Hexadecane Degradation Research Articles

Dynamics of microbial-induced oil degradation at the microscale.

Microbial-induced oil degradation (MIOD) has a wide range of applications, such as microbial enhanced oil recovery and bioremediation of oil pollution. However, our understanding of MIOD is still far from complete. Particularly, how is the dynamics of degradation process at the microscale level with a single-cell resolution remains to be disclosed. In this work, using hexadecane droplets in water as a model system, we have studied the dynamics of hexadecane degradation by different strains, including Pseudomonas aeruginosa PAO1, IMP68, O-2-2, and Dietzia sp. DQ12-45-1b, at the microscale. Based on visualization of MIOD, the dynamics of MIOD can be characterized by a three-stage process, including adhesion, adaptation, and degradation stages. Although different strains showed similar three-stage dynamics of MIOD, the effective degradation rate varied and followed an order of PAO1 > O-2-2 > IMP68 > DQ12-45-1b under aerobic conditions. Different oxygen conditions were also tested, and the dynamics of MIOD was slowed down under anaerobic conditions in comparison to under aerobic conditions. Further investigations at the degradation stage revealed that biofilms formed at the oil-water interface enhanced oil degradation, but a prerequisite for such enhanced degradation is proper stimulation of biofilm cells in the course of biofilm formation. The findings in this work provided a detailed picture on the dynamics of MIOD at the microscale and would be beneficial for better applications of MIOD.IMPORTANCEMicrobial-induced oil degradation is environmental friendly and economic and has become a promising technique in the fields of enhanced oil recovery and remediation of crude oil-polluted environments. For better applications of microbial-induced oil degradation, understanding the degradation dynamics particularly at the microscale is crucial. In this study, we investigated the degradation dynamics of hexadecane oil droplets incubated with different strains, including Pseudomonas aeruginosa PAO1, O-2-2, IMP68, and Dietzia sp. DQ12-45-1b at the microscale by employing microdroplet-based methods and bacterial tracking techniques. The findings in this study provided a detailed picture on the dynamics of microbial-induced oil degradation at the microscale, which will deepen our understandings on the biodegradation mechanisms of alkanes and shed insights for developing more effective biodegradation techniques.

Multi-omics association study of hexadecane degradation in haloarchaeal strain Halogranum rubrum RO2-11

Haloarchaea with the capacity to degrade alkanes is promising to deal with petroleum pollution in hypersaline environments. However, only a limited number of haloarchaeal species are investigated, and their pathway and mechanism for alkane degradation remain unclear. In this study, Halogranum rubrum RO2-11, a haloarchaeal strain, verified the ability to degrade kerosene and hexadecane in 184 g/L NaCl, with 53% and 52% degradation rates after 9 and 4 days, respectively. Genome sequencing and gene annotation indicated that strain RO2-11 possesses a complete potential alkane-degrading pathway, of which alkane hydroxylases may include CYP450, AlmA, and LadA. Transcriptome and metabolome analyses revealed that the upregulation of related genes in TCA cycle, lysine biosynthesis, and acetylation may help improve hexadecane degradation. Additionally, an alternative degrading pathway of hexadecane based on dual-terminal β-oxidation may occur in strain RO2-11. It is likely to be the first report of alkane degradation by the genus Halogranum, which may be helpful for applications of oil-pollution bioremediation under high-salt conditions.

Anaerobic hexadecane degradation by a thermophilic Hadarchaeon from Guaymas Basin.

