Bacteria stretch and bend oil to feed their appetite.

Microbes reshape oil droplets to speed biodegradation.

Alcanivorax borkumensis biofilms enhance oil degradation by interfacial tubulation.

During the consumption of alkanes, Alcanivorax borkumensis will form a biofilm around an oil droplet, but the role this plays during degradation remains unclear. We identified a shift in biofilm morphology that depends on adaptation to oil consumption: Longer exposure leads to the appearance of dendritic biofilms optimized for oil consumption effected through tubulation of the interface. In situ microfluidic tracking enabled us to correlate tubulation to localized defects in the interfacial cell ordering. We demonstrate control over droplet deformation by using confinement to position defects, inducing dimpling in the droplets. We developed a model that elucidates biofilm morphology, linking tubulation to decreased interfacial tension and increased cell hydrophobicity.

Alcanivorax xiamenensis sp. nov., a novel marine hydrocarbonoclastic bacterium isolated from surface seawater.

A novel Alcanivorax-related strain, designated 6-D-6T, was isolated from the surface seawater collected around Xiamen Island. The novel strain is Gram-stain-negative, rod-shaped and motile, and grows at 10-45 °C, pH 6.0-9.0 and in the presence of 0.5-15.0 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that it belongs to the genus Alcanivorax, with the highest sequence similarity to Alcanivorax dieselolei B5T (99.9 %), followed by Alcanivorax xenomutans JC109T (99.5 %), Alcanivorax balearicus MACL04T (99.3 %) and other 13 species of the genus Alcanivorax (93.8 %-95.6 %). The digital DNA-DNA hybridization and average nucleotide identity values between strain 6-D-6T and three close type strains were 40.1-42.9/90.6-91.4 %, and others were below 22.9/85.1 %, respectively. The novel strain contained major cellular fatty acids of C16 : 0 (31.0 %), C19 : 0 ω8c cyclo (23.5 %), C17 : 0 cyclo (9.7 %), C12 : 0 3OH (8.6 %), summed feature 8 (7.6 %) and C12 : 0 (5.4 %). The genomic G+C content of strain 6-D-6T was 61.38 %. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two unidentified phospholipids and one amino-group-containing phospholipid were detected. On the basis of phenotypic and genotypic characteristics, strain 6-D-6T represents a novel species within the genus Alcanivorax, for which the name Alcanivorax xiamenensis sp. nov. is proposed. The type strain is 6-D-6T (=MCCC 1A01359T=KCTC 92480T).

Alcanivorax quisquiliarum sp. nov., isolated from anaerobic fermentation liquid of food waste by high-throughput cultivation.

Strain CY1518T was isolated from an anaerobic fermentation liquid of food waste treatment plant in Beijing, PR China, and characterized to assess its taxonomy. Cells of CY1518T were Gram-stain-negative, oxidase-negative, catalase-positive and ellipsoidal. Growth occurred at 20-42 °C (optimum, 37 °C), pH 6.0-10.0 (optimum, pH 8) and with 0-6.0 % (w/v) NaCl (optimum, 1.5%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CY1518T belongs to the genus Alcanivorax, with the highest sequence similarity to Alcanivorax pacificus W11-5T (95.97 %), followed by Alcanivorax indicus SW127T (95.08%). The similarity between strain CY1518T and other strains of Alcanivorax was less than 95 %. The genomic DNA G+C content of strain CY1518T was 60.88 mol%. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain CY1518T and the closely related taxa A. pacificus W11-5T and A. indicus SW127T were 77.61, 78.03 and 21.2 % and 74.15, 70.02 and 19.3%, respectively. The strain was able to use d-serine, Tween 40 and some organic acid compounds for growth. The polar lipids comprised aminophospholipid, diphosphatidylglycerol, glycolipid, an unknown polar lipid, phosphatidylethanolamine, phosphatidylglycerol and phospholipid. The principal fatty acids (>5 %) were C19 : 0 cyclo ω8c (36.3%), C16 : 0 (32.3%), C12 : 0 3-OH (8.3%) and C12 : 0 (7.6%). Based on its phenotypic, genotypic and genomic characteristics, strain CY1518T represents a novel species in the genus Alcanivorax, for which the name Alcanivorax quisquiliarum sp. nov. is proposed. The type strain is CY1518T (=GDMCC 1.2918T=JCM 35120T).

Phylogenomic analysis of the genus Alcanivorax: proposal for division of this genus into the emended genus Alcanivorax and two novel genera Alloalcanivorax gen. nov. and Isoalcanivorax gen. nov.

