含油污泥

REGULAR ARTICLE

Rhizobacteria (Pseudomonas sp.SB)assist phytoremediation of oily-sludge-contaminated soil by tall fescue (Testuca arundinacea L.)

Wuxing Liu &Jianying Sun &Linlin Ding &

Yongming Luo &Mengfang Chen &Caixian Tang

Received:15January 2013/Accepted:5April 2013#Springer Science+Business Media Dordrecht 2013

Abstract

Background and aims The objectives of this study were to examine the effect of direct inoculation of seeds with the rhizobacteria Pseudomonas sp.SB on the growth of tall fescue and phytodegradation effi-ciency in an oily-sludge-contaminated soil.

Methods SB isolated from rhizosphere soil of tall fescue was evaluated for their plant-growth-promoting characters and ability to produce biosurfactant.A pot experiment was conducted to study the effect of inoculation of SB on phytoremediation.

Results SB reduced the surface tension of culture me-dia and produced indole acetic acid,siderophores,and 1-aminocyclopropane-1-carboxylate deaminase.Inoc-ulation of SB increased shoot and root dry weights of

tall fescue in oily-sludge-contaminated soil by 28%and 19%,respectively.Over 120days,the content of total petroleum hydrocarbon in soil decreased by 33.9%,68.0%,and 84.5%,and of polycyclic aro-matic hydrocarbons (PAHs)by 32.9%,40.9%,and 46.2%,respectively,in the no-plant control,tall fescue,and tall fescue +SB treatments.Inoculation of SB also increased the activity and biodiversity of soil microbial communities in the planted treatments.

Conclusions SB could produce biosurfactant and exhibited a number of characters of plant-growth-promoting rhizobacteria.Inoculation of SB to tall fescue led to more effective remediation of oily-sludge-contaminated soils.

Keywords Biosurfactant-producing bacterium .PGPR .Phytoremediation .Oily sludge

Introduction

The widespread extraction,refining,processing,trans-portation,and utilization of petroleum are posing an ever increasing risk of soil contamination with petro-leum hydrocarbons (Tahhan et al.2011).In addition to accidental contamination,improper disposal of oily sludge generated in oil refineries and petrochemical industries often leads to soil contamination and poses a serious threat to soil and groundwater as many of the constituents of oily sludge are carcinogenic and potent immunotoxicants (Propst et al.1999).

Plant Soil

DOI

10.1007/s11104-013-1717-x

Responsible Editor:Peter Christie.

W.Liu (*):J.Sun :L.Ding :Y .Luo :M.Chen Key Laboratory of Soil Environment and Pollution Remediation,Institute of Soil Science,Chinese Academy of Sciences,Nanjing 210008,China

e-mail:liuwuxin@https://www.sodocs.net/doc/269788398.html, Y .Luo

Institute of Coastal Zone Research,Chinese Academy of Sciences,Yantai 264003,China

C.Tang

Department of Agricultural Science,La Trobe University,Melbourne Campus,

Bundoora,Victoria 3086,Australia

Oily-sludge-contaminated soils are currently treated using physical,chemical,and biological processes. Physical or chemical methods include incineration,chlo-rination,ozonation,and combustion.Many of these technologies,however,are either costly or do not result in complete removal of contaminants.On the other hand, biological treatment(“bioremediation”)is widely used (Huang et al.2005;Liu et al.2010;Vasudevan and Rajaram2001;Wang et al.2012)and appears to be among the most promising methods for remediating a wide range of organic contaminants,particularly petro-leum hydrocarbons though some fractions of them with high molecular weight are fairly resistant to bioremedi-ation,requiring long periods of time for assimilation. Phytoremediation is one of bioremediation technologies. It uses plants and their associated microbes for environ-mental cleanup and is generally considered as an envi-ronmental friendly and gentle management option for polluted soils as it uses solar-driven biological processes to treat pollutants(Wenzel2009).Phytoremediation can be applied at moderate contamination levels and biotoxicity or after the application of other remediation measures as a further step to degrade residual pollutants (Merkl et al.2005).The successful application of phytoremediation of petroleum-contaminated soils has been shown in a number of studies(Gerhardt et al.2009; Huang et al.2005;Muratova et al.2008).However,there are also many limitations in phytoremediation.One of the factors limiting biodegradation of oil pollutants is that many hydrocarbon compounds are less readily accessible to microorganisms in the soil due to their low aqueous solubility and strong binding/sorption onto soil matrix.Low biomass production and slow growth of the plants due to the phytotoxicity of hydrocarbons in soil also limit effective remediation(Huang et al.2004, 2005).