Hadarchaeota inhabit subsurface and hydrothermally heated environments, but previous to this study, they had not been cultured. Based on metagenome-assembled genomes, most Hadarchaeota are heterotrophs that grow on sugars and amino acids, or oxidize carbon monoxide or reduce nitrite to ammonium. A few other metagenome-assembled genomes encode alkyl-coenzyme M reductases (Acrs), β-oxidation, and Wood-Ljungdahl pathways, pointing toward multicarbon alkane metabolism. To identify the organisms involved in thermophilic oil degradation, we established anaerobic sulfate-reducing hexadecane-degrading cultures from hydrothermally heated sediments of the Guaymas Basin. Cultures at 70°C were enriched in one Hadarchaeon that we propose as Candidatus Cerberiarchaeum oleivorans. Genomic and chemical analyses indicate that Ca. C. oleivorans uses an Acr to activate hexadecane to hexadecyl-coenzyme M. A β-oxidation pathway and a tetrahydromethanopterin methyl branch Wood-Ljungdahl (mWL) pathway allow the complete oxidation of hexadecane to CO2. Our results suggest a syntrophic lifestyle with sulfate reducers, as Ca. C. oleivorans lacks a sulfate respiration pathway. Comparative genomics show that Acr, mWL, and β-oxidation are restricted to one family of Hadarchaeota, which we propose as Ca. Cerberiarchaeaceae. Phylogenetic analyses further indicate that the mWL pathway is basal to all Hadarchaeota. By contrast, the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex in Ca. Cerberiarchaeaceae was horizontally acquired from Bathyarchaeia. The Acr and β-oxidation genes of Ca. Cerberiarchaeaceae are highly similar to those of other alkane-oxidizing archaea such as Ca. Methanoliparia and Ca. Helarchaeales. Our results support the use of Acrs in the degradation of petroleum alkanes and suggest a role of Hadarchaeota in oil-rich environments.

Non-syntrophic Methanogenic Hydrocarbon Degradation by an Archaeal Species

Methanogenic hydrocarbon biodegradation alters the composition of many subsurface oil reservoirs (Jones et al. 2007). This process reduced the crude oil quality by removing alkanes and thus increasing the oil viscosity. The process has been described for syntrophic associations of hydrocarbon-degrading bacteria and methanogenic archaea (Zengler et al. 1999, Dolfing et al. 2007). However, recent culture-independent studies suggest that the archaeon ‘Candidatus Methanoliparum’ may combine alkane degradation and methanogenesis (Laso-Pérez et al. 2019, Borrel et al. 2019). Here we cultured Ca. Methanoliparum from a subsurface oil reservoir. To study this culture, situ hybridization, metagenomics and metatranscriptomics were combined with stable isotope probing and metabolite analyses for describing its functioning and assessing its potential role in reservoir chemistry. Incubated an anoxic oily sludge of the Shengli oilfield with sulfate-free medium, we established a methanogenic culture. This culture consumed various different long-chain alkanes, but also alkyl-benzenes and alkyl-cycloalkanes, and produced methane and CO2 as products (Fig. 1a-b). Our analyses revealed that our culture is dominated by a single archaeon, Ca. Methanoliparia (green). To study the specific turnover of n-alkanes, the cultures were supplemented with 1,2-13C-labelled or unlabelled n-hexadecane (Fig. 2). Within 100 days of incubation, both compounds were quantitatively converted into methane and carbon dioxide. In the 13C-labelling experiment, around 0.46 mmol of 13CH4 and around 0.15 mmol of 13CO2 were produced, which was equal to 85% to 92% of the stoichiometric conversion of the supplemented labelled hexadecane according to 4C16H34 + 30H2O -- 49CH4 + 15CO2 (Fig. 2a-d). We examined the functioning of Ca. Methanoliparum in the hexadecane-degrading culture using amplicon sequencing, metagenomics and metatranscriptomics. In the archaeal domain, the relative abundance of Ca. Methanoliparum in the hexadecane-degrading cultures comprised up to 75% of the total abundance according to analysis of archaeal 16S rRNA genes. Furthermore, Ca. Methanoliparum accounted for approximately 34–40% of the total microbial community as determined by metagenomic read recruitment estimation (Fig. 2e-f). We analysed the gene expression patterns of Ca. Methanoliparum during methanogenic hexadecane degradation (Fig. 3). The genes encoding the methanogenic hexadecane degradation pathway ranked among the top 10% to 25% of all Ca. M. thermophilum transcribed genes. Moreover, genes of Ca. M. thermophilum encoding ACR and MCR ranked among the top 2% of all transcribed genes within the whole community (Fig. 3b). The MAGs of Ca. M. thermophilum also showed the highest transcription among all described MAGs (Fig. 3c). These analyses indicate that Ca. M. thermophilum performs both the degradation of hexadecane and the formation of methane. We searched the cell extracts of the hexadecane-degrading cultures for hexadecyl-CoM formation using Q-Exactive Plus Orbitrap masss pectrometry. The unlabelled hexadecane culture contained a prominent mass peak of m/z = 365.21868 that matches the mass produced by synthesized authentic standard of hexadecyl-CoM. Fragmentation of both peaks yielded hexadecyl-thiol (m/z = 257.23080, C16H33S−), ethenesulfonate (m/z = 106.98074, C2H3SO3−) and bisulfite (m/z = 80.96510, HSO3−). Moreover, cultures supplied with 1,2-13C-hexadecane produced a peak at m/z = 367.22524 for 1,2-13C-hexadecyl-CoM and the fragment 259.23721 for 1,2-13C-hexadecyl-thiol, with a mass shift of 2 units compared with the unlabelled group. These analyses confirmed the activation of n-hexadecane as hexadecyl-CoM (Fig. 4). Here we demonstrate the activation of different hydrocarbon classes by ACRs of Ca. Methanoliparum, expanding the substrate range of this enzyme to an unforeseen number of compounds. Ca. Methanoliparum couples the degradation of long-chain alkanes and alkyl-substituted hydrocarbons to methane formation, proposed as alkylotrophy. Its metabolic pathways represent an additional mode of methanogenesis, adding to CO2 reduction, methylotrophy, methyl reduction, acetate fermentation and the recently reported methoxydotrophy. Ca. Methanoliparum grows in a wide temperature range, at least between 35 and 55 °C, covering the temperature range of most biodegraded oil reservoirs. Indeed, sequences of Ca. Methanoliparum are present in various anoxic hydrocarbon-rich environments worldwide. Thus, the demonstration of the unique features of Ca. Methanoliparum in hydrocarbon conversion may fundamentally change our view of crude oil transformation and biogeochemical processes in subsurface oil reservoirs. Future studies with Ca. Methanoliparia cultures will resolve the biochemical mechanisms of methanogenic hydrocarbon degradation in archaea, and will be helpful for the application of microbial-enhanced energyrecovery from depleted oil reservoirs.