The members of the genus Alcanivorax are key players in the removal of petroleum hydrocarbons from polluted marine environments. More than half of the species were described in the last decade using 16S rRNA gene phylogeny and genomic-based metrics. However, the 16S rRNA gene identity (<94 %) between some members of the genus Alcanivorax suggested their imprecise taxonomic status. In this study, we examined the taxonomic positions of Alcanivorax species using 16S rRNA phylogeny and further validated them using phylogenomic-related indexes such as digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), average amino acid identity (AAI), percentage of conserved proteins (POCP) and comparative genomic studies. ANI and dDDH values confirmed that all the Alcanivorax species were well described at the species level. The phylotaxogenomic analysis showed that Alcanivorax species formed three clades. The inter-clade values of AAI and POCP were less than 70 %. The pan-genome evaluation depicted that the members shared 1223 core genes and its number increased drastically when analysed clade-wise. Therefore, these results necessitate the transfer of clade II and clade III members into Isoalcanivorax gen. nov. and Alloalcanivorax gen. nov., respectively, along with the emended description of the genus Alcanivorax sensu stricto.

The effect of Alcanivorax borkumensis SK2, a hydrocarbon-metabolising organism, on gas holdup in a 4-phase bubble column bioprocess.

To design bioprocesses utilising hydrocarbon-metabolising organisms (HMO) as biocatalysts, the effect of the organism on the hydrodynamics of bubble column reactor (BCR), such as gas holdup, needs to be investigated. Therefore, this study investigates the first use of an HMO, Alcanivorax borkumensis SK2, as a solid phase in the operation and hydrodynamics of a BCR. The study investigated the gas holdup in 3-phase and 4-phase systems in a BCR under ranges of superficial gas velocities (UG) from 1 to 3 cm/s, hydrocarbon (chain length C13-21) concentrations (HC) of 0, 5, and 10% v/v and microbial concentrations (MC) of 0, 0.35, 0.6 g/l. The results indicated that UG was the most significant parameter, as gas holdup increases linearly with increasing UG from 1 to 3 cm/s. Furthermore, the addition of hydrocarbons into the air-deionized water -SK2 system showed the highest increase in the gas holdup, particularly at high UG (above 2 cm/s). The solids (yeast, cornflour, and SK2) phases had differing effects on gas holdup, potentially due to the difference in surface activity. In this work, SK2 addition caused a reduction in the fluid surface tension in the bioprocess which therefore resulted in an increase in the gas holdup in BCR. This work builds upon previous investigations in optimising the hydrodynamics for bubble column hydrocarbon bioprocesses for the application of alkane bioactivation.

High abundance of hydrocarbon-degrading Alcanivorax in plumes of hydrothermally active volcanoes in the South Pacific Ocean.

Species within the genus Alcanivorax are well known hydrocarbon-degraders that propagate quickly in oil spills and natural oil seepage. They are also inhabitants of the deep-sea and have been found in several hydrothermal plumes. However, an in-depth analysis of deep-sea Alcanivorax is currently lacking. In this study, we used multiple culture-independent techniques to analyze the microbial community composition of hydrothermal plumes in the Northern Tonga arc and Northeastern Lau Basin focusing on the autecology of Alcanivorax. The hydrothermal vents feeding the plumes are hosted in an arc volcano (Niua), a rear-arc caldera (Niuatahi) and the Northeast Lau Spreading Centre (Maka). Fluorescence in situ hybridization revealed that Alcanivorax dominated the community at two sites (1210-1565 mbsl), reaching up to 48% relative abundance (3.5 × 104 cells/ml). Through 16S rRNA gene and metagenome analyses, we identified that this pattern was driven by two Alcanivorax species in the plumes of Niuatahi and Maka. Despite no indication for hydrocarbon presence in the plumes of these areas, a high expression of genes involved in hydrocarbon-degradation was observed. We hypothesize that the high abundance and gene expression of Alcanivorax is likely due to yet undiscovered hydrocarbon seepage from the seafloor, potentially resulting from recent volcanic activity in the area. Chain-length and complexity of hydrocarbons, and water depth could be driving niche partitioning in Alcanivorax.