One of the effective ways to increase the bioavail-ability of petroleum hydrocarbon pollutants in soil is using surfactants to enhance their desorption and solu-bilization,thereby facilitating microbial degradation (Kuyukina et al.2005).Biosurfactants are structurally diverse group of surface-active substances produced by living cells and have desirable characteristics such as biodegradability,low toxicity,and ecological accept-ability.Though most of biosurfactants are produced by microorganisms,including rhamnolipids,surfactin, sophorolipids,and so on,there are also plant-derived biosurfactants such as saponins.They can also be pro-duced from renewable and cheaper substrates than many synthetic surfactants(Banat et al.2000).A number of studies have been successfully carried out on soil biore-mediation using biosurfactants(Cameotra and Singh 2008;Lai et al.2009;Liu et al.2012;Urum et al.2003).

Successful plant growth in petroleum-contaminated soils is also critical for the optimum performance of phytoremediation.Plant-growth-promoting rhizobacteria (PGPR)have been used to enhance the tolerance of plants against toxicity through the production of plant-growth-promoting factors such as siderophores,indole-3-acetic acid(IAA),and1-aminocyclopropane-1-carboxylate (ACC)deaminase(Glick et al.2007).Therefore,the application of rhizobacteria with PGPR characters is a promising approach to improving the efficiency of phytoremediation.However,to the best of our knowl-edge,no report has been published on the utilization of biosurfactant-producing bacteria with PGPR characters to assist phytoremediation petroleum-contaminated soils.

Tall fescue(Testuca arundinacea L.)which can sig-nificantly increase the efficiency of hydrocarbon degra-dation in the soil(Huang et al.2005)was used in this study.The aims of this work were(1)to characterize the biosurfactant-producing Pseudomonas sp.SB which has the characters of PGPR,(2)to determine the impact of the strain inoculation on tall fescue growth and re-moval rate of total petroleum hydrocarbon(TPH)and polycyclic aromatic hydrocarbons(PAHs)in an oily-sludge-contaminated soil,and(3)to examine changes in the functional diversity indices of the microbial com-munity and the acute biological toxicity of the contam-inated soil after phytoremediation.

Materials and methods

Soil characterization

The oily-sludge-contaminated soil was collected from the surface layer(0–20cm)in Jingmeng city,Hubei Province,China(31°02′N,112°11′E).Soil samples were air-dried,ground to pass through a2-mm sieve, and stored in plastic bags until use.Hydrolyzable nitro-gen,cation exchange capacity(CEC),pH,and available phosphorus and potassium were measured using stan-dard methods(Lu1999)before phytoremediation,and the soil had the following properties(dry-weight basis): pH(1:2.5soil/water)6.45,hydrolyzable N558mg kg?1, available P171mg kg?1,available K174mg kg?1,and cation exchange capacity23cmol kg?1.

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Bacterium and surface tension measure

Pseudomonas sp.SB was isolated from rhizosphere soil of tall fescue(Testuca arundinacea L.)grown in a petroleum-contaminated soil and stored in the labora-tory.The isolated strain was inoculated into50ml of the LB medium and incubated at25°C with shaking at 180rpm for48h.The surface tension and the optical density at600nm of the culture medium were mea-sured every4h.The surface tension was measured by the ring method at25°C using a Model ZL-2digital tensiometer(Boshan Tongye Analytical Instrument, Shandong,China).

ACC deaminase,phosphate solubilization,IAA,

and siderophore assay

The activity of ACC deaminase of cell-free extracts was determined by estimating the amount ofα-ketobutyrate (α-KB)generated by the enzymatic hydrolysis of ACC. The amount ofα-KB was determined by comparing the absorbance at540nm of a sample to a standard curve of α-KB(Zhang et al.2011).Total protein was determined by the method of Bradford(1976)using bovine serum albumin as a standard.After determining the amount of protein andα-KB,the enzyme activity was expressed as molarα-KB per milligram per hour(Jalili et al.2009). Mineral phosphate solubilization was assayed on Pikovskaya agar plates containing insoluble tricalcium phosphate Amprayn et al.(2012).The plates were incu-bated at28°C,and development of a clear zone around the colony was evaluated at day5.

Bacterial IAA was measured as previously described (Glickmann and Dessaux1995).Single bacterial colony was inoculated into the TSB medium and grown over-night with shaking at200rpm in a water bath at30°C. The absorbance at600nm of culture was adjusted to approximately1.2,and5ml of each culture was inocu-lated into5ml of the DF medium[with(NH4)2SO4] containing L-tryptophan(200μg ml?1).The cultures were grown with shaking at200rpm at30°C for40h (Reed et al.2005).Siderophore secretion by the Pseu-domonas sp.SB was detected by the method of Payne (1994).

Pot experiment

A pot experiment was conducted in a glasshouse with natural light.The experiment consisted of three treatments in four replicates with a fully randomized design.The treatments were(1)no-plant control,(2) tall fescue,and(3)tall fescue plus Pseudomonas sp. SB(tall fescue+SB).Plastic pots were filled with 2.0kg air-dry soil,and water content of the soil was maintained at about80%field capacity during the experimental period.All treatments received80mg P kg?1as KH2PO4before the experiment.Other nutri-ents were at adequate ranges and thus were not applied.

