The prospects for peroxidase-based biorefining of petroleum fuels

Peroxidases have many potential uses for biotechnological processes. In this review, peroxidase-catalyzed reactions potentially applicable to the petroleum industry are described. Although peroxidases are attractive catalysts for desulfurization, aromatic oxidation and asphaltene transformation, there are important issues that must be overcome before any industrial application can be considered. The opportunities and challenges of enzymatic petroleum biorefining are documented and discussed, with emphasis on the available tools to design a biocatalyst with appropriate performance for the oil and other industries.     

石油生物催化脱硫的研究进展

石油生物催化脱硫技术是新兴的极具潜力的石油非加氢脱硫技术,在降低轻质油品生产成本、提高油品质量和环境保护等方面显示出潜在的优势,被誉为21世纪的石油脱硫技术。本文主要对石油生物催化脱硫技术特点、各种降解路线和研究现状进行了综述,指出了石油生物催化脱硫技术存在的问题,并提出了进一步研究发展的方向。     

The extent and significance of petroleum hydrocarbon contamination in Crater Lake,Oregon

In order to evaluate hydrocarbon inputs to Crater Lake from anthropogenic and natural sources, samples of water, aerosol, surface slick and sediment were collected and analyzed by gas chromatography-mass spectrometry (GC-MS) for determination of their aliphatic and aromatic hydrocarbon concentrations and compositions. Results show that hydrocarbons originate from both natural (terrestrial plant waxes and algae) and anthropogenic (petroleum use) sources and are entering the lake through direct input and atmospheric transport. The concentrations of petroleum hydrocarbons range from low to undetectable. The distributions and abundances of n-alkanes, polycyclic aromatic hydrocarbons (PAH) and unresolved complex mixture (UCM) from petroleum are similar for all surface slick sampling sites. The estimated levels of PAH in surface slicks range from 7–9 ng/m² which are low. Transport of petroleum-derived hydrocarbons from the lake surface has resulted in their presence in some sediments, particularly near the boat operations mooring (total petroleum HC = 1440 μg/kg, dry wt. compared to naturally derived n-alkanes, 240 μg/kg, dry wt.). The presence of biomarkers such as the tricyclic terpanes, hopanes and steranes in shallow sediments further confirms petroleum input from boat traffic. In the deep lake sediments, petroleum hydrocarbon concentrations were very low (16 μg/kg, dry wt.). Very low concentrations of PAH were detected in shallow sediments (17–40 μg/kg at 5 m depth near the boat operations) and deep sediments (3–15 μg/kg at 580 m depth). The individual PAH concentrations in sediments (μg/kg or ppb range) are at least three orders of magnitude less than reported threshold effects levels (mg/kg or ppm range, test amphipod Hyalella azteca). Therefore, no adverse effects are expected to occur in benthic biota exposed to these sediments. Boating activities are leaving a detectable level of petroleum in surface waters and lake sediments but these concentrations are very low.     

Protein-coated Microcrystals for use in Organic Solvents: Application to Oxidoreductases

Protein-coated microcrystals (PCMC), a biocatalyst preparation previously demonstrated to display substantially increased transesterification activity of proteases and lipases in organic solvents when compared to their as received counterparts [Kreiner M, Moore BD, Parker MC (2001) Chem. Commun. 12:1096--1097], was applied to oxidoreductases. Horse liver alcohol dehydrogenase (HLADH), catalase (CAT), soybean peroxidase and horseradish peroxidase were immobilised onto K₂SO₄ crystals and dehydrated in a 1-step process, resulting in PCMC. These PCMC preparations showed enhanced activity in different organic solvents in most types of reactions tested. The highest activation was observed with HLADH (50-fold as active as enzyme as received) and CAT (25-fold).     

Attenuation of petroleum hydrocarbons by weathering: a case study

Possible alterations in the distribution and composition of total petroleum hydrocarbon (TPH), polycyclic aromatic hydrocarbons (PAHs), and benzene, toluene, ethyl benzene, and xylene isomers (BTEX) in the released oil at Idu-Ekpeye in Niger Delta (Nigeria) were studied within two seasonal variations of two months and six months, with a view to assessing the level of attenuation of these hydrocarbons in impacted soils. Although there were significant contaminations in the kerosene range (n-C10-n-C14) two months after, especially of the n-C12 and n-C13 fractions, the complete disappearance of the n-C8 to n-C23 hydrocarbons, including the acyclic isoprenoids (pristane and phytane), and the reduced amounts of PAHs, and BTEX, six months after, provided substantial evidence of attenuation as indicated in the reduction in total hydrocarbon content (THC) from 61.17 to 42.86%. Soil physicochemical properties such as pH, moisture content, heavy metal, TOC, and TOM, all provided corroborative evidence of hydrocarbon attenuation. The pristane/phytane ratio of the spill samples suggests that the spilled oil was genetically oxic.     

