were added to water samples from a coastal marina to stimulate diesel fuel biodegradation. Diesel fuel was added in concentrations approximating those found in a spill and biodegradation of straight chain aliphatic constituents was estimated by measuring mineralization of ¹⁴C hexadecane added to diesel fuel. All treatments that included POC showed stimulation of biodegradation. However, the addition of inert particles (glass fiber filters and nylon screening) caused no stimulation of biodegradation. The addition of nitrogen and phosphorus alone did not result in stimulation of biodegradation, but nitrogen and Spartina (although not phosphorus and Spartina) did result in stimulation above that of Spartina alone. Maximum biodegradation rates were obtained by the addition of the Spartina POC, ammonium, and phosphate. The addition of mannitol, a labile DOC source with POC and phosphate resulted in a decrease in diesel fuel biodegradation as compared to POC and phosphate alone. The seasonal pattern of diesel fuel biodegradation showed a maximum in the summer and a minimum in the winter. Therefore, of the treatments tested, the most effective for bioremediation of diesel fuel in marine waters is the addition of POC, nitrogen, and phosphorus.     

Enhanced biodegradation of diesel fuel through the addition of particulate organic carbon and inorganic nutrients in coastal marine waters

Diesel fuel pollution in coastal waters, resulting from recreational boating and commercial shipping operations, is common and can adversely affect marine biota. The purpose of this study was to examine the effect of additions of particulate organic carbon (POC) in the form of naturally-occurring marsh grass (Spartina alterniflora), inorganic nutrients (nitrogen and phosphorus), inert particles, and dissolved organic carbon (DOC) on diesel fuel biodegradation and to attempt to formulate an effective bioremedial treatment for small diesel fuel spills in marine waters. Various combinations of treatments were added to water samples from a coastal marina to stimulate diesel fuel biodegradation. Diesel fuel was added in concentrations approximating those found in a spill and biodegradation of straight chain aliphatic constituents was estimated by measuring mineralization of ¹⁴C hexadecane added to diesel fuel. All treatments that included POC showed stimulation of biodegradation. However, the addition of inert particles (glass fiber filters and nylon screening) caused no stimulation of biodegradation. The addition of nitrogen and phosphorus alone did not result in stimulation of biodegradation, but nitrogen and Spartina (although not phosphorus and Spartina) did result in stimulation above that of Spartina alone. Maximum biodegradation rates were obtained by the addition of the Spartina POC, ammonium, and phosphate. The addition of mannitol, a labile DOC source with POC and phosphate resulted in a decrease in diesel fuel biodegradation as compared to POC and phosphate alone. The seasonal pattern of diesel fuel biodegradation showed a maximum in the summer and a minimum in the winter. Therefore, of the treatments tested, the most effective for bioremediation of diesel fuel in marine waters is the addition of POC, nitrogen, and phosphorus.     

Influence of high salinities on the degradation of diesel fuel by bacterial consortia

Microbial communities from three Argentinean saline soils were extracted and tested for their ability to degrade diesel fuel in liquid culture at salinities between 0% and 25%. In each case, the degradation process was continuously monitored by measuring oxygen consumption. Two communities (CR1 and CR2) showed nearly equal degrees of degradation across a salinity range of 0%-10% (the former degrading about 63% of the diesel fuel and the latter about 70% after 53 and 80 d, respectively). Furthermore, the degree of degradation was not significantly lower in the presence of 17.5% salt (58% and 65% degraded, respectively). A third community (El Zorro) showed a maximum turnover at 5% salt (79% diesel fuel degraded) and significant degradation (66%) at a salinity of 10%. However, the degree of degradation by this community clearly dropped at 0% and 15% salt. None of the communities were able to degrade diesel fuel in the presence of 25% salt, but the living cell counts showed that components of the microbial population survived the long-term exposure. The surviving portion is obviously sufficient to allow substantial restoration of the original community, as verified by the BIOLOG method. Isolates of the CR1 community were identified as members of the genera Cellulomonas, Bacillus, Dietzia, and Halomonas. In light of our investigations, the bioremediation of contaminated saline soils should be quite possible if the salinity of the soil water is lower than 15% or if it is reduced below this limit by the addition of water.     

