Marine biodegradation of plastic films by Alcanivorax under various ambient temperatures: Bacterial enrichment, morphology alteration, and release of degradation products.

The global ocean has been receiving massive amounts of plastic wastes. Marine biodegradation, influenced by global climate, naturally breaks down these wastes. In this study, we systematically compared the biodegradation performance of petroleum- and bio-based plastic films, i.e., low-density polyethylene (LDPE), polylactic acid (PLA), and polyhydroxyalkanoates (PHAs) under three ambient temperatures (4, 15, and 22 °C). We deployed the our previously isolated cold-tolerant plastic-degrading Alcanivorax to simulate the accelerated marine biodegradation process and evaluated the alteration of bacterial growth, plastic films, and released degradation products. Notably, we found that marine biodegradation of PHA films enriched more bacterial amounts, induced more conspicuous morphological damage, and released more microplastics (MPs) and dissolved organic carbon (DOC) under all temperatures compared to LDPE and PLA. Particularly, MPs were released from film edges and cracks with a mean size of 2.8 µm under all temperatures. In addition, the degradation products released by biodegradation of PHA under 22 °C induced the highest acute toxicity to Vibrio fischeri. Our results highlighted that: (1) marine biodegradation of plastics would release millions of MPs per cm2 exposed surface area even in cold environments within 60 days; (2) different marine biodegradation scenarios of these plastics may raise disparate impacts and mitigation-related studies.

Marinicella marina sp. nov. and Marinicella gelatinilytica sp. nov., isolated from coastal sediment, and genome analysis and habitat distribution of the genus Marinicella.

Three Marinicella strains, X102, S1101T and S6413T, were isolated from sediment samples from different coasts of Weihai, PR China. All strains were Gram-stain-negative, rod-shaped and non-motile. The predominant fatty acids of all strains were iso-C15 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c) and the major polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Strains X102 and S1101T shared 100 % 16S rRNA gene sequence similarity, and strains S1101T/X102 and S6413T had 95.4 % similarity. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strains S1101T and X102 were 99.9 and 99.2 %, respectively. Strain S1101T had ANI values of 69.1-72.9% and dDDH values of 17.9-20.5 % to members of the genus Marinicella. Strain S6413T had ANI values of 69.1-77.5% and dDDH values of 17.6-21.5 % to members of the genus Marinicella. The results of phylogenetic and comparative genomic analysis showed that the three strains belong to two novel species in the genus Marinicella, and strains X102 and S1101T represented one novel species, and strain S6413T represented another novel species. The result of BOX-PCR and genomic analysis showed that X102 and S1101T were not the same strain. The phylogenetic analyses and genomic comparisons, combined with phylogenetic, phenotypic and chemotaxonomic features, strongly supported that the three strains should be classified as representing two novel species of the genus Marinicella, for which the names Marinicella marina sp. nov. and Marinicella gelatinilytica sp. nov. are proposed, respectively. The type strains of the two novel species are S1101T (=KCTC 92642T=MCCC 1H01359T) and S6413T (=KCTC 92641T=MCCC 1H01362T), respectively. In addition, all previously described isolates of Marinicella were isolated from marine environments, but our study showed that Marinicella is also distributed in non-/low-saline habitats (e.g. animal gut, soil and indoor surface), which broadened our perception of the environmental distribution of Marinicella.

Alcanivorax bacteria as important polypropylene degraders in mesopelagic environments.

IMPORTANCE: PP biodegradation has not been clearly shown (it has been uncertain whether the PP structure is actually biodegraded or not). This is the first report on the obvious biodegradation of PP. At the same time, this study shows that Alcanivorax bacteria could be major degraders of PP in mesopelagic environments. Moreover, PP biodegradation has been investigated by using solid PP as the sole carbon source. However, this study shows that PP would not be used as a sole carbon and energy source. Our data thus provide very important and key knowledge for PP bioremediation.

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.

Bacteria stretch and bend oil to feed their appetite.

Microbes reshape oil droplets to speed biodegradation.

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).

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.

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.

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.

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.

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.