The Heterogeneity of the 7S Soybean Protein by Sepharose Gel Chromatography and Disc Gel Electrophoresis

Two kinds of N-acetylmuramidase, M-1 and M-2 enzymes, that were isolated from the cultural broth of Stm. globisporus 1829, were remarkably different in amino acid composition, immunological properties and modes of lytic action from each other. The M-1 enzyme was composed of 186 amino acid residues of which two moles were of half cystine, while the M-2 enzyme was composed of 99 amino acid residues with no cysteine. The hydrolyzing action of the M-2 enzyme was suppressed by the presence of an O-acetyl group on muramic acid residues in the peptidoglycan moiety, while that of the M-l enzyme was independent of the presence of O-acetyl groups. However, the hydrolyzing activity of both enzymes was enhanced when some muramic acid residues were substituted with stem peptides containing alanine, isoglutamine and lysine.     

Hemin-mediated oxidative inactivation of 3-hydroxy-3-methylglutaryl CoA reductase

Summary Addition of hemoglobin, methemoglobin, hemin or hematin in the assay mixture of rat liver 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase inhibited the activity of the enzyme. The inhibition by hemin was rapid, without any apparent dependence on time of preincubation. At 20 M hemin, a maximum of about 50% inhibition was obtained in the case of the microsomal enzyme while the solubilized enzyme showed almost 80%6 inhibition. Dithiothreitol at high concentrations or either of the two substrates of the enzyme (HMGCoA and NADPH) could afford partial protection when added before hemin. The K_m for both the substrates increased in the presence of hemin. The inhibition by hemin appeared to be irreversible, the presence of KCN or NaN₃ being the only means of preventing the inhibition. Molecular oxygen was required for the inhibition. Oxygen radicals and H₂O₂, however, did not seem to be involved. This offered a clue that an oxidation reaction of the reductase protein may be the likely mechanism of its inactivation. The enzyme protein did not, however, get degraded under the conditions of inhibition.Abbreviations HMGCoA 3-Hydroxy-3-methylglutaryl coenzyme - DTT Dithiothreitol - DTNB 5,5-Dithiobis-(2-nitrobenzoic acid) - SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

A systematic survey for thermophilic fermentative bacteria and archaea in high temperature petroleum reservoirs

Abstract: Production waters from 36 high temperature petroleum reservoirs were examined for the presence of thermophilic, fermentative microorganisms. The direct supplementation of production waters with glucose and either yeast extract, peptone, tryptone or casamino acid resulted in the isolation of thermophilic, fermentative microorganisms from 47% of the petroleum reservoirs examined. Three distinctive morphological groups were isolated from the production waters of petroleum reservoirs with depths ranging from 396–3048 metres, temperatures ranging from 21–130°C, salinities ranging from 2.8–128 gl⁻¹ and pHs ranging from 6.0–8.5. Group 1 were pleomorphic rod-shaped bacteria, Group 2 were sheathed rod-shaped bacteria, and Group 3 were coccoid archaea. Partial characterisation of strains from one seawater-flooded petroleum reservoir and three non-waterflooded petroleum reservoirs tentatively identified some strains in Group 1 as members of the genera Thermoanaerobacter and Thermoanaerobacterium , Group 2 as members of the Thermotogales order, and Group 3 as members of the genus Thermococcus . Production water salinity determined the type of microorganisms that were isolated. Group 1 organisms were found primarily in petroleum reservoirs with salinities less than 30 g/l, while Group 2 and 3 organisms were found to dominate in more saline reservoirs. The successful isolation of thermophilic, fermentative microorganisms from petroleum reservoirs decreased significantly with increasing salinity and temperature. These findings support the existence of a deep biosphere where fermentative microorganisms are widespread.     

Suicide-Peroxide inactivation of microperoxidase-11: A kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H₂O₂. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H₂O₂]>>[AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, α_i, and the apparent inactivation rate constant, k_i, were obtained as 0.282 ± 0.006 min^{? 1} and 0.497 ± 0.013 min^{? 1} at [H₂O₂] = 1.0 mM, 27°C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).     