湛江海域浮游藻类生态及其多样性

近岸海域富营养化导致浮游藻类大量繁殖,影响海域生系统平衡。对湛江海域浮游藻类种类组成、数量分布、群落结构等进行冬、夏二季调查,得出湛江海域以硅藻门（Ｂａｃｌｌａｒｉｏ－ｐｈｙｔａ）为主要优势类群。夏季种类多样性高于冬季;赤潮生物种种类、数量均夏季高于冬季。     

Biotechnological processes for biodiesel production using alternative oils

As biodiesel (fatty acid methyl ester (FAME)) is mainly produced from edible vegetable oils, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. The use of waste lipids such as waste frying oils, waste fats, and soapstock has been proposed as low-cost alternative feedstocks. Non-edible oils such as jatropha, pongamia, and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast, and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstocks for FAME production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole-cell catalysts, avoiding soap production as the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered, and the purification of FAME is simplified using biological catalysts.     

Molecular characterization of putative biocorroding microbiota with a novel niche detection of Epsilon- and Zetaproteobacteria in Pacific Ocean coastal seawaters

Submerged metal surfaces in marine waters undergo rapid microbial colonization and biocorrosion, causing huge damage to marine engineering facilities and significant financial losses. In coastal areas, an accelerated and particularly severe form of biocorrosion termed accelerated low water corrosion (ALWC) is widespread globally. While identification of biocorroding microorganisms and the dynamics of their community structures is the key for understanding the processes and mechanisms leading to ALWC, neither one is presently understood. In this study, analysis of constructed clone libraries and qPCR assays targeting group-specific 16S rRNA or functional marker genes were used to determine the identity and abundance of putative early carbon steel surface-colonizing and biocorroding microbes in coastal seawater. Diverse microbial groups including 10 bacterial phyla, archaea and algae were found to putatively participate in the surface-colonizing process. Analysis of the community structure of carbon steel surface microbiota revealed a temporal succession leading to ALWC. By extending the current state of knowledge, our work demonstrates the global importance of Alphaproteobacteria (mainly Rhodobacterales), Gammaproteobacteria (mainly Alteromonadales and Oceanospirillales), Bacteroidetes (mainly Flavobacteriales) and microalgae as the pioneer and sustaining surface colonizers that contribute to initial formation and development of surface biofilms. We also discovered Epsilonproteobacteria and the recently described Zetaproteobacteria as putative corrosion-causing microorganisms during early steps of the ALWC process. Hence, our study reports that Zetaproteobacteria may be ubiquitous also in non-hydrothermal coastal seawaters and that ALWC of submerged carbon steel surfaces in coastal waters may involve a highly diverse, complex and dynamic microbial consortium. Our finding that Epsilon- and Zetaproteobacteria may play pivotal roles in ALWC provides a new starting point for future investigation of the ALWC process and mechanism in marine environments. Further studies of Epsilon- and Zetaproteobacteria in particular may thus help with the design of effective corrosion prevention and control strategies.     

Survival of human enteroviruses in the Hawaiian ocean environment: evidence for virus-inactivating microorganisms.

The stability of certain human enteroviruses in the Hawaiian ocean environment was examined. The present data indicated that the time for 90% reduction of poliovirus type 1 at 24 +/- 1 degree C in seawater samples obtained from different sites in Hawaii ranged from 24 to 48 h, and complete inactivation occurred within 72 to 96 h. The accumulated evidence also strongly indicated that a virus-inactivating agent(s) of a microbiological nature was present in both clean and sewage-polluted seawaters, but not in fresh, mountain stream waters. The antiviral activity was lost when the seawater samples were subjected to boiling, autoclaving, or filtration through a 0.22- or 0.45-micrometer, but not a 1.0-micrometer, membrane filter. That the antiviral activity of the seawater was related to the growth activities of microorganisms was corroborated by the observed effects of added nutrients, a lower temperature of incubation, and the presence of certain antibiotics. Other enteric viruses, such as coxsackie virus B-4 and echo virus-7, were also shown to be similarly inactivated in seawater.     