Petroleum Hydrocarbon Degradation and Treatment of Automobile Service Station Wastewater by Halophilic Consortia Under Saline Conditions

The halophilic consortia were enriched from water samples of Abhor, Red Sea, Jeddah, Saudi Arabia for the degradation of phenanthrene, fluorene, hexadecane, pyrene, and treatment of automobile service station wastewater under saline conditions (4%). Complete degradation of phenanthrene and fluorene was recorded up to a concentration of 500 mg.L-1 in 12 days, when the concentration was raised to 800 mg.L-1, the percentage of degradation of the two compounds was recorded by 84 and 90% within 14 days, while when the concentration increased to 1000 mg.L-1, a significant decline was recorded. Pyrene degradation was studied under saline conditions, where it recorded a degradation rate of 92 and 81% at a concentration of 50 and 100 mg.L-1 in 10 and 12 days, respectively, while when increasing the concentration, a severe decrease in the percentage of degradation was recorded that reached 57 and 44% at concentration 200 and 300 mg.L-1, respectively .Hexadecane recorded complete degradation at a concentration of 0.5 and 1%, within 4 and 6 days, respectively, while at a concentration of 1.5%, the rate of deterioration was 88% in 10 days. Record 93% removal of COD in CSTR within 40th day, when treatment of automobile service station wastewater with halophilic bacterial consortia. The existing bacterial strains were classified as potentially responsible for petroleum hydrocarbon degradation and treatment of automobile service station wastewater such as Ochrobactrum, Propionispira, Martelella, Bacillus, Marinobacter, and Azospira. The present study recommends that the hydrophilic consortia can be used in the treatment of automobile service station wastewater under saline conditions.

Hydrocarbons Biodegradation by Rhodococcus: Assimilation of Hexadecane in Different Aggregate States.

The aim of our study was to reveal the peculiarities of the adaptation of rhodococci to hydrophobic hydrocarbon degradation at low temperatures when the substrate was in solid states. The ability of actinobacteria Rhodococcus erythropolis (strains X5 and S67) to degrade hexadecane at 10 °C (solid hydrophobic substrate) and 26 °C (liquid hydrophobic substrate) is described. Despite the solid state of the hydrophobic substrate at 10 °C, bacteria demonstrate a high level of its degradation (30–40%) within 18 days. For the first time, we show that specialized cellular structures are formed during the degradation of solid hexadecane by Rhodococcus at low temperatures: intracellular multimembrane structures and surface vesicles connected to the cell by fibers. The formation of specialized cellular structures when Rhodococcus bacteria are grown on solid hexadecane is an important adaptive trait, thereby contributing to the enlargement of a contact area between membrane-bound enzymes and a hydrophobic substrate.