Uniform seeds of tall fescue were surface-disinfected by immersing in0.3%sodium hypochlorite for5min and rinsing with sterilized distilled H2O.An aliquot of50 seeds were then sown in each pot of the planted treat-ments.For the tall fescue+SB treatment,the strain SB in the exponential phase in the LB medium were collected by centrifugation at9,000×g for15min at4°C,washed with sterile distilled water,and centrifuged.Bacterial inoculum was prepared by resuspending pelleted cells in sterile distilled water to obtain an inoculum density of109colony-forming units(cfu)ml?1.Bacterial suspen-sion(10ml pot?1)was mixed with the soil before sowing seeds.Plants were harvested at day120.

TPH analysis

Sub-samples(10g)of freeze-dried and pulverized soil were mixed with an equal volume of anhydrous Na2SO4.Then the mixtures were placed in a thimble and extracted in a Soxhlet apparatus with100ml tetrachloromethane for24h and analyzed by FT-IR after passing the extract through a florisil column.The TPH content of the soil before remediation is 1,851mg kg?1.

PAHs analysis

An aliquot of2g freeze-dried soil samples was mixed with an equal volume of anhydrous Na2SO4.Then the mixtures underwent Soxhlet extraction with dichloromethane(DCM)for24h.The extracts were reduced to2ml using rotary evaporation followed by further concentration under a weak stream of nitrogen. The residue was dissolved in2ml of cyclohexane and 0.5ml of the solute and extracted.The extracts were then purified by a silica gel column(8mm×220mm) and washed with a mixture of hexane and DCM(1:1). The first1ml of elute was discarded because it contained non-polar saturated hydrocarbons and was

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less retained than PAHs by silica gel.The second 2-ml aliquot of elute was collected,dried by sparging with N2,and dissolved in1ml hexane (Ping et al.2007).

Sixteen US Environmental Protection Agency (USEPA)priority PAH compounds were identified and quantified using GC–MS.An Agilent7890N GC sys-tem and Agilent5795B series MSD equipped with G6500-CTC autosampler was used.The GC–MS oper-ating conditions were as follows:DB-5silica column (30m×0.25mm×0.25μm);injection temperature was set at250°C;splitless mode.The column temperature was programmed as follows:50°C hold for1min, rising at25°C min?1to200°C,rising at8°C to 280°C,rising at1°C to283°C,and rising at2°C to 290°C.Helium was used as the carrier gas with flow rate at1.0ml min?1.Peaks was verified and quan-tified based on key fragment ions,retention times compared to those of external PAH standards,and/or mass spectra.The MS operating conditions were as follows:ionization voltage70eV,transfer line tem-perature260°C,ion source230°C,and mass scan range m/z60–640amu.

Microbial counts and community-level physiological profiling(CLPP)analysis

Soil samples were taken on days60and120for analyses of heterotrophic bacteria,TPH degraders and PAH degraders,and on day120for determination of the CLPP of the soil microbial communities.The heterotrophic bacteria were enumerated on nutrient agar plates.TPH degraders and PAH degraders were enumerated by the most-probable-number(MPN) technique(Wrenn and Venosa1996).The CLPP of the soil microbial communities was analyzed with gram-negative(GN)microplates(BIOLOG Inc.).Al-iquots of10?3dilutions were used to inoculate GN plates(150μl per well)and incubated at30°C.The plates were read every12h(OD590)over132h using a BIOLOG automated plate reader.All wells were blanked to the control well A1.For the BIOLOG data, average well color development(AWCD)was calcu-lated as described by Garland and Mills(1991).The Shannon index,Shannon evenness,McIntosh index, McIntosh evenness,and Gini coefficient of soil micro-bial communities were used to measure species meta-bolic diversity(Harch et al.1997;Yang and Yao 2000).Toxicity assay

The biotoxicity of the contaminated soil before and after remediation was studied according to the methods of P?aza et al.(2005a).Briefly,DCM and dimethylsulfoxide(DMSO)were used to extract the soil.The biological toxicity of the DCM/DMSO ex-tracts and DMSO(control)were tested based on the measurement of reduction in light emission by Photobacterium phosphoreum T3under toxic stress. All bioassays were carried out in triplicate and light output was measured using a portable luminometer (Model DXY-2).Toxicity results were reported as the effective concentration promoting a50%(EC50) reduction in light emitted by the bacteria.

Statistical analysis

All experiments were conducted with four replicates or otherwise specified.The data collected were ana-lyzed statistically using SPSS16.0software.Duncan’s multiple range tests was used to compare the means of the treatments,variability in the data was expressed as the standard deviation,and a P<0.05was considered to be statistically significant.

Results

Bacterial growth and biosurfactant production

Growth curves were obtained for the strains Pseudo-monas sp.SB in order to establish the relation between cell growth and surface activity of the biosurfactant in time(Fig.1).The results showed the absorbance (growth)and surface tension of the LB medium plot-ted versus time for the strain.Surfactant production was directly proportional to cell growth as the surface tension decreased with increasing cell density.The lowest surface tension(27.3mN m?1)and highest absorbance(1.59)was obtained at16h.Therefore, 16h incubation is sufficient for surfactant production and cell growth in the following experiments.

