Hydrocarbon-Degrading Bacteria and Paraffin from Polluted Seashores 9 Years after the Nakhodka Oil Spill in the Sea of Japan

Pollution of petroleum hydrocarbons, in particular oil spills, has attracted much attention in the past and recent decades. Oil spills influence natural microbial community, and physical and chemical properties of the affected sites. The biodegradation of hydrocarbons by microorganisms is one of the primary ways by which oil spill is eliminated from contaminated sites. One such spill was that of the Russian tanker the Nakhodka that spilled heavy oil into the Sea of Japan on January 2, 1997. The impact of the Nakhodka oil spill resulted in a viscous sticky fluid fouling the shores and affected natural ecosystems. This paper describes the weathering of hydrocarbon-degrading bacteria （genus Pseudomonas） and crystallized organic compounds from the Nakhodka oil spill-polluted seashores after nine years. The Nakhodka oil has hardened and formed crust of crystalline paraffin wax as shown by XRD analysis （0.422, 0.377, and 0.250 nm d-spacing） in association with graphite and calcite after 9 years of bioremediation. Anaerobic reverse side of the oil crust contained numerous coccus typed bacteria associated with halite. The finding of hydrocarbon-degrading bacteria and paraffin wax in the oil crust may have a significant effect on the weathering processes of the Nakhodka oil spill during the 9- year bioremediation.     

Landfarmed oil sludge as a carbon source for Canavalia ensiformis during phytoremediation

Petroleum exploitation in oilfields, especially drilling, generates an oily sludge mixed with hydrocarbons and mineral solids. This oily sludge is sometimes treated by bioremediation and phytoremediation. This investigation established that landfarmed oil sludge provided adequate soil conditions to grow jack beans (Canavalia ensiformis) that in turn rhizo- and phytoremediated residual aliphatic and aromatic hydrocarbons in the soil. Landfarming oily sludge adequately reduced jack bean phytotoxicity. Rhizo- and phytodegradation reduced total petroleum hydrocarbons by 57.38 % during 4 months of growing jack beans. Aliphatic hydrocarbons were detected in the roots but not in the aerial parts. Polycyclic aromatic hydrocarbons were translocated to the roots, stems, leaves, and beans, requiring successive cropping to manage all risks associated with some aromatic hydrocarbons found such as: acenaphthylene, anthracene, pyrene, benzo(a)anthracene, benzo(k)fluoranthene, and benzo(a)pyrene. Landfarming and phytoremediation, perhaps with successive crops, holds the promise of providing inexpensive management of extensive oily wastes when sufficient land is available.     

Population structures shift during the biodegradation of crude and fuel oil by an indigenous consortium

Petroleum and fuel oil are complex mixtures of recalcitrant hydrocarbons. The biodegradation of these hydrocarbons needs the action of a vast variety of enzymatic capacities. A microbial consortium offers the capability to degrade complex substrates through the assembly of different biochemical reactions, providing a metabolic versatility superior to axenic cultures. In this work, the microbial population dynamics, taxonomy, and the catabolic capacity of a stabilized consortium exposed to fuel and crude oil was analyzed through metagenomics. The stabilized consortium degraded 59% of crude oil components after 8 days, and 34% of fuel oil components after 130 days. Population dynamics analysis indicates that in fuel oil the biodiversity richness was higher; however, denaturing gradient gel electrophoresis similarity dendrogram shows significant changes in the microbial population during crude oil degradation. Taxonomy studies indicate a great genera divergence; only eight microbial genera were common in both samples. In crude oil, the Limnobacter sp. was the most abundant specie (15.6%), while Sphingomonas wittichii (7.9%) and Novosphingobium aromaticivorans (7.6%) were abundant in fuel oil. These microorganisms have been reported to participate in the degradation of aliphatic and aromatic hydrocarbons. Functional analysis suggests that fuel and crude oil components changed the interactions between the consortium members affecting the collective metabolic functionality.     