Complete Genome Analysis of Rhodococcus opacus S8 Capable of Degrading Alkanes and Producing Biosurfactant Reveals Its Genetic Adaptation for Crude Oil Decomposition.

Microorganisms capable of decomposing hydrophobic substrates in cold climates are of considerable interest both in terms of studying adaptive reactions to low temperatures and in terms of their application in biotechnologies for cleaning up oil spills in a crude-oil polluted soil. The aim of this work was to investigate the genome of Rhodococcus opacus S8 and explore behavior traits of this strain grown in the presence of hexadecane. The genome size of strain S8 is 8.78 Mb, of which the chromosome size is 7.75 Mb. The S8 strain contains 2 circular plasmids of 135 kb and 105 kb and a linear plasmid with a size of 788 kb. The analysis of the genome revealed the presence of genes responsible for the degradation of alkanes and synthesis of biosurfactants. The peculiarities of morphology of microbial cells when interacting with a hydrophobic substrate were revealed. An adaptive mechanism responsible in the absence of oxygen for maintaining the process of degradation of hexadecane is discussed. The data obtained show that the strain S8 has great potential to be used in biotechnologies.

Bioelectroremediation of hexadecane in electrical cells containing Aspergillus niger immobilized in alginate

Bioelectroremediation (BER) uses electrical current to stimulate catabolism of environmental pollutants, such as petroleum spills. However, applying current to soil may have adverse effects on the microorganisms involved in petroleum degradation. We identify and evaluate the BER capabilities of a strain of Aspergillus niger obtained from petroleum-contaminated soil. Spores of this strain immobilized in alginate spheres (2 g) were mixed with 100 g of hexadecane-contaminated sandy loam soil and exposed to 5, 10, or 15 mA direct current in a 200 cm3 cell with copper electrodes. Soil hexadecane concentration was measured by gas chromatography. More than 94% of hexadecane was removed from the soil within 12 days for the currents tested, and the A. niger grew to 6 x106 CFU g-1 in 15 days at 10 mA current. The maximum hexadecane degradation was achieved using a 10-mA current for 20 days, but more than 99% of the hexadecane was removed by the fifth day. These results suggest that the use of spore-containing alginate beads promotes growth and petroleum biodegradation of A. niger exposed to electrical currents.
 Keywords: Aspergillus, bioelectroremediation, hexadecane, petroleum, soil.

Promotion effect of extracellular abiotic degradation of hexadecane by co-existence of oxalic acid in the culture medium of Pseudomonas aeruginosa NY3

Oxalic acid, known as the smallest organic acid produced during biodegradation of persistent organic pollutants (POPs), is ubiquitously existed. But its effect on the biodegradation of POPs was still unclear. This study investigated the promotion mechanism of hexadecane degradation by P. aeruginosa NY3 in the presence of oxalic acid. RT-qPCR and enzyme activity measurements showed that the expression of putative AlkB encoding genes and the activity of AlkB monooxygenase were suppressed when oxalic acid was in the presence of culture medium; meanwhile, degradation ability of hexadecane by cell-free extracellular fluid was greatly promoted. Further study showed that, the promotion mechanism of extracellular degradation of hexadecane by oxalic acid was through its improvement on the secretion of phenazine-1-carboxylic acid (PCA) which reacted with iron ions and oxalic ions to produce hydroxyl radical and resulted in the abiotic degradation of hexadecane in the environment of culture medium. During all the degradation process, the concentration of oxalic acid was nearly unchanged. These results indicated that an abiotic degradation of hexadecane promoted by the coexistence of oxalic acid in the extracellular fluid of P. aeruginosa NY3. • Oxalic acid suppressed expression of the key genes and activity of the corresponding enzyme. • Oxalic acid increased abiotic degradation of hexadecane by extracellular fluid. • Oxalic acid promoted the secretion of PHCs by P. aeruginosa NY3, especially for PCA. • Oxalic acid reacted with metal ions and PHCs to produce hydroxyl radical.