Plant-growth-promotion features of Pseudomonas sp.SB

Pseudomonas sp.SB was found to exhibit a number of traits which are important for plant-growth-promoting activity.The production of ACC deaminase was

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2.04M α-KB mg ?1h ?1in the DF medium.The strain also had the capacity to produce IAA (16.0mg l ?1)in the LB medium when the medium was supplemented with L -tryptophan.Orange halos around the colonies of SB on the blue agar were formed,indicating siderophore excretion of the strain.Clear zone around the colony of the strain appeared in the SRSM agar plate after 5days,indicating that the strain was able to solubilize P (Jin-Hee et al.2011).Plant growth

The inoculation of Pseudomonas sp.SB improved the growth of tall fescue.It increased shoot and root dry weights by 28%and 19%,respectively,by the end of the experiment (Table 2).Degradation of TPH and PAHs

The TPH contents of the soil after 120days are shown in Table 1.The TPH decreased by 33.8%,68.0%,and 84.5%in non-planted control,tall fescue,and tall fescue +SB treatments,respectively.The content of TPH was significantly lower (P <0.05)in two treat-ments with plants than the control,and the inoculation of BS further decreased TPH contents.

Sixteen USEPA PAHs were detected in the initial soil (Table 2).After 120days,total PAHs decreased by 31.7%,40.7%,and 46.2%in the control,tall fescue,and tall fescue +SB treatments,https://www.sodocs.net/doc/269788398.html,-pared with the tall fescue treatment,the inoculation of BS further decreased the contents of total PAHs and individual PAHs with high molecular weights (HMW)(five to six rings)such as benzo[k ]fluoranthene,

dibenz[ah ]anthracene,and benzo[ghi ]perylene.For example,the removal efficiency of benzo[a ]pyrene,a strongly carcinogenic compound,was 43.7%,43.7%,and 61.3%in the soil of the control,tall fescue,and tall fescue+SB treatments,respectively.Microbial populations

To reveal the treatment effects on microbial commu-nities in the soil,heterotrophic bacteria,TPH de-graders,and PAH degraders were quantified at days 60and 120.The counts of heterotrophic bacteria,TPH degraders,and PAH degraders were all the highest in the tall fescue +SB treatment and lowest in the no-plant control at day 60.Heterotrophic bacteria showed the same trend at day 120,but the counts of TPH and PAHs degraders decreased over time and were lower in the plant treatments than in the control at day 120(Table 3).

CLPP of the sludge microbial communities

AWCD and diversity index were analyzed using BIOLOG data to detect treatment difference in micro-bial communities.The activity of soil microbial com-munities in the soil evaluated by AWCD increased over time and was highest in the tall fescue+SB treat-ment and lowest in the controls (Fig.2).The absor-bance of each well in the BIOLOG plates at 72h was used to calculate diversity indices of the microbial community (Table 4).The Shannon index,Gini coef-ficient,and McIntosh evenness were significantly higher in the planted treatments than in the control.The inoculation of SB further increased the Shannon evenness,McIntosh Index,and Gini

coefficient.

Table 1Effect of growing tall fescue for 120days and inocu-lation of Pseudomonas sp.SB on plant biomass and concentra-tion of total petroleum hydrocarbon (TPH)in soil Treatments

Shoot dry weight(g)Root dry weight (g)TPH

(mg kg ?1)No-plant control –

–

1,224±16a Tall fescue 9.64±0.50b 2.29±0.27b 593±63b Tall fescue +SB

13.38±1.34a

2.84±0.19a

286±22c

The data represent the mean ±standard deviation of four repli-cates,and data followed by different letters indicate a significant difference at p <0.05according to Duncan ’s multiple range tests

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Biotoxicity assay

Biotoxicity of the soil was determined before and after phytoremediation by P.phosphoreum T3.EC50values were ranked as follows:Before remediation(Before Rem)

Table2Removal of polycyclic aromatic hydrocarbon(PAHs)after120days of growing tall fescue with or without inoculation of Pseudomonas sp.SB

PAHs Initial No-plant control Tall fescue Tall fescue+SB

(μg kg?1soil)Concentration

(μg kg?1soil)%Removal a Concentration

(μg kg?1soil)

%Removal Concentration

(μg kg?1soil)