新古尼异虫草石油醚提取物的成分分析

【背景】新古尼异虫草具有多种药理作用,是一种具有开发潜力的新资源。但该虫草仍有很多活性物质值得探寻,目前对该虫草活性物质的研究多集中于大分子或极性物质,对小分子或弱极性物质的研究关注甚少。【目的】研究新古尼异虫草石油醚提取物中非极性/弱极性小分子化合物,以期完善该虫草中活性物质的化学指纹图谱库。【方法】利用石油醚对新古尼异虫草进行索氏提取,借助傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FTIR)和气相色谱-质谱(GC-MS)联用技术鉴定分析石油醚萃取物中的物质组成。【结果】FTIR表明该虫草石油醚萃取物中含有C-H、C=O、C-O和C=C等官能团,经GC-MS进一步分析鉴定出109种化合物,主要包括烷烃、芳烃、烯烃、酸、酯、醇、胺等化合物,且首次检出甾类、芳烃类、烷烃类、酰胺类、烯烃类和酚类非极性/弱极性的小分子化合物,其中亚油酸及其同分异构体的相对含量最高(38.33%)。【结论】从新古尼异虫草中提取得到多种小分子活性成分,补充了该虫草中的物质组成,为其高附加值利用提供数据支撑。     

石油污染盐碱土壤棉花根际微生物与石油烃降解关系

【目的】探讨棉花(Gossypium spp.)生长对石油烃(TPH)污染盐碱土壤微生物群落结构的影响,揭示根际微生物与TPH降解的相关关系。【方法】利用磷脂脂肪酸(PLFA)方法解析根际土壤活性微生物群落随棉花生长的动态变化特征。【结果】根际土壤先后出现了21种PLFAs,包括：饱和脂肪酸(SAT),标识除放线菌之外的细菌;甲基支链末端型饱和脂肪酸(TBSAT),标识除放线菌之外的革兰氏阳性(G+)细菌;标识真菌的多不饱和脂肪酸(PUFA);标识放线菌的甲基支链中间型饱和脂肪酸(MBSAT);标识革兰氏阴性(G?)细菌的单不饱和脂肪酸(MONO)和环丙基脂肪酸(CYCLO)。棉花根际与未栽种棉花的对照(CK)相比,根际土壤微生物PLFAs种类在苗期、蕾期、吐絮期分别增加了100%、83.3%、20.0%,生物量分别增加了53.9%、6.60倍和60.7%;土壤TPH降解率分别提高13.0%、28.0%和30.6%。相关性分析表明：根际土壤TPH降解与根际土壤微生物总生物量具有低度正相关关系(|r|=0.5),但与a14:0、a16:0、i15:0标记的G+细菌生物量高度正相关(|r|≥0.8)。【结论】棉花生长对石油污染盐碱土壤活性微生物群落结构具有显著(p<0.05)的影响,且加速了土壤TPH的降解。该结果将为今后更好地开展石油污染盐碱土壤的生物修复技术研究提供理论依据。     

石油烃污染及修复过程中的微生物分子生态学研究进展

针对环境中广泛存在的石油烃污染问题,从分子生态学的角度总结石油烃降解过程中的微生物生态学研究进展。着重介绍分子生态学的研究方法及与石油烃降解相关的降解基因和基因芯片的最新研究进展,同时对存在的问题和今后的研究方向进行总结。     

Isolation and characterization of Sphingomonas sp. GTIN11 capable of carbazole metabolism in petroleum

A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to metabolize the nitrogen-containing heterocycle carbazole. The culture was identified as a Sphingomonas sp. and was given the designation GTIN11. A cloned 4.2kb DNA fragment was confirmed to contain genes responsible for carbazole degradation. DNA sequence analysis revealed that the fragment contained five open reading frames (ORFs) with the deduced amino acid sequence showing homology to; carbazole terminal dioxygenase (ORF1), 2,3-dihydroxybiphenyl dioxygenase subunits (ORF2 and ORF3), meta-cleavage compound hydrolases (ORF4), and ferrodoxin component of bacterial multicomponent dioxygenases (ORF5). The percent similarity was 61% of these proteins or less to known proteins. The specific activity of Sphingomonas sp. GTIN11 for the degradation of carbazole at 37 degrees C was determined to be 8.0 micromol carbazole degraded/min/g dry cell. This strain is unique in expressing the carbazole degradation trait constitutively. Resting cells of Sphingomonas sp. GTIN11 removed 95% of carbazole and 50% of C1-carbazoles from petroleum in a 16-h treatment time.     