High-Resolution Small RNAs Landscape Provides Insights into Alkane Adaptation in the Marine Alkane-Degrader Alcanivorax dieselolei B-5

Alkanes are widespread in the ocean, and Alcanivorax is one of the most ubiquitous alkane-degrading bacteria in the marine ecosystem. Small RNAs (sRNAs) are usually at the heart of regulatory pathways, but sRNA-mediated alkane metabolic adaptability still remains largely unknown due to the difficulties of identification. Here, differential RNA sequencing (dRNA-seq) modified with a size selection (~50-nt to 500-nt) strategy was used to generate high-resolution sRNAs profiling in the model species Alcanivorax dieselolei B-5 under alkane (n-hexadecane) and non-alkane (acetate) conditions. As a result, we identified 549 sRNA candidates at single-nucleotide resolution of 5'-ends, 63.4% of which are with transcription start sites (TSSs), and 36.6% of which are with processing sites (PSSs) at the 5'-ends. These sRNAs originate from almost any location in the genome, regardless of intragenic (65.8%), antisense (20.6%) and intergenic (6.2%) regions, and RNase E may function in the maturation of sRNAs. Most sRNAs locally distribute across the 15 reference genomes of Alcanivorax, and only 7.5% of sRNAs are broadly conserved in this genus. Expression responses to the alkane of several core conserved sRNAs, including 6S RNA, M1 RNA and tmRNA, indicate that they may participate in alkane metabolisms and result in more actively global transcription, RNA processing and stresses mitigation. Two novel CsrA-related sRNAs are identified, which may be involved in the translational activation of alkane metabolism-related genes by sequestering the global repressor CsrA. The relationships of sRNAs with the characterized genes of alkane sensing (ompS), chemotaxis (mcp, cheR, cheW2), transporting (ompT1, ompT2, ompT3) and hydroxylation (alkB1, alkB2, almA) were created based on the genome-wide predicted sRNA-mRNA interactions. Overall, the sRNA landscape lays the ground for uncovering cryptic regulations in critical marine bacterium, among which both the core and species-specific sRNAs are implicated in the alkane adaptive metabolisms.

Responses of Alcanivorax species to marine alkanes and polyhydroxybutyrate plastic pollution: Importance of the ocean hydrocarbon cycles.

Understanding microbial responses to hydrocarbon and plastic pollution are crucial for limiting the detrimental impacts of environmental contaminants on marine ecosystems. Herein, we reported a new Alcanivorax species isolated from the North Atlantic Ocean capable of degrading alkanes and polyhydroxybutyrate (PHB) plastic (one of the emerging bioplastics that may capture the future plastic market). The whole-genome sequencing showed that the species harbors three types of alkane 1-monooxygenases (AlkB) and one PHB depolymerase (PhaZ) to initiate the degradation of alkanes and plastics. Growth profiling demonstrated that n-pentadecane (C15, the main alkane in the marine environment due to cyanobacterial production other than oil spills) and PHB could serve as preferential carbon sources. However, the cell membrane composition, PhaZ activity, and expression of three alkB genes were utterly different when grown on C15 and PHB. Further, Alcanivorax was a well-recognized alkane-degrader that participated in the ocean hydrocarbon cycles linking with hydrocarbon production and removal. Our discovery supported that the existing biogeochemical processes may add to the marine ecosystem's resilience to the impacts of plastics.

The Glycine-Glucolipid of Alcanivorax borkumensis Is Resident to the Bacterial Cell Wall.

The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

A mechanistic understanding of polyethylene biodegradation by the marine bacterium Alcanivorax.

Polyethylene (PE) is one of the most recalcitrant carbon-based synthetic materials produced and, currently, the most ubiquitous plastic pollutant found in nature. Over time, combined abiotic and biotic processes are thought to eventually breakdown PE. Despite limited evidence of biological PE degradation and speculation that hydrocarbon-degrading bacteria found within the plastisphere is an indication of biodegradation, there is no clear mechanistic understanding of the process. Here, using high-throughput proteomics, we investigated the molecular processes that take place in the hydrocarbon-degrading marine bacterium Alcanivorax sp. 24 when grown in the presence of low density PE (LDPE). As well as efficiently utilising and assimilating the leachate of weathered LDPE, the bacterium was able to reduce the molecular weight distribution (Mw from 122 to 83 kg/mol) and overall mass of pristine LDPE films (0.9 % after 34 days of incubation). Most interestingly, Alcanivorax acquired the isotopic signature of the pristine plastic and induced an extensive array of metabolic pathways for aliphatic compound degradation. Presumably, the primary biodegradation of LDPE by Alcanivorax sp. 24 is possible via the production of extracellular reactive oxygen species as observed both by the material's surface oxidation and the measurement of superoxide in the culture with LDPE. Our findings confirm that hydrocarbon-biodegrading bacteria within the plastisphere may in fact have a role in degrading PE.