短期原油污染致使冻土活动层细菌群落显著改变——以加格达奇冻土活动层为例

生物降解与土著微生物群落结构、功能及其变化密切相关. 目前, 对于东北冻土土壤中的适冷降解菌了解不足. 新建成的中俄输油管道穿越中国东北的多年冻土区, 为相关研究提供了契机. 实验利用454高通量测序分析了加格达奇冻土活动层土壤在受控原油污染前后的微生物群落结构. 结果显示: 污染后的Proteobacteria和Firmicutes相对丰度显著升高, 优势类群包括Alicyclobacillus、Sphingomonas、Nevskia以及Bacillus. 群落以芳烃降解菌或者耐受油污环境的细菌为主. 这种变化与原油(尤其是芳烃)组分的生态毒害作用有关. 较高浓度的原油污染下, 群落中可耐受油污环境的细菌丰度相对更高.     

Fast bioremediation of petroleum-contaminated soils by a consortium of biosurfactant/bioemulsifier producing bacteria

Biodegradation of petroleum hydrocarbons as a decontamination mechanism is a relatively slow process. This study aimed to investigate the impact of a tailored consortium of bacteria with higher capacities in biosurfactant production and biodegradation on the acceleration of the biodecontamination process. To this end, 18 biosurfactant producing bacteria were isolated from the crude oil-contaminated soil samples of Isfahan refinery, and the activity of the produced biosurfactants was measured in terms of surface tension reduction and emulsification E24 test. Then, the isolates screened for the biodegradation of kerosene hydrocarbons and chemical structure of the purified biosurfactants produced by the most efficient isolates were partially characterized. Next, the isolates were sorted based on their surfactant activity and biodegradation efficiency, and the higher ranked bacteria thus selected were utilized to form an efficient consortium removing hydrocarbons from the oil-contaminated soil samples in a slurry phase system. The consortium consisted of Bacillus subtilis tb1 and Pseudomonas aeruginosa species having the highest biodegradation capabilities and surface activities. The results revealed that the hydrocarbon removal efficiency of the consortium was at least 25 % higher than single species, and the final removal efficiency for the consortium could be reached in a considerably shorter time.     

Biodegradation of phthalic acid esters in sewage sludge by composting with pig manure and rice straw

To comparatively study the biodegradation of phthalic acid esters (PAEs) in sewage sludge by composting with pig manure or with rice straw, four composting modes were designed: Mode 1 (sewage sludge + pig manure + intermittent aeration), Mode 2 (sewage sludge + pig manure + continuous aeration), Mode 3 (sewage sludge + rice straw + intermittent aeration) and Mode 4 (sewage sludge + rice straw + continuous aeration). Physicochemical parameters of composts were measured according to standard methods and PAEs were analyzed by gas chromatography coupled with mass spectrometry (GC/MS). The biodegradation of each PAE was also discussed. The results showed that Mode 1 was the best mode to biodegrade PAEs, which might be related to the wide class of indigenous microbial communities in pig manure and high efficiency of intermittent ventilation for providing oxygen. During the biodegradation process, di(2-ethylhexyl) phthalate was the most abundant and decisive for the removal of total PAEs. It showed a first-order kinetic degradation model. In conclusion, composting with pig manure could be suggested as an effective detoxification process for the removal of PAEs from sewage sludge, providing a safe end product.     

Isolation,biodegradation ability and molecular detection of hydrocarbon degrading bacteria in petroleum samples from a Brazilian offshore basin

The detection of microorganisms with potential for biodeterioration and biodegradation in petroleum fields is of great relevance, since these organisms may be related to a decrease in petroleum quality in the reservoirs or damage in the production facilities. In this sense, petroleum formation water and oil samples were collected from the Campos Basin, Brazil, with the aim of isolating microorganisms and evaluating their ability to degrade distinct classes of hydrocarbon biomarkers (9,10-dihydrophenanthrene, phytane, nonadecanoic acid and 5α-cholestane). Twenty eight bacterial isolates were recovered and identified by sequencing their 16S rRNA genes. Biodegradation assays revealed that bacterial metabolism of hydrocarbons occurred through reactions based on oxidation, carbon–carbon bond cleavage and generation of new bonds or by the physical incorporation of hydrocarbons into microbial cell walls. Based on the biodegradation results, selective PCR-based systems were developed for direct detection in petroleum samples of bacterial groups of interest, namely Bacillus spp., Micrococcus spp., Achromobacter xylosoxidans, Dietzia spp. and Bacillus pumilus. Primer sets targeting 16S rRNA genes were designed and their specificity was confirmed in silico (i.e. computational analysis) and in PCR reactions using DNA from reference strains as positive and negative controls. Total DNA from oil was purified and the amplification tests revealed the presence of the target bacteria in the samples, unraveling a significant potential for petroleum deterioration in the reservoirs sampled, once proper conditions are present for hydrocarbon degradation. The application of molecular methods for rapid detection of specific microorganisms in environmental samples would be valuable as a supporting tool for the evaluation of oil quality in production reservoirs.     