Genetic engineering of grass cell wall polysaccharides for biorefining

Grasses represent an abundant and widespread source of lignocellulosic biomass, which has yet to fulfil its potential as a feedstock for biorefining into renewable and sustainable biofuels and commodity chemicals. The inherent recalcitrance of lignocellulosic materials to deconstruction is the most crucial limitation for the commercial viability and economic feasibility of biomass biorefining. Over the last decade, the targeted genetic engineering of grasses has become more proficient, enabling rational approaches to modify lignocellulose with the aim of making it more amenable to bioconversion. In this review, we provide an overview of transgenic strategies and targets to tailor grass cell wall polysaccharides for biorefining applications. The bioengineering efforts and opportunities summarized here rely primarily on (A) reprogramming gene regulatory networks responsible for the biosynthesis of lignocellulose, (B) remodelling the chemical structure and substitution patterns of cell wall polysaccharides and (C) expressing lignocellulose degrading and/or modifying enzymes in planta. It is anticipated that outputs from the rational engineering of grass cell wall polysaccharides by such strategies could help in realizing an economically sustainable, grass‐derived lignocellulose processing industry.     

Autochthonous death assemblages from chemoautotrophic communities at petroleum seeps: Palaeoproduction,energy flow,and implications for the fossil record

Death assemblages produced by chemoautotrophic communities at cold seeps represent a type of autochthonous accumulation that is difficult to differentiate from other heterotrophic autochthonous communities using taphonomic characteristics. We test the hypothesis that cold‐seep assemblages can be discriminated by unique biological or community attributes rather than taphonomic attributes. To test this hypothesis, we compared several cold seeps on the Louisiana upper continental slope to heterotrophic sites on the Louisiana slope and to a putative seep site in the middle‐late Campanian Pierre Shale near Pueblo, Colorado. Seep assemblages are characterized by a unique tier and guild structure, size‐frequency composition, and animal density that together identify the palaeoenergetics structure of these communities and distinguish them from the other assemblages of the shelf and slope. All seep assemblages were dominated by primary consumers, whereas the heterotrophic assemblage was dominated by carnivores. Carnivore dominance seems to be typical of shelf (or euhaline) death assemblages. Seep assemblages, in contrast, retain the theoretically‐expected rarity of predaceous forms in fossil assemblages. Epifauna and semi‐infauna dominate the tier structure of the heterotrophic assemblage as is typical for continental shelf and slope assemblages. The infaunal tier was unusually well represented in most petroleum seep assemblages. Local enrichment of food resources and the dominance of shelled primary consumers explain the guild and tier structure of seep assemblages. Hindcasting of energy demand (palaeoingestion) and an estimate of sedimentation rate confirms that energy demand by the community exceeds the supply from planktonic rain in seep communities. Thus, seep assemblages can be recognized using biological attributes where taphonomic analysis is ambiguous.     

The Origin of the Oxidative Burst in Plants

A large number of publications recently have drawn strong analogies between the production of active oxygen species in plant cells and the “oxidative burst” of the phagocyte, even to the point of constructing elaborate models involving receptor mediated G-protein activation of a plasmalemma NADPH oxidase in plant cells. However there are potentially other active oxygen species generating systems at the plant cell surface. The present work examines these alternatives with particular emphasis on the rapid production of active oxygen species, in common with a number of other systems, by suspension-cultured cells of French bean on exposure to an elicitor preparation from the fungal pathogen Colletotrichum lindemuthianum. The cells show a rapid increase in oxygen uptake which is followed shortly afterwards by the appearance of a burst of these active oxygen species, as measured by a luminescence assay, which is probably all accounted for by hydrogen peroxide. An essential factor in this production of H₂O₂ appears to be a transient alkalinization of the apoplast where the pH rises to 7.0-7.2. Dissipation of this pH change with a number of treatments, including ionophores and strong buffers, substantially inhibits the oxidative burst. Little evidence was found for enhanced activation of a membrane-bound NADPH oxidase. However the production of H₂O₂ under alkaline conditions can be modelled in vitro with a number of peroxidases, one of which, an M_r 46,000 wall-bound cationic peroxidase, is able to sustain H₂O₂ production at neutral pH unlike the other peroxidases which only show low levels of this reaction under such conditions and have pH optima at values greater than 8.0. On the basis of such comparative pH profiles between the cells and the purified peroxidase and further inhibition studies a direct production of H₂O₂ from the wall peroxidase in French bean cells is proposed. These experiments may mimic some of the responses to plant pathogens, particularly the hypersensitive response, which is an important feature of resistance. A cell wall peroxidase-origin for the oxidative burst is clearly different from a model consisting of receptor activation of a plasmaiemma-localised NADPH oxidase generating superoxide. An alternative simple and rapid mechanism thus exists for the generation of H₂O₂ which does not require such multiple proteinaceous components.     