%Removal

Naphthalene747±31a329±26c55.9±3.5176±7b76.4±0.991±2d87.9±0.2 Acenaphthylene106±14a61±5bc42.1±4.251±1b51.4±0.544±6c58.4±5.3 Acenaphthene150±22a94±7b37.5±4.680±1b46.9±0.573±8b51.8±5.3 Fluorene505±17a362±20b28.3±4.0292±18c42.2±3.5292±27c42.2±5.3 Phenanthrene288±24a299±21bc0255±6c11.7±2.0264±17bc8.7±6.0 Anthracene93±7a51±9b44.8±10.057±4b38.5±4.459±4b36.4±4.1 Fluoranthene157±8a118±9b25.3±5.4119±3b24.5±2.0114±3b27.3±2.0 Pyrene185±13a143±5b22.8±2.6141±8b23.6±4.0117±6c36.7±3.1 Benz[a]anthracene151±12a96±13b36.3±8.894±4b37.7±3.084±2b44.5±1.2 Chrysene824±18a442±40b46.4±4.9361±9c56.2±1.2301±11d63.5±1.3 Benzo[b]fluoranthene612±15a232±23b62.1±3.9190±22b69.0±3.6250±84b59.2±13.7 Benzo[k]fluoranthene411±14a175±6b57.5±1.3171±5b58.5±1.1104±4c74.7±1.0 Benz[a]pyrene426±6a240±5b43.7±1.1240±6b43.7±1.4165±3c61.3±0.8 Indeno[123-cd]pyrene357±23a227±17b36.5±4.8160±23c55.1±6.3135±8c62.2±2.2 Dibenz[ah]anthracene7,599±115a5,614±159b26.1±2.15,066±271c33.3±3.64,741±90c37.6±1.2 Benzo[ghi]perylene339±20a203±9b40.2±2.7202±4b40.4±1.1130±3c61.6.6±0.8 Total12,950±65a8,684±101b32.9±0.87,655±331c40.9±2.56,964±179d46.2±1.4

The data represent the mean±standard deviation of four replicates,and data followed by different letters in the same row indicate a significant difference at p<0.05according to Duncan’s multiple range tests

a%Removal was calculated as(PAH concentration after120days×100)/initial concentration

Table3Heterotrophic bacteria,TPH degraders,and PAH degraders in the soil after growing tall fescue for60and120days with or without inoculation of Pseudomonas sp.SB

60Days120Days

Control Tall fescue Tall fescue+SB Control Tall fescue Tall fescue+SB

Heterotrophic bacteria

(log cfu g?1dry soil)

8.28±0.06b8.49±0.04a8.57±0.05a8.27±0.04c8.44±0.05b8.65±0.03a

TPH degraders

(log MPN g?1dry soil)

4.98±0.04c 6.22±0.09b 6.42±0.08a

5.2±0.17a 4.50±0.13b 4.44±0.07b

PAH degraders

(log MPN g?1dry soil)

4.82±0.05b

5.27±0.12a 5.43±0.06a 4.30±0.30a 3.76±0.05b 3.69±0.17b

The data represent the mean±standard deviation of four replicates,and data followed by different letters in the same row at the same sampling day indicate a significant difference at p<0.05according to Duncan’s multiple range tests

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Discussion

Mass transfer from adsorbed or insoluble phases to the aqueous phase is considered to be the rate-limiting step in biodegradation of organic contaminants be-cause the compounds must be released to the aqueous phase prior to entering the microbial cell and subse-quent intracellular transformation by the necessary catabolic enzymes (Dean et al.2001).Compounds present in oily sludge are inherited from petroleum hydrocarbons,and most of them are insoluble.Fur-thermore,oily sludge undergoes several aging and weathering processes that eventually lead to the com-pounds having low water solubility.As shown in Table 2,the HMW PAHs (five-and six-ring PAHs)with low water solubility accounted for 75%of total PAHs in the oily contaminated soil used in this exper-iment.There are several reports on improving oily sludge degradation by addition of biosurfactant or chemical surfactant to increase the bioavailability of

the hydrocarbons (Ahn et al.2010;Benincasa 2007;Cameotra and Singh 2008;Lai et al.2009).However,the effect of inoculating biosurfactant-producing bacteria which can produce surfactants successively is relatively less studied.

This study showed that Strain SB produced the biosurfactant that reduces the surface tension of medium to 27.3mN m ?1.The biosurfacant is expected to en-hance desorption of TPHs and PAHs and increased their removal from the soil.It was evident that SB inoculation significantly (P <0.05)increased the removal of TPHs and PAHs in the sludge-contaminated soil.For HMW PAHs such as benzo[ghi ]perylene,benz[a ]pyrene,and benzo[k ]fluoranthene,their removal rates were 21.2%,17.6%,and 16.3%higher in the BS inoculated treat-ment than in the tall fescue alone treatment,respectively.These PAHs all have large K ow (above 6.0)and low water solubility.The bioavailability could be the rate-determining factor for their degradation.One possible reason why biosurfactant-producing bacteria enhanced the removal of TPHs and PAHs is that the inoculation could increase the rate of solubilization and desorption of TPHs and PAHs by enhancing the rate of

mass

Fig.2Changes in average well color development (AWCD)of soil microbial community during incubation.Error bars :±1standard deviation (n =4)

Table 4Functional diversity indices of microbial community in soils of no-plant control and after 120days of growing tall fescue with or without inoculation of Pseudomonas sp.SB Treatments Shannon index Shannon evenness Gini coefficient McIntosh index McIntosh evenness No-plant control 3.971±0.015b 0.901±0.007b 0.978±0.001c 11.211±2.463b 0.955±0.004b Tall fescue 4.193±0.071a 0.912±0.014b 0.982±0.001b 11.972±0.439b 0.973±0.006a Tall fescue +SB

4.322±0.040a

0.959±0.004a

0.986±0.001a

16.782±1.342a

0.984±0.002a

The data represent the mean ±standard deviation of four replicates,and data followed by different letters in the same column indicate a significant difference at p <0.05according to Duncan ’s multiple range

tests

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transfer from adsorbed phases to soil solution(Juhasz and Naidu2000).