Induced degradation of crude oil mediated by microbial augmentation and bulking agents

Different bacterial and fungal strains, isolated from petroleum hydrocarbon-contaminated soil, were tested, in isolation as well as in combination, for their ability to degrade total petroleum hydrocarbon (TPH) in soil samples spiked with crude oil (2, 5 or 10 %, w/w) for 30 days. The selected combination of bacterial and fungal isolates, i.e., Pseudomonas stutzeri BP10 and Aspergillus niger PS9, exhibited the highest efficiency of TPH degradation (46.7 %) in soil spiked with 2 % crude oil under control condition. Further, when this combination was applied under natural condition in soil spiked with 2 % (w/w) crude oil along with inorganic fertilizers (NPK) and different bulking agents such as rice husk, sugarcane, vermicompost or coconut coir, the percent degradation of TPH was found to be maximum (82.3 %) due to the presence of inorganic fertilizers and rice husk as bulking agent. Further, results showed that the presence of NPK and bulking agents induced the activity of degradative enzymes, such as catalase (0.718 m mol H₂O₂ g^?1), laccase (0.77 µmol g^?1), dehydrogenase (37.5 µg g^?1 h^?1), catechol 1, 2 dioxygenase (276.11 µ mol g^?1) and catechol 2, 3 dioxygenase (15.15 µ mol g^?1) as compared to control (without bioaugmentation). It was inferred that the selected combination microbes along with biostimulants could accentuate the crude oil degradation as evident from the biostimulant-induced enhanced activity of degradative enzymes.     

氧化镁烧结-电感耦合等离子体质谱法测定砂岩型铀矿中的痕量铼

砂岩型铀矿中铀的平均含量为635μg/g,而铼的含量仅为0.3~1.9μg/g,由于铼的含量低,准确测定高含量铀矿样中的低含量铼仍是分析化学的一个难题。本文建立了电感耦合等离子体质谱法测定砂岩型铀矿中痕量铼的方法。样品经过氧化镁烧结,热水浸提以及采用103Rh作内标元素等方式消除了铀、钼及其他基体元素对测定铼的干扰。在最佳条件下,痕量铼的检出限可低至0.12 ng/g,回收率达99.8%。本方法通过多种标准样品验证,铼的测定值与标准值吻合,对铼含量在0.06~180.57μg/g范围内的实际样品进行测定,相对标准偏差均小于1.5%,能够满足砂岩型铀矿等高含量铀矿中痕量铼的测定需求。     

石油污染地下水中细菌分子生态学研究

采集了某废弃炼油厂的石油污染地下水样品,提取水中微生物总DNA,构建细菌16S rDNA克隆文库,并通过16S rDNA序列的系统发育分析,对样品中的细菌种群多样性以及群落结构进行了研究。结果表明,文库中阳性克隆的16S rDNA序列分属11个细菌类群,分别为Betaproteobacteria(381％),Alphaproteobacteria(353％),Gamaproteobacteria(51％),Deltaroteobacteria(52％),Bacteroidetes(40％),Verrucomicrobia(25％),Epsilonproteobacteria(19％),Nitrospira(13％),Planctomycetes(13％),Candidate Division OD1(13％),Unclassified Bacteria(38％)。在这一生态系统中,Betaproteobacteria和Alphaproteobacteria类细菌占据主导地位,二者所占比例均在三分之一以上。群落中最主要的降解菌是噬氢菌属(Hydrogenophaga)和鞘氨醇单胞菌属(Sphingomonas)细菌,它们在文库中所占比例达239％和195％。该石油污染地下水样品中细菌与许多其他已知的降解菌亲缘关系较近,如Sulfuricurvum kujiense、Trichlorobacter thiogenes、Rhodoferax ferrireducens以及红细菌属(Rhodobacter)、甲基单胞菌属(Methylomonas)和涅瓦菌属(Nevskia)细菌等。此外,文库中克隆的16S rDNA序列与许多类似的污染场地中发现的环境克隆相似性很高,如煤焦油污染的地下水、苯污染的地下水、原油污染的土壤、溴甲烷和氯甲烷污染的土壤、抗生素生产废水以及活性污泥等,证明该石油污染地下水中有大量降解菌群的存在。石油污染物的种类对降解菌群的组成有一定的选择作用。     