The abundance of nahAc genes correlates with the 14C-naphthalene mineralization potential in petroleum hydrocarbon-contaminated oxic soil layers

In this study, we evaluated whether the abundance of the functional gene nahAc reflects aerobic naphthalene degradation potential in subsurface and surface samples taken from three petroleum hydrocarbon contaminated sites in southern Finland. The type of the contamination at the sites varied from lightweight diesel oil to high molecular weight residuals of crude oil. Samples were collected from both oxic and anoxic soil layers. The naphthalene dioxygenase gene nahAc was quantified using a replicate limiting dilution-polymerase chain reaction (RLD-PCR) method with a degenerate primer pair. In the non-contaminated samples nahAc genes were not detected. In the petroleum hydrocarbon-contaminated oxic soil samples nahAc gene abundance [range 3 x 10(1)-9 x 10(4) copies (g dry wt soil)(-1)] was correlated (Kendall non-parametric correlation r2=0.459, p<0.01) with the aerobic 14C-naphthalene mineralization potential (range 1 x 10(-5)-0.1 d(-1)) measured in microcosms at in situ temperatures (8 degrees C for subsurface and 20 degrees C for surface soil samples). In these samples nahAc gene abundance was also correlated with total microbial cell counts (r2=0.471, p<0.01), respiration rate (r2=0.401, p<0.01) and organic matter content (r2=0.341, p<0.05). NahAc genes were amplified from anoxic soil layers indicating that, although involved in aerobic biodegradation of naphthalene, these genes or related sequences were also present in the anoxic subsurface. In the samples taken from the anoxic layers, the aerobic 14C-naphthalene mineralization rates were not correlated with nahAc gene abundance. In conclusion, current sequence information provides the basis for a robust tool to estimate the naphthalene degradation potential at oxic zones of different petroleum hydrocarbon-contaminated sites undergoing in situ bioremediation.     

Oxidative potential of smoke from burning wood and mixed biomass fuels

Abstract

More than half the world's population still rely on burning biomass fuels to heat and light their homes and cook food. Household air pollution, a common component of which is inhalable particulate matter (PM), emitted from biomass burning is associated with increased vulnerability to respiratory infection and an enhanced risk of developing chronic obstructive pulmonary disease. In the light of an emerging hypothesis linking chronic PM exposure during childhood and increased vulnerability to respiratory diseases in adulthood, in a chain of events involving oxidative stress, reduced immunity and subsequent infection, the aim of this study was to characterise the oxidative potential (OP) of PM collected during the burning of wood and mixed biomass, whilst cooking food in the Kathmandu Valley, Nepal. Our assessments were based on the capacity of the particles to deplete the physiologically relevant antioxidants from a validated, synthetic respiratory tract lining fluid (RTLF). Incubation of mixed biomass and wood smoke particles suspensions with the synthetic RTLF for 4 h resulted in a mean loss of ascorbate of 64.76 ± 16.83% and 83.37 ± 14.12% at 50 μg/ml, respectively. Reduced glutathione was depleted by 49.29 ± 15.22% in mixed biomass and 65.33 ± 13.01% in wood smoke particles under the same conditions. Co-incubation with the transition metal chelator diethylenetriaminepentaacetate did not inhibit the rate of ascorbate oxidation, indicating a negligible contribution by redox-active metals in these samples. The capacity of biomass smoke particles to elicit oxidative stress certainly has the potential to contribute towards negative health impacts associated with traditional domestic fuels in the developing world.     

石油烃和酚类物质在土中的生物降解与土壤酶活性

本文通过模拟实验,研究了不同条件下石油烃和酚类物质在土中的降解进程及其与土壤酶活性的关系,并在此基础上,对所述污染物的土地处理提出了若干建议。