Ability of Mangrove Fungi in Biodegradation of Hexadecane

Oil pollution, especially in the marine environment, has become a serious environmental problem. Hexadecane (HXD) is a major alkane component and it is present in the aliphatic fragment of crude oil, which can be used by fungi as a sole carbon source. Biosurfactant which is produced by fungi facili tates HXD degradation. This study investigated the ability of mangrove fungi to be used as HXD and produce biosurfactant. The medium used to determine the ability of fungi to use hexadecane is MSM-HXD 2%, whereas Hua medium is used for determining the potential for producing biosurfactants. Biosurfactant production by fungi strains was indicated by oil displacement test, the reduction in surface tension and emulsion index. The results showed that 13 out of 16 species of fungi can use HXD as a sole carbon source. Inonotus radiatus LM 3020 is observed to be the most capable isolate to use HXD with a growth ratio of 6.0, and can produce biosurfactant with a positive oil displacement test (ODT) value whose minimum surface tension was 54.01 dyne/cm but the emulsion index was found to be zero.

Role of Iron Speciation in Oxidation and Deposition at the Hexadecane–Iron Interface

Interactions between iron surfaces and hydrocarbons are the basis for a wide range of materials synthesis processes and novel applications, including sensing. However, in diesel engines these interactions can lead to deposit formation that reduces performance, lowers efficiency, and increases emissions. Here, we present a global study to understand deposition at iron-hexadecane interfaces. We use a combination of spectroscopy, microscopy, and mass spectrometry to investigate surface reactions, bulk chemistry, and deposition processes. A dynamic equilibrium between the oxidation products, both at the surface and in solution, determines the deposition at the surface. Considering the solution and the surface in parallel, we find that the iron speciation affects the morphology, composition, and quantity of the deposit at the surface, as well as the oxidation of hexadecane. Fe(II) and Fe(III) both promote the decomposition of peroxides-intermediates in the oxidation of hexadecane-but through noncatalytic and catalytic mechanisms, respectively. In contrast, Fe(0) is proposed to initiate hexadecane autoxidation during its oxidation to Fe(III). We find that in all cases, the surfaces exclusively contain Fe(III) following heat treatment with hexadecane. Upon subsequent exposure at room temperature, Fe(III) species are found to promote oxidation; this finding is particularly concerning for hybrid vehicles where longer time periods are expected between engine operation. Our work provides a foundation for the development of strategies that disrupt the role of iron in the degradation of hexadecane to ultimately reduce oxidation and deposition in diesel engines.

Enhanced biodegradation of n-Hexadecane in solid-phase of soil by employing immobilized Pseudomonas Aeruginosa on size-optimized coconut fibers

In this research, biodegradation of hexadecane as a model contaminant in solid soil using both free and immobilized Pseudomonas Aeruginosa, capable of producing biosurfactant, was investigated. Coconut fibers in three mesh sizes were used as a cellulosic biocarrier for immobilization procedure. Bioremediation experiments were monitored for 60 days after incubation at 27 °C in small columns, containing contaminated solid soil, with the capability of aeration from bottom to top. The difference in the number of immobilized bacteria cells on the fibers with different particle sizes, emphasizes the importance of choosing an optimized carrier size. Enhancement in hexadecane degradation up to 50 % at the end of experiments was achieved by immobilized Pseudomonas Aeruginosa on the fibers with a mesh size between 8 and 16 compared to inoculation of free bacteria cells into the soil. Effect of mixing the pretreated fibers with soil and inoculating free cells into this mixture was also investigated compared to free cell experiments without fiber, which led to 28 % decrease in hexadecane degradation. Obtained kinetic equations for experiments confirm the impact of immobilization of bacteria on the enhancement of biodegradation rate and reduction of the half-life of the contaminant is soil.