石油沥青质的吸附、沉淀机理及其影响因素

对于原油中石油沥青质的存在状态尚无统一认识,存在多种石油胶体体系理论模型。沥青质有两种方式从石油中分离出来,即吸附和沉淀,它们具有完全不同的机理。吸附的沥青质不能被溶解,要通过解吸才能消除,且解吸过程相对溶解而言很慢。沉淀是由于石油胶体体系的变化引起的,而吸附则是由石油胶体体系以及体系外的水介质环境和周围岩石矿物特征等所控制的。影响吸附的主要因素有:矿物本身的化学性质和结构特征、矿物质表面水层的性质、原油组成。影响沉淀的因素主要有:原油的组成、温度和压力、石油所处的状态、孔隙特征。低渗储层比高渗储层更容易导致沥青质的沉淀。     

Improved assessment of biodegradation extent and prediction of petroleum quality

Biodegradation of crude oil causes volumetrically important compositional changes, which lead to significant deterioration in quality, in particular during the early stages of alteration. To better understand these effects we focussed on a detailed assessment of light to moderate levels of alteration. We investigated a suite of 40 crude oil samples from five different petroleum systems to evaluate the extent of alteration occurring in reservoirs. Based on a comprehensive geochemical characterization, five individual crude oil sequences were defined, where compositional variability is mainly due to microbial activity in the reservoir. In particular, samples from the Gullfaks field (offshore Norway) and from a petroleum system offshore Angola illustrate that conventional molecular biodegradation parameters, such as the Pr/n-C₁₇ and Ph/n-C₁₈ alkane ratios are not suitable for defining the extent of biodegradation in petroleum reservoirs. Here, we suggest a new molecular biodegradation parameter, the degradative loss, that can be used to quantify depletion in individual crude oil constituents. The approach allows improved assessment of the extent of biodegradation in crude oil samples by means of the mean degradative loss. It is demonstrated that crude oil quality, as assessed from API gravity, can be predicted directly from the molecular composition of crude oils. Our data clearly indicate that the degradation patterns of light hydrocarbons and n-alkanes differ in different petroleum systems. This suggests that microbial communities are different and therefore generate different molecular degradation patterns which have to be evaluated individually for each system.     

Degradation Kinetics of Petroleum Contaminants in Soft-Water Systems

On the basis of site investigation and sample collection of petroleum contaminants in the soil-water-crop system in the Shenyang-Fushun sewage irrigation area, the physical-chemical-biological compositions of the unsaturated zone is analyzed systematically in this paper. At the same time, the degradation kinetics of residual and aqueous oils is determined through biodegradation tests. The studies show that dominant microorganisms have been formed in the soils after long-term sewage irrigation. The microorganisms mainly include bacteria, and a few of fungus and actinomycetes.After a 110-days‘ biodegradation test, the degradation rate of residual oil is 9.74%--10.63%, while the degradation rate of aqueous oil reaches 62.43 %. This indicates that the degradation rate of low-carbon aqueous oil is higher than that of highcarbon residual oil. In addition, although microbial degradation of petroleum contaminants in soils is suitable to the firstorder kinetics equation, the half-lives of aqueous oil, No. 20 heavy diesel and residual oil in the surface soils (L2-1, S1-1 and X1-1) are 1732 h, 3465 h and 17325 h, respectively.     

岩盐中合成烃包裹体与母油的地球化学特征及其在油源对比中的意义

油气包裹体广泛应用于油气成藏研究,但油气包裹体能否继承母油荧光及地球化学特征等,尚缺乏直接的实验证据。本文在开放体系下用NaCl挥发结晶法对一轻质原油进行合成烃类包裹体实验,通过镜下观察和激光剥蚀色谱-质谱技术分析合成的烃包裹体和母油的荧光特征、成分特征及地化特征,研究两者的异同点。结果表明,在NaCl合成烃类包裹体中共发现三种相态的包裹体:纯液相烃包裹体、气液两相烃包裹体和气油水三相包裹体;共有绿黄色荧光、蓝色荧光两种不同颜色的荧光特征。相对于母油,合成包裹体的饱和烃和芳烃类化合物中的轻质组分含量较低,可以推测在母油被捕获成为包裹体的过程中,不同的化学成分存在差异性捕获。但合成包裹体的Pr/Ph等地化参数和不同系列化合物相对含量与母油相差不大,能很好地反映母油的沉积环境等地化特征。因此,包裹体成分信息可以应用于油源对比分析。     