Apart from the produced biosurfactant,Pseudomonas sp.SB used in this experiment also exhibited a number of characters of PGPR and improved the development of the root and shoot system of tall fescue.It produced IAA and siderophore,and had the P-solubilization ability.Espe-cially,the bacteria with ACC-deaminase activity metab-olize ACC(an immediate precursor of ethylene in plants) intoα-ketobutyric acid and ammonia,thus regulating the biosynthesis of ethylene and promoting plant growth.

The degradation of most hydrocarbons is believed to enhance through a rhizosphere effect.Plants exude organ-ic compounds through their roots,which increase the density,diversity,and activity of specific microorganisms which in turn degrade hydrocarbons in the surrounding rhizosphere(Phillips et al.2006).Hence,extensive root growth is a prerequisite of maximizing the effectiveness of phytoremediation processes.Reduced root growth in contaminated soils might be owing to production of eth-ylene induced by toxicity stress(Arshad et al.2007). Huang et al.(2004;2005)demonstrated that biomass accumulation and root growth of tall fescue were severely decreased by TPHs in soil,although tall fescue is relative-ly tolerant of organic contaminants.Merkl et al.(2005) also showed that root biomass was positively correlated with phytoremediation activity in a hydrocarbon-contaminated soil.

In phytoremediation,hydrocarbons are degraded mainly by soil microbial communities.Therefore,the phytoremediation potential can be characterized by enumerating cultivable soil heterotrophic and pollutant-degrading bacteria in the rhizosphere(Muratova et al.2008).This present study showed that tall fescue planting and SB inoculation promoted TPH and PAH degraders at60days,although the TPH and PAHs de-graders were lower than that of control at120days (Table3).The heterotrophic bacterial counts in the two planted treatments were always higher than in the control plot throughout the experiment(Table3).The reason might be that most of the TPH and PAHs were degraded in two planted treatments and their concentration was very low and hard to support the degraders’growth at the end of the experiment.

Wuensche et al.(1995)demonstrated that substrate utilization patterns as recorded with the BIOLOG sys-tem are suitable for rapidly assessing the dynamics of autochthonous communities and evaluating their biodegradative potential in soil.The highest activity of soil microbial communities evaluated by AWCD in the tall fescue+SB treatment indicates that tall fescue and Pseudomonas sp.SB inoculation had important influences on the microbial activity.

There were also similar effects of growing tall fescue and SB inoculation on microbial metabolic diversity reflected by Shannon index,McIntosh index, and Gini coefficient.The Shannon index provides the information on the distribution of C source utilization by microbial communities and potential metabolic diversity of the communities while the McIntosh index provides information on species richness based on the number of species and their abundances.Gini coefficient quantifies the inequality of use of carbon sources (richness and evenness)(Harch et al.1997).Thus, it can be concluded that phytoremediation not only decreased TPH content but also increased microbial activity and community diversity in this oily-sludge-contaminated soil.It must be emphasized that CLPP is not a culture-independent method and is somewhat biased towards fast-growing and easily cultivable species(Smalla et al.1998).

Petroleum contains hundreds of individual com-pounds with varying degrees of toxicity,mutagenicity, and carcinogenicity,and its composition varies with sites and source strata(Liu et al.2009).Therefore, ecotoxicity bioassays should be used as supplementary tools for monitoring the remediating effectiveness of petroleum-contaminated sites(P?aza et al.2008;P?aza et al.2005b).In this study,the toxicities of the soil as a function of petroleum biodegradation activity were also determined.P.phosphoreum T3was used to test the biotoxicity of the oily sludge before and after bioreme-diation.The EC50of the soils increased by41%, 218%,and550%in the control,tall fescue,and tall fescue+SB treatments,respectively,at the end of the experiment.Clearly,the EC50of the SB-inoculated treatment was the highest,indicating that the soil of this treatment had the lowest toxicity.This ecotoxicity decrease of the soils might be due to the efficient conversion of the toxic raw-petroleum material to less or non-toxic intermediates and by-products during biodegradation(P?aza et al.2008). Conclusion

In this study,the biosurfactant-producing strain Pseudomonas sp.SB was isolated from rhizosphere

Plant Soil

of tall fescue grown in a petroleum-contaminated soil. This strain can produce biosurfactants thereby decreasing the surface tension of the culture medium to27.3mN m?1 after16h of growth.This strain also produced IAA, siderophores,and ACC deaminase.The inoculation of the SB strain promoted the growth of tall fescue and significantly enhanced the degradation of TPH and PAHs.It also increased the microbial activity and diver-sity in the soil.Furthermore,the inoculation of the SB markedly decreased the biotoxicity of the contaminated soil.Thus biosurfactant-producing Pseudomonas sp.SB which has the characters of PGPR may be a suitable bioinoculant to assist phytoremediation of oily-sludge-contaminated soils,but validation under field conditions is needed.