Alternative fate of glyoxylate during acetate and hexadecane metabolism in Acinetobacter oleivorans DR1

The glyoxylate shunt (GS), involving isocitrate lyase (encoded by aceA) and malate synthase G (encoded by glcB), is known to play important roles under several conditions including oxidative stress, antibiotic defense, or certain carbon source metabolism (acetate and fatty acids). Comparative growth analyses of wild type (WT), aceA, and glcB null-strains revealed that aceA, but not glcB, is essential for cells to grow on either acetate (1%) or hexadecane (1%) in Acinetobacter oleivorans DR1. Interestingly. the aceA knockout strain was able to grow slower in 0.1% acetate than the parent strain. Northern Blot analysis showed that the expression of aceA was dependent on the concentration of acetate or H2O2, while glcB was constitutively expressed. Up-regulation of stress response-related genes and down-regulation of main carbon metabolism-participating genes in a ΔaceA mutant, compared to that in the parent strain, suggested that an ΔaceA mutant is susceptible to acetate toxicity, but grows slowly in 0.1% acetate. However, a ΔglcB mutant showed no growth defect in acetate or hexadecane and no susceptibility to H2O2, suggesting the presence of an alternative pathway to eliminate glyoxylate toxicity. A lactate dehydrogenase (LDH, encoded by a ldh) could possibly mediate the conversion from glyoxylate to oxalate based on our RNA-seq profiles. Oxalate production during hexadecane degradation and impaired growth of a ΔldhΔglcB double mutant in both acetate and hexadecane-supplemented media suggested that LDH is a potential detoxifying enzyme for glyoxylate. Our constructed LDH-overexpressing Escherichia coli strain also showed an important role of LDH under lactate, acetate, and glyoxylate metabolisms. The LDH-overexpressing E. coli strain, but not wild type strain, produced oxalate under glyoxylate condition. In conclusion, the GS is a main player, but alternative glyoxylate pathways exist during acetate and hexadecane metabolism in A. oleivorans DR1.

Functional and Genomic Characterization of a Pseudomonas aeruginosa Strain Isolated From the Southwestern Gulf of Mexico Reveals an Enhanced Adaptation for Long-Chain Alkane Degradation

Indigenous bacterial populations play an important role in the restoration of crude oil-polluted marine environments. The identification and characterization of these bacteria are key in defining bioremediation strategies for the mitigation of possible future oil spills. In this work, we characterized Pseudomonas aeruginosa strain GOM1, which was isolated from the water column in the southwestern Gulf of Mexico. Phylogenetic analysis revealed that GOM1 strain was most closely related to P. aeruginosa WC55, a strain isolated from the northern Gulf of Mexico after the Deepwater Horizon oil spill. The hydrocarbon-degrading capacity of P. aeruginosa GOM1 was investigated using various approaches. This strain degraded 96% of the aliphatic fraction (C12-C38) of crude oil during a 30-day incubation period, exhibiting a high activity on long-chain alkanes, and expressing alkane hydroxylases AlkB1, AlkB2 and AlmA. Addition of nitrogen and phosphate to seawater culture medium enhanced hexadecane degradation by GOM1. Additionally, the strain exhibited high surfactant/rhamnolipid production and emulsifying activity when grown in a complex medium in the presence of hexadecane. Comparisons of growth kinetics, hydrocarbon degradation and gene expression between GOM1 and the closely related P. aeruginosa laboratory strain PAO1 revealed that the marine isolate is better adapted to degrade alkanes. Taken together, our results place P. aeruginosa GOM1 as a potentially effective candidate to be included in a consortium for use in the bioremediation of oil-polluted sites.

Effect of the Structural and Regulatory Heat Shock Proteins on Hydrocarbon Degradation by Rhodococcus pyridinivorans 5Ap

The role of heat shock proteins in ability of Rhodococcus pyridinivorans 5Ap to degrade hydrocarbons at different temperatures was studied. The presence of the Сpn60.1–Сpn10 chaperons and of the Hrc regulatory protein was found to be required for hexadecane degradation at 42°C. When genetic determinants responsible for synthesis of these proteins were inactivated, the efficiency of hexadecane degradation decreased 1.7 and 2.7 times, respectively. Mutations in the cpn and hrcA genes did not affect the viability of R. pyridinivorans 5Ар: the original strain and the mutants exhibited the same growth rates at all temperatures in the minimal medium with succinate and in full-strength medium. In the absence of the Сpn60.1–Сpn10 heat shock proteins, the growth rate at 42°C decreased in the case of minimal agar media with kerosene, diesel fuel, acetone, naphthalene, 2-methylnaphthalene, or phenanthrene.