中国石油安全评价及建议

石油安全对一个国家的经济发展具有重大意义。随着中国经济的持续快速增长,以及受国内石油资源短缺、石油地缘政治等因素的影响,国内石油供需矛盾进一步加剧,石油安全问题日益显现。为科学地评价中国石油安全的程度,先从国内资源禀赋、国内生产能力、国际市场可得性和国家应急保障能力4个方面设计了石油安全评价指标体系;然后,再采用德尔菲法、主成分分析法,选取国内原油储采比、国内原油储量替代率、国内石油消费对外依存度、国内石油进口集中度、国际原油价格和国内石油储备水平6个要素指标,构成一个新的综合指标,即石油安全度;最后,以石油安全度为工具,对中国近年来石油安全的形势进行了定量评价。     

地质微生物作用与油气资源

地质微生物作用与油气资源的生成、保存、破坏、开采和勘探的各个环节都有着密切的联系。微生物(细菌)本身不仅是良好的成烃母质,还是促使有机物转化为石油烃的主要营力。微生物作用使沉积有机质向更有利于生烃的物质转化,并使有机质中表征成熟度的一些指标在尚未受热成熟作用前就显示成熟特征。沉积环境中微生物的发育对有机质的贡献与保存有积极作用,细菌对有机质的降解也有利于有机质中成烃组分的保存与富集。微生物作用生成的天然气、煤层气和甲烷水合物在世界各地已成为有商业开采价值的资源。然而,微生物对已成藏的油气资源又具有破坏作用。储层中喜氧与厌氧菌的存在,不仅破坏石油储量,还使原油质量明显下降。     

Distribution of time lags between successive petroleum exploration activities

Four time lags are studied: (1) drilling activity behind seismic activity; (2) oil and gas reserves discovered behind drilling activity; (3) oil and gas production behind reserves discovered; and (4) oil and gas production behind drilling activity. A ridge regression method is used to estimate the distributions of these lags for 50 countries and regions active in petroleum exploration. A complete review of obtained results is given as well as an interpretation of estimated models and failed ridge regressions. Five types of distributions have been found: decreasing monotonically, unimodal, bimodal, uniform, and increasing monotonically. The distributions allow estimating the capital freezing in the exploration process and assessing the resulting policy made by all companies active in a country. For example, the actual exploration history in Poland is analyzed with the use of distributed time-lag approach and net present-value concept. An overinvestment in drilling in comparison with petroleum infrastructure has been stated on the basis of the excessive lagging of production behind drilling activity. A direction of further lag studies is also suggested.     

Solubility of crude oil in methane as a function of pressure and temperature

The solubility of a 44° API (0.806 sp. gr.) whole crude oil has been measured in methane with water present at temperatures of 50 to 250°C and pressures of 740 to 14,852 psi, as have the solubilities of two high molecular weight petroleum distillation fractions at temperatures of 50 to 250°C and pressures of 4482 to 25,266 psi. Both increases in pressure and temperature increase the solubility of crude oil and petroleum distillation fractions in methane, the effect of pressure being greater than that of temperature. Unexpectedly high solubility levels (0.5–1.5 grams of oil per liter of methane—at laboratory temperature and pressure) were measured at moderate conditions (50–200°C and 5076–14504 psi). Similar results were found for the petroleum distillation fractions, one of which was the highest molecular weight material of petroleum (material boiling above 266°C at 6 microns pressure). Unexpectedly mild conditions (100°C and 15,200 psi; 200°C and 7513 psi) resulted in cosolubility of crude oil and methane. Under these conditions, samples of the gas-rich phase gave solubility values of 4 to 5 g/l, or greater.Qualitative analyses of the crude-oil solute samples showed that at low pressure and temperature equilibration conditions, the solute condensate would be enriched in C₅–C₁₅ range hydrocarbons and in saturated hydrocarbons in the C₁₅₊ fraction. With increases in temperature and especially pressure, these tendencies were reversed, and the solute condensate became identical to the starting crude oil.The data of this study, compared to that of previous studies, shows that methane, with water present, has a much greater carrying capacity for crude oil than in dry systems. The presence of water also drastically lowers the temperature and pressure conditions required for cosolubility.The data of this and/or previous studies demonstrate that the addition of carbon dioxide, ethane, propane, or butane to methane also has a strong positive effect on crude oil solubility, as does the presence of fine grained rocks.The n-paraffin distributions (as well as the overall composition) of the solute condensates are controlled by the temperature and pressure of solution and exsolution, as well as by the composition of the original starting material. It appears quite possible that primary migration by gaseous solution could ‘strip’ a source rock of crude-oil like components leaving behind a bitumen totally unlike the migrated crude oil. The data of this study demonstrate previous criticisms of primary petroleum migration by gas solution are invalid; that primary migration by gaseous solution cannot occur because methane cannot dissolve sufficient volumes of crude oil or cannot dissolve the highest molecular weight components of petroleum (tars and asphaltenes).     