Development of an enzyme assay for rapid assessment of Escherichia coli in seawaters

AIMS: An analytical protocol has been developed and applied for the detection of glucuronidase activity in marine waters as a rapid alternative approach to assess the microbiological quality of seawaters. METHODS AND RESULTS: The fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide is cleaved to a fluorescent product, methylumbelliferone, by the enzyme beta-glucuronidase, specific to Escherichia coli and closely related enterobacterial species (Shigella). The results suggest that this test is related to E. coli numbers, as estimated by immunofluorescence, more significantly than to faecal coliform numbers, obtained from culture media. CONCLUSIONS: The determination of the potential rate of glucuronidase activity may be used as a diagnostic tool for the indirect estimation of the presence of E. coli in seawaters. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be particularly useful in the early warning of seawater pollution, allowing the screening of coastal areas with different contamination levels in reduced time.     

Survival of human enteroviruses in the Hawaiian ocean environment: evidence for virus-inactivating microorganisms

The stability of certain human enteroviruses in the Hawaiian ocean environment was examined. The present data indicated that the time for 90% reduction of poliovirus type 1 at 24 +/- 1 degree C in seawater samples obtained from different sites in Hawaii ranged from 24 to 48 h, and complete inactivation occurred within 72 to 96 h. The accumulated evidence also strongly indicated that a virus-inactivating agent(s) of a microbiological nature was present in both clean and sewage-polluted seawaters, but not in fresh, mountain stream waters. The antiviral activity was lost when the seawater samples were subjected to boiling, autoclaving, or filtration through a 0.22- or 0.45-micrometer, but not a 1.0-micrometer, membrane filter. That the antiviral activity of the seawater was related to the growth activities of microorganisms was corroborated by the observed effects of added nutrients, a lower temperature of incubation, and the presence of certain antibiotics. Other enteric viruses, such as coxsackie virus B-4 and echo virus-7, were also shown to be similarly inactivated in seawater.     

Relationships Between Environmental Factors, Bacterial Indicators, and the Occurrence of Enteric Viruses in Estuarine Sediments

Current standards for evaluation of the public health safety of recreational and shellfish-harvesting waters are based upon bacteriological analysis, but do not include an evaluation of the number of viruses. The objective of this study was to determine the occurrence of enteric viruses in estuarine sediments and to find a relationship, if any, between the presence of viruses in seawater or sediment or both and various biological and physicochemical characteristics of the environment. Viruses were found in greater numbers in sediment than in overlying seawater on a volume basis. Several types of enteroviruses were isolated: coxsackievirus types A16, B1, and B5, echovirus type 1, and poliovirus type 2. On several occasions, viruses were isolated from sediments when overlying seawaters met bacteriological water quality standards for recreational use. Statistical analysis of the relationship between viruses in seawater or in sediment and other variables measured yielded only one significant association: the number of viruses in sediment was found to be positively correlated with the number of fecal coliforms in sediment. No other physical, chemical, or biological characteristic of seawater or sediment that was measured showed statistically significant association with viral numbers. No correlation was found between bacterial indicators and virus in the overlying waters. The data indicated that evaluation of the presence of bacteria and viruses in sediment may provide additional insight into long-term water quality conditions and that indicator bacteria in water are not reflective of the concentration of enteric viruses in marine waters.     

Biomonitoring of the genotoxic potential (micronucleus and erythrocyte nuclear abnormalities assay) of the Admiralty Bay water surrounding the Brazilian Antarctic Research Station “Comandante Ferraz,” King George Island

The micronucleus (Mn) and other kinds of erythrocyte nuclear abnormality (ENA) assays were used to assess the genotoxic potential of the seawater in front of the Brazilian Antarctic Research Station “Comandante Ferraz” (King George Island). In two consecutive summers, the fish Trematomus newnesi was exposed to the seawater in front of the fuel storage tanks and the sewage discharge outlet of the Station in laboratory bioassays and in situ caging assays. After the exposure, frequencies of ENA and Mn were scored on blood smears. Significantly higher Mn and ENA frequencies in relation to those of the controls were observed in fish exposed to both types of water, in both types of experiment. Seawater in front of the fuel tanks and sewage discharge outlet at the Station was found to induce the formation of micronuclei and other ENAs, indicators of genotoxicity. The need for further investigation and the suitability of the methods for screening mutagenic pollution in polar waters are discussed.