Heterogeneous Fenton-like catalyst for treatment of rhamnolipid-solubilized hexadecane wastewater

The treatment of wastewater containing hydrophobic organic pollutants solubilized by surfactants is of great environmental importance. In this work, the removal of rhamnolipid-solubilized hexadecane via a salicylic acid-methanol-acetone modified steel converter slag (SMA-SCS) catalyzed Fenton-like process was studied. First, we investigated the adsorption of rhamnolipid and hexadecane onto SCS and SMA-modified SCS. Compared to that of SCS, SMA-SCS exhibited better adsorption performance with maximum adsorption capacities of 0.23 and 0.28 mg/g for hexadecane and rhamnolipid, respectively. Degradation experiments showed that hexadecane was more readily degraded by the Fenton-like process than rhamnolipid. Up to 81.1% of hexadecane removal was achieved over 20 g/L of SMA-SCS within 24 h, whereas only 36% of rhamnolipid was degraded. On the other hand, the results indicated that increased rhamnolipid concentration had a negative effect on the degradation of hexadecane. During the oxidation reaction, the pH value of solution remained between 6.0 and 6.72. All these results demonstrated that the SMA-SCS/H2O2 Fenton-like process could be a cost-effective and promising approach for the treatment of surfactant-solubilized hydrophobic organic compounds.

Phormidium Improves Seed Germination and Growth Parameters of Trifolium alexandrinum in Hexadecane-contaminated Soil

Petroleum is known as the main source of fuel and one of the most important environmental pollutants. Petroleum contaminated soil negatively influences plant growth and human health. Bioremediation of petroleum-contaminated soil could be achieved by adding cyanobacteria and photosynthetic microalgae in the soil. In the current study, the beneficial effects of cyanobacterium Phormidium sp. ISC108 treatment on seed germination and plant growth parameters were investigated on berseem clover plants exposed to 1% hexadecane- contaminated soil. For this purpose, after cultivation of berseem clover seeds in soil with 0 and 1% hexadecane, they were irrigated every two days by 3 ml Phormidium (OD600 = 0.7) and water for the cyanobacteria treatment and control, respectively. After 30 days, the biodegradability of hexadecane in the soil around the root was measured by gas chromatography mass-spectrometry (GC-MS). The results showed that Phormidium treatment accelerated germination of berseem clover seeds in both control and hexadecane- contaminated soil. Improvement of plant growth indices such as leaf area, plant fresh weight and leaf RWC due to cyanobacteria treatment was observed in hexadecane-contaminated soil. Hexadecane levels in the soil around the root of the plants irrigated by cyanobacteria were significantly decreased. In addition, the hexadecane degradation in the soil around the roots of berseem clover plant was increased in both control (water) treatment. In conclusion, due to positive effects of Phormidium treatment on seed germination, plant growth of berseem clover and hexadecane degradation, it can be effectively used to enhance plant capacity to cope with hexadecane toxicity in petroleum-contaminated soils.

Biofilm mediated synergistic degradation of hexadecane by a naturally formed community comprising Aspergillus flavus complex and Bacillus cereus group

BackgroundThe hydrophobic nature of hydrocarbons make them less bioavailable to microbes, generally leading to low efficiency in biodegradation. Current bioremediation strategies for hydrocarbon contamination, uses induced mixed microbial cultures. This in-vitro study demonstrates the utilization of naturally occurring communities in suitable habitats for achieving highly efficient, synergistic degradation of hydrocarbons in a simple community structure without additives.MethodsEnrichment media supplemented with 1% (7652.53 mg/L) hexadecane (HXD) as the sole carbon source were inoculated with samples of soil with waste polythene, collected from a municipal landfill in order to isolate microbial communities. Gas Chromatography-Mass Spectrometry (GC-MS) analysis was performed on HXD grown co-cultures and individual counterparts after 14 days incubation and percentage degradation was calculated. Microbes were identified using 16S rRNA gene and Internal Transcribed Spacer region sequencing. Biofilm formation was confirmed through scanning electron microscopy, in the most efficient community.ResultsThree mixed communities (C1, C2 and C3) that demonstrated efficient visual disintegration of the HXD layer in the static liquid cultures were isolated. The C1 community showed the highest activity, degrading > 99% HXD within 14 days. C1 comprised of a single fungus and a bacterium and they were identified as a Bacillus sp. MM1 and an Apsergillus sp. MM1. The co-culture and individual counterparts of the C1 community were assayed for HXD degradation by GC-MS. Degradation by the fungal and bacterial monocultures were 52.92 ± 8.81% and 9.62 ± 0.71% respectively, compared to 99.42 ± 0.38% by the co-culture in 14 days. This proved the synergistic behavior of the community. Further, this community demonstrated the formation of a biofilm in oil-water interface in the liquid medium. This was evidenced from scanning electron microscopy (SEM) showing the Bacillus cells attached on to Aspergillus mycelia.ConclusionsThis study demonstrates the utilization of naturally formed fungal-bacterial communities for enhanced biodegradation of hydrocarbons such as hexadecane and reports for the first time, synergistic degradation of hexadecane through biofilm formation, by a community comprising of Bacillus cereus group and Aspergillus flavus complex.