Pilot-scale production and in-situ application of petroleum-degrading enzyme cocktail from Alcanivorax borkumensis.

Petroleum degrading enzymes can be used as an alternative way to improve petroleum bioremediation approaches. Alcanivorax borkumensis is an alkane-degrading bacteria that can produce petroleum degrading enzymes such as alkane hydroxylase and lipase. In this study, pilot-scale Alcanivorax borkumensis fermentation was developed for producing large volumes of petroleum degrading enzymes cocktail (∼900 L). Different process conditions, such as inoculum age 72 h and size 4% v/v, temperature 30 ± 1 °C, agitation speed at 150 rpm and, fermentation period 3 days were determined as the optimum for producing alkane hydroxylase and lipase activity. The oxygen transfer capacity was studied for obtaining better bacterial growth and higher enzyme activities in bioreactor process optimization as well as scale-up. Results showed that the maximum values of oxygen mass transfer coefficient (kLa), oxygen uptake rate (OUR), oxygen transfer rate (OTR), alkane hydroxylase, lipase, and cell count were 196.95 h-1, 0.92 mmol O2/L/h, 1.8 mmol O2/L/h, 222.49 U/mL, 325 U/mL, and 8.6 × 1010 CFU/mL, respectively. Compared with the bench-scale bioreactors, the 150 L fermenter showed a better oxygen transfer rate which affected the cell growth that doubled the number and enzymes production that increased. Then, the enzyme cocktail was used for a field test in a diesel source zone using a 5-spot well pattern. The results showed a significant reduction in concentrations of C10 - C50 (from 36% to > 99%) after one injection of enzyme cocktail, mainly for the contaminated soils located in the saturated zone of the unconfined aquifer. This study confirmed the scaling-up ofalkane-degrading enzyme production to an industrial-scale and its application for effective bioremediation of petroleum contaminated sites.

Mesopelagic microbial community dynamics in response to increasing oil and Corexit 9500 concentrations.

Marine microbial communities play an important role in biodegradation of subsurface plumes of oil that form after oil is accidentally released from a seafloor wellhead. The response of these mesopelagic microbial communities to the application of chemical dispersants following oil spills remains a debated topic. While there is evidence that contrasting results in some previous work may be due to differences in dosage between studies, the impacts of these differences on mesopelagic microbial community composition remains unconstrained. To answer this open question, we exposed a mesopelagic microbial community from the Gulf of Mexico to oil alone, three concentrations of oil dispersed with Corexit 9500, and three concentrations of Corexit 9500 alone over long periods of time. We analyzed changes in hydrocarbon chemistry, cell abundance, and microbial community composition at zero, three and six weeks. The lowest concentration of dispersed oil yielded hydrocarbon concentrations lower than oil alone and microbial community composition more similar to control seawater than any other treatments with oil or dispersant. Higher concentrations of dispersed oil resulted in higher concentrations of microbe-oil microaggregates and similar microbial composition to the oil alone treatment. The genus Colwellia was more abundant when exposed to multiple concentrations of dispersed oil, but not when exposed to dispersant alone. Conversely, the most abundant Marinobacter amplicon sequence variant (ASV) was not influenced by dispersant when oil was present and showed an inverse relationship to the summed abundance of Alcanivorax ASVs. As a whole, the data presented here show that the concentration of oil strongly impacts microbial community response, more so than the presence of dispersant, confirming the importance of the concentrations of both oil and dispersant in considering the design and interpretation of results for oil spill simulation experiments.

Isolation of bacteria able to degrade poly-hydroxybutyrate-co-hydroxyhexanoate, and the inhibitory effects of the degradation products on shrimp pathogen Vibrio penaeicida.