Bioemulsification activity assessment of an indigenous strain of halotolerant Planococcus and partial characterization of produced biosurfactants

Halotolerant bacteria are regarded as effective oil-scavengers in the polluted saltern and seawater. In this regard, a halotolerant Planococcus was isolated from oil-contaminated area of Dezful north springs, Iran, due to its capacity in biosurfactant (BS) production. To facilitate hydrocarbons degradation, in the current study, the efficiency of BS production as function of growth rate of the halotolerant Planococcus was investigated in the vicinity of heavy crude oil by emulsification index (E24). Subsequently, the BS characterization was made by thin-layer chromatography (TLC), gas chromatography (GC) and infrared spectra analysis, and the stability was determined by E24 value measurement over a certain pH (5–9), temperature (20–100 °C) and salt concentration (0–10 % w/v) ranges. The BS production was found to be growth-associated. Detection of a unique band on TLC and GC chromatogram showed the extensive refining capacity of the BS purification, using the medium supernatant under acetone alkaline precipitation followed by oil dissolution from the sediment by carbon tetrachloride. Accordingly, it was clarified that the BS ultimately accumulated outside the cells. The glycolipid quality of the BS was further determined by the routine chemical characterization on TLC and by IR spectra analysis. Moreover, there was no protein detected by lowery total protein assay. Finally, the optimal temperature, pH and NaCl concentration to reach highest E24 values (85.7, 77.0, and 79.0 %) were found at respective 40 °C, pH = 9 and 0 % w/v. Our results revealed the practically potential of strain Dezful Isolate for BS large-scale production as environmentally friendly oil-eliminating agents.     

Bioremediation of heavily hydrocarbon-contaminated drilling wastes by composting

Oil-based drilling cuttings comprise a large and hazardous waste stream generated by oil and gas wells drilling operations. Oil-based cuttings are muddy materials with high contents of salts and hydrocarbons. Composting strategies have shown to be effective in the biodegradation of petroleum hydrocarbons, and it offers numerous advantages in comparison with other bioremediation methods. In order to assess the effectiveness of drilling cuttings bioremediation by composting with food and garden wastes, an experiment was conducted in 60-L reactors for 151 days. Four treatments were carried out: only oil-based cuttings, two proportions (in a volume basis) of organic wastes to drilling cuttings (33 and 75 %) and only organic wastes (as a traditional composting reference), with pine-tree woodchips as bulking agent. High degradation percentages of total hydrocarbons (≈82 %), n-alkanes (≈96 %) and the 16 USEPA-listed polycyclic aromatic hydrocarbons (≈93 %) were reached in the treatment with 75 % of organic wastes, and applying 33 % of organic wastes was not more effective than not applying organic wastes for the drilling cuttings hydrocarbons biodegradation. Furthermore, in the treatment with 75 % of organic wastes, alkanes half-life and polycyclic aromatic hydrocarbons half-life were about 10 times and four times lower, respectively, than those in the treatment with 33 % of organic wastes. Possibly, lower hydrocarbons and salts initial concentrations (i.e., lower toxicity), higher microbial counts, adequate nutrient proportions and water content supported a high biological activity with a consequent elevated biodegradation rate in the treatment with 75 % of organic wastes.