Anaerobic degradation of hexadecane and phenanthrene coupled to sulfate reduction by enriched consortia from northern Gulf of Mexico seafloor sediment

To advance understanding of the fate of hydrocarbons released from the Deepwater Horizon oil spill and deposited in marine sediments, this study characterized the microbial populations capable of anaerobic hydrocarbon degradation coupled with sulfate reduction in non-seep sediments of the northern Gulf of Mexico. Anaerobic, sediment-free enrichment cultures were obtained with either hexadecane or phenanthrene as sole carbon source and sulfate as a terminal electron acceptor. Phylogenetic analysis revealed that enriched microbial populations differed by hydrocarbon substrate, with abundant SSU rRNA gene amplicon sequences from hexadecane cultures showing high sequence identity (up to 98%) to Desulfatibacillum alkenivorans (family Desulfobacteraceae), while phenanthrene-enriched populations were most closely related to Desulfatiglans spp. (up to 95% sequence identity; family Desulfarculaceae). Assuming complete oxidation to CO2, observed stoichiometric ratios closely resembled the theoretical ratios of 12.25:1 for hexadecane and 8.25:1 for phenanthrene degradation coupled to sulfate reduction. Phenanthrene carboxylic acid was detected in the phenanthrene-degrading enrichment cultures, providing evidence to indicate carboxylation as an activation mechanism for phenanthrene degradation. Metagenome-assembled genomes (MAGs) revealed that phenanthrene degradation is likely mediated by novel genera or families of sulfate-reducing bacteria along with their fermentative syntrophic partners, and candidate genes linked to the degradation of aromatic hydrocarbons were detected for future study.

Removal of hexadecane by hydroponic root mats in partnership with alkane-degrading bacteria: bacterial augmentation enhances system’s performance

In this study, degradation of hexadecane was investigated in the contaminated water by employing plant–bacteria partnership in hydroponic root mat system. A halophyte grass, Leptochloa fusca, was planted in the presence of three hydrocarbons-degrading bacteria, namely Acinetobacter sp. ACRH82, Acinetobacter sp. BRRH61, and Bacillus niabensis ACSI85. The strains were screened based on their in vitro potential of hexadecane degradation as well as plant growth-promoting activities. Hydroponic systems containing vegetation or bacterial consortium separately attenuated a good proportion of hydrocarbons, chemical oxygen demand, biochemical oxygen demand, and total dissolved solids. Nevertheless, combined application of vegetation and bacteria significantly enhanced the system performance; i.e., hydrocarbons degradation was recorded up to 92%, chemical oxygen demand was reduced up to 95%, biochemical oxygen demand up to 84%, and total dissolved solids up to 47%. The inoculated bacteria displayed highest persistence in the roots followed by shoots and then in the wastewater. The biotoxicity assay revealed that hydroponic root mat containing plant–bacteria partnership was highly efficient in reducing the toxicity level. Conclusively, the present study implies that application of L. fusca in partnership with the hydrocarbon-degrading bacteria in hydroponic root mat is a suitable choice for the phytoremediation of hexadecane. Similar systems may be exploited for the enhanced degradation of other long-chain saturated alkanes from oil-contaminated wastewaters.