Poly-hydroxybutyrate-co-hydroxyhexanoate (PHBH) is a biodegradable, water-insoluble polymer produced by specific bacteria. The monomers of PHBH are the hydroxyalkanoic acids 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HH). Previously, we reported that 3HB and 3HH showed marked antibacterial activities against the shrimp pathogenic bacterium Vibrio penaeicida, and that addition of 5% (w/w) PHBH to the standard aquaculture diet significantly increased survival rate in kuruma shrimp (Marsupenaeus japonicus) after challenge by V. penaeicida, which we attributed to the degradation of PHBH to its monomers in the shrimp gut. In the present study, we isolated four strains of bacteria with high PHBH-degrading activity and evaluated their inhibitory effects on V. penaeicida with PHBH: one strain from shrimp gut contents (E1; Pseudoalteromonas shioyasakiensis/P. mariniglutinosa), two strains from coastal surface seawater (F1; P. shioyasakiensis/P. mariniglutinosa, and F5; Alcanivorax dieselolei/A. xenomutans), and one strain that was a contaminant in commercial PHBH powder (Y1; Bacillus pseudofirmus). Strains E1, F1, and Y1 showed strong PHBH-degrading activity within 24 h of inoculation to PHBH-containing agar plates. Although none of the isolates alone had any effect on the growth of V. penaeicida, when cultured with E1 or F1 and PHBH, the growth of V. penaeicida was markedly suppressed. Incubation with E1 and PHBH resulted in a gradual reduction in the concentration of V. penaeicida from 2 days after the start of incubation until the concentration was 1.2% of that in the control (V. penaeicida alone). Incubation with F1 and PHBH resulted in a rapid reduction in the concentration of V. penaeicida from 2 days after the start of incubation until the concentration was only 0.32% of that of the control. Compared with strains E1 and F1, Y1 showed similar PHBH-degrading activity but did not show any suppressive effect on the growth of V. penaeicida until 5 days after the start of incubation. In addition, this suppressive effect was relatively weak compared with that of the other two strains, suggesting that Y1 can quickly degrade PHBH but that it takes several days to produce monomers. Together, these results suggest that addition to the aquaculture diet of PHBH and PHBH-degrading bacteria that rapidly degrade PHBH to its monomers may speed up degradation of PHBH to its monomers in the shrimp gut, and that it would increase resistance to infection mortality by V. penaeicida in kuruma shrimp.

New insights into the structure and function of the prokaryotic communities colonizing plastic debris collected in King George Island (Antarctica): Preliminary observations from two plastic fragments.

In Antarctic regions, the composition and metabolic activity of microbial assemblages associated with plastic debris ("plastisphere") are almost unknown. A macroplastic item from land (MaL, 30 cm) and a mesoplastic from the sea (MeS, 4 mm) were collected in Maxwell Bay (King George Island, South Shetland) and analyzed by Fourier transform infrared spectroscopy in attenuated total reflectance geometry (FTIR-ATR), which confirmed a polystyrene foam and a composite high-density polyethylene composition for MaL and MeS, respectively. The structure and function of the two plastic-associated prokaryotic communities were studied by complementary 16S ribosomal RNA gene clone libraries, total bacterioplankton and culturable heterotrophic bacterial counts, enzymatic activities of the whole community and enzymatic profiles of bacterial isolates. Results showed that Gamma- and Betaproteobacteria (31% and 28%, respectively) dominated in MeS, while Beta- and Alphaproteobacteria (21% and 13%, respectively) in MaL. Sequences related to oil degrading bacteria (Alcanivorax,Marinobacter) confirmed the known anthropogenic pressure in King George Island. This investigation on plastic-associated prokaryotic structure and function represents the first attempt to characterize the ecological role of plastisphere in this Antarctic region and provides the necessary background for future research on the significance of polymer type, surface characteristics and environmental conditions in shaping the plastisphere.

Genome sequence of an obligate hydrocarbonoclastic bacterium Alcanivorax marinus NMRL4 isolated from oil polluted seawater of the Arabian Sea.

Alcanivorax belongs to the hydrocarbonoclastic group of bacteria that are known for their preferential growth on alkanes and other related compounds. Here we report the genomic features of Alcanivorax marinus strain NMRL4 (=MCC 4632) isolated from oil polluted seawater of the Arabian Sea. Its 4,062,055 bp genome with 66.1% GC content encodes for 3935 coding sequences. The genome annotations of strain NMRL4 revealed the presence of multiple hydrocarbon degradation genes suggestive of its wider hydrocarbon substrate range. The strain encodes for three alkane monooxygenases, two cytochrome P450 and two flavin binding monooxygenases for degradation of short and long-chain alkanes. The genome shows capabilities for scavenging of nutrients, biofilm formation at oil-water interfaces, chemotaxis, motility and habitat specific adaptation. The genomic insights showed that the strain NMRL4 is an ideal candidate for bioremediation of pollutant petroleum hydrocarbons from the marine environment.

Column tests for evaluation of the enzymatic biodegradation capacity of hydrocarbons (C10-C50) contaminated soil.