Acknowledgments We thank the National Natural Science Foundation of China(41001182),Jiangsu Provincial Natural Science Foundation of China(BK2012891),and the Environmental Protection Public Welfare Special Fund for Scientific Research (201009015)for financial support.

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热解技术含油污泥无害化处理与资源化利用

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陕西省含油污泥处理处置企业监督管理指南 （试行） 为落实《中华人民共和国环境保护法》、《中华人民共和国固体废物污染环境防治法》、《关于办理环境污染刑事案件适用法律若干问题的解释》、《危险废物经营许可证管理办法》及《陕西省固体废物污染环境防治条例》等法律法规，强化我省含油污泥处理处置企业危险废物管理的主体责任，规范我省含油污泥处理处置企业的监督管理，特制定《陕西省含油污泥处理处置企业监督管理指南（试行）》（以下简称“指南”）。 指南在分析我省含油污泥处理处置企业项目构成及主要工艺、废物产生环节与规律的基础上，明确该行业环境监督管理的要点和方法，指导企业规范处理处置危险废物，供环境保护主管部门开展日常管理与监督检查时参考使用。 1适用范围 本指南适用于我省各级环境保护行政主管部门对本行政区域内含油污泥处理处置企业的日常环境管理和监督检查。 2监管依据 2.1法律、法规 2.1.1《中华人民共和国环境保护法（修订）》 2.1.2《中华人民共和国大气污染防治法（修订）》 2.1.3《中华人民共和国水污染防治法（修订）》 2.1.4《中华人民共和国固体废物污染环境防治法（修订）》 2.1.5《国家危险废物名录》（环境保护部、国家发展和改革委员会、公安部令第39号，2016年） 2.1.6《危险废物转移联单管理办法》（国家环境保护总局令第5号，1999年） 2.1.7《危险废物经营许可证管理办法》（国务院令第408号，2004年） 2.1.8《陕西省煤炭石油天然气开发环境保护条例（2007年修正本）》（陕西省

人民代表大会常务委员会公告第78号，2007年） 2.1.9《陕西省固体废物污染环境防治条例》（陕西省人民代表大会常务委员会公告第29号，2015年） 2.2政策 2.2.1《危险废物污染防治技术政策》（环发[2001]199号） 2.2.2 《关于进一步加强危险废物和医疗废物监管工作的意见》（环发[2011]19 号） 2.2.3《石油天然气开采业污染防治技术政策》（环境保护部公告2012年第18号） 2.2.4《关于进一步规范油泥、泥浆等危险废物的无害化处置和综合利用工作的通知》（陕环函[2010]766号） 2.2.5《关于进一步加强危险废物规范化管理工作的通知》（陕环办发[2012]144号） 2.2.6《危险废物规范化管理指标体系》（环办[2015]99号） 2.3标准、规范 2.3.1《危险废物鉴别标准》（GB5085-2007） 2.3.2《一般工业固体废物贮存、处置场污染控制标准》（GB18599-2001及其修改单） 2.3.3《危险废物贮存污染控制标准》（GB18597-2001及其修改单） 2.3.4《危险废物焚烧污染控制标准》（GB18484-2001） 2.3.5《危险废物填埋污染控制标准》（GB18598-2001及其修改单） 2.3.6《恶臭污染物排放标准》（GB14554-93） 2.3.7《含油污泥处置利用控制限值》（DB61/T1025-2016） 2.3.8《危险废物收集贮存运输技术规范》(HJ2025-2012) 2.3.9《危险废物处置工程技术导则》（HJ2042-2014） 3术语和定义 下列术语和定义适用于本指南：