Though many studies pertaining to soil bioremediation have been performed to study the microbial kinetics in shake flasks, the process efficiency in column tests is seldom. In the present study, soil columns tests were carried out to study the biodegradation of soil contaminated with a high concentration of diesel (≈19.5 g/kg) petroleum hydrocarbons expressed as C10-C50. Experiments were done with crude enzymatic cocktail produced by the hydrocarbonoclastic bacterium, Alcanivorax borkumensis. A. borkumensis was grown on a media with 3% (v/v) motor oil as the sole carbon and energy source. The effects of the enzyme concentration, treatment time and oxidant on the bioremediation efficiency of C10-C50 were investigated. A batch test was also carried out in parallel to investigate the stability of the enzymes and the effect of the biosurfactants on the desorption and the bioconversion of C10-C50. Batch tests indicated that the biosurfactants significantly affected the desorption and alkane hydroxylase and lipase enzymes, maintained their catalytic activity during the 20-day test, with a half-life of 7.44 days and 8.84 days, respectively. The crude enzyme cocktail, with 40 U/mL of lipase and 10 U/mL of alkane hydroxylase, showed the highest conversion of 57.36% after 12 weeks of treatment with a degradation rate of 0.0218 day-1. The results show that the soil column tests can be used to optimize operating conditions for hydrocarbon degradation and to assess the performance of the overall bioremediation process.

Alcanivorax profundimaris sp. nov., a Novel Marine Hydrocarbonoclastic Bacterium Isolated from Seawater and Deep-Sea Sediment.

Two novel Alcanivorax-related strains, designated ST75FaO-1T and 521-1, were isolated from the seawater of the South China Sea and the deep-sea sediment of the West Pacific Ocean, respectively. Both strains are Gram-stain-negative, rod-shaped, and non-motile, and grow at 10-40 °C, pH 5.0-10.0, in the presence of 1.0-15.0% (w/v) NaCl. Their 16S rRNA gene sequences showed 99.9% similarity. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that both strains belong to the genus Alcanivorax, and share 92.9-98.1% sequence similarity with all valid type strains of this genus, with the highest similarity being to type strain Alcanivorax venustensis DSM 13974T (98.0-98.1%). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity values between strains ST75FaO-1T and 521-1 were 75.7% and 97.1%, respectively, while the corresponding values with A. venustensis DSM 13974T were only 25.4-25.6% and 82.4-82.7%, respectively. The two strains contained similar major cellular fatty acids including C16:0, C18:1 ω7c/ω6c, C19:0 cyclo ω8c, C16:1 ω7c/ω6c, C12:0 3-OH, and C12:0. The genomic G + C content of strains ST75FaO-1T and 521-1 were 66.3% and 66.1%, respectively. Phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids, and one unidentified polar lipid were present in both strains. On the basis of phenotypic and genotypic characteristics, the two strains represent a novel species within the genus Alcanivorax, for which the name Alcanivorax profundimaris sp. nov. is proposed. The type strain is ST75FaO-1T (= MCCC 1A17714T = KCTC 82142T).

Alcanivorax limicola sp. nov., isolated from a soda alkali-saline soil.

An alkaliphilic and aerobic bacterium, designated as strain JB21T, was isolated from a soda alkali-saline soil sample in Heilongjiang, Northeast China. Strain JB21T is a Gram-stain-negative, rod-shaped, non-motile and amylase-positive bacterium. Growth occurred at 15-45 °C (optimum, 35-37 °C), in the presence of 0-15.0% (w/v) NaCl (optimum, 1.0%) and at pH 6.5-10.5 (optimum, pH 8.5-9.5). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JB21T was most closely related to type strains of the genus Alcanivorax, with the highest sequence similarity to Alcanivorax indicus SW127T (96.3%), and shared 95.4-93.1% sequence identity with other valid type strains of this genus. The major cellular fatty acids identified were C16:0 and summed feature 8 (C18:1ω6c and/or C18:1ω7c). The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol and one unidentified phospholipid. The genomic G + C content of strain JB21T was 61.3 mol%. The digital DNA-DNA hybridization (dDDH) estimation and average nucleotide identity (ANI) between strain JB21T and type strains of the genus Alcanivorax were 18.3-23.2% and 69.2-79.0%, respectively. On the basis of its phenotypic and phylogenetic characteristics, we suggest the creation of a new species within the Alcanivorax genus, named Alcanivorax limicola sp. nov., type strain JB21T (= CGMCC 1.16632T = JCM 33717T).