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龙源期刊网 https://www.sodocs.net/doc/269788398.html, 浅析含油污泥处理 作者：柴源 来源：《山东工业技术》2019年第16期 摘要：含油污泥属于危险废物，主要来自于石油行业。本文针对石油行业含油污泥处理 现状，通过已应用的含油污泥处理技术对比，对其应用的含油污泥的类别进行分析，选择最佳的处理技术。 关键词：含油污泥;对比;分析;处理技术 DOI：10.16640/https://www.sodocs.net/doc/269788398.html,ki.37-1222/t.2019.16.027 0 前言 含油污泥（砂）成分非常复杂，是一种含泥砂、原油、污水等多相稳定胶态悬浮体系。 其危害环境的主要成分是烃类、石油类、各类化学处理剂残留物等。含油污泥处理应遵循“减量化、无害化、资源化”的原则，采取“科学、安全、无害化”的技术方法。有力、有序、有效的开展含油污泥的处理、处置工作。以保护环境、消除风险和综合利用为最终目的。 1 含油污泥处置总体技术路线 （1）含油污泥来源。1）落地油泥。落地油泥多从油田的井下“测井”、“试井”、“修井”和采油设施的“清淤”得到的固体含油污泥，其特点是：砂石泥土含量大;塑料杂草等纤维物质多;石油含量高（20%左右）;化学反应随产地、堆放时间变得极其复杂。2）清罐油泥。采油生产中处理含油污水设施产生的罐底泥、池底泥清罐油泥。其特点是：由水包油、油包水的微细悬浮物组成，比重比水重;稠油、胶质、蜡质含量高;含水高达95%～99.7%，浓缩、过滤困难。3）浮渣。采油生产中处理含油污水设施产生气浮泥其特点是：由水包油、油包水的微细悬浮物组成，比重接近水;含水高达90%以上。 （2）含油污泥处理。含油污泥采用预处理工艺进行分选和清洗，分离出的油水进入联合站现有油水处理系统进行处理，分离出的固相与其它含油污泥进入深度减量化处理工艺。深度减量化处理分别采用精细化学热洗技术、土壤修复技术、间接热解吸技术、水洗法油泥处理技术等。分离出的剩余固相含油率小于2%，符合《陆上石油天然气开采含油固体废弃物资源化综合利用及污染控制技术要求》SY/T7301-2016标准，用于铺垫油田通井路、井场，或者采用非烧结技术制砖，用于建设井场围堰，最终实现含油污泥的资源化利用。 2 精细化学热洗技术 （1）处理油泥类型。含油污泥化学热洗技术广泛应用于含油量高，低乳化原油和油砂。

含油污泥来源与处理方法方案

前言 含油污泥是在石油开采、运输、炼制及含油污水处理过程中产生的含油固体废物。污泥中一般含油率在10～50%，含水率在40～90%，我国石油化学行业中，平均每年产生80万t罐底泥、池底泥，胜利油田每年产生含油污泥在10万吨以上，大港油田每年产生含油污泥约15万吨，河南油田每年产生5×104m3含油污泥。含油污泥中含有大量的苯系物、酚类、蒽、芘等有恶臭的有毒物质，含油污泥若不加以处理，不仅污染环境，而且造成资源的浪费。含油污泥的处理一直是困扰油田的一大难题。 1．含油污泥来源 含油污泥的来源主要有以下几种途径： 1.1 原油开采产生含油污泥 原油开采过程中产生的含油污泥主要来源于地面处理系统，采油污水处理过程中产生的含油污泥，再加上污水净化处理中投加的净水剂形成的絮体、设备及管道腐蚀产物和垢物、细菌(尸体)等组成了含油污泥。此种含油污泥一般具有含油量高、粘度大、颗粒细、脱水难等特点,它不仅影响外输原油质量,还导致注水水质和外排污水难以 达标。

1.2 油田集输过程产生含油污泥 胜利油田含油污泥的主要来源于接转站、联合站的油罐、沉降罐、污水罐、隔油池底泥、炼厂含油水处理设施、轻烃加工厂、天然气净化装置清除出来的油沙、油泥，钻井、作业、管线穿孔而产生的落地原油及含油污泥。油品储罐在储存油品时，油品中的少量机械杂质、沙粒、泥土、重金属盐类以及石蜡和沥青质等重油性组分沉积在油罐底部，形成罐底油泥。 中原油田污泥产生主要是一次沉降罐、二次沉降罐、洗井水回收罐的排污。含油污泥本身成分复杂，含有大量的老化原油、蜡质、沥青质、胶体和固体悬浮物、细菌、盐类、酸性气体、腐蚀产物等，污水处理过程中还加入了大量的凝聚剂、缓蚀剂、阻垢剂、杀菌剂等水处理药剂。 在3~6年的油罐定期清洗中，罐底含油污泥量约占罐容的1%左右。罐底含油污泥的特点是碳氢化合物（油）含量极高。据调查测试发现，油罐底泥中大约25%为水，5%的无机沉淀物如泥沙，70%左右为碳氢化合物，其中沥青质占7.8%，石蜡占6%，污泥灰分含量4.8%。 1.3 炼油厂污水处理场产生的含油污泥 炼油厂污水处理场的含油污泥主要来源于隔油池底泥、浮选池浮渣、原油罐底泥等，俗称“三泥”，这些含油污泥组成各异，通常含

试论含油污泥如何实现无害化处理

试论含油污泥如何实现无害化处理 油田以及炼化企业在生产的过程中往往会产生大量的含油污泥，因此如何更加有效地开展含油污泥的无害化处理显得尤为重要，也成为了近年来相关研究的重点。本文对于含油污泥无害化处理的技术原理进行探討，对于当前含油污泥无害化处理技术进行分析，其中包括了堆肥法处理技术，固化处理技术以及高温处理技术等。通过对于含油污泥无害化处理技术的优缺点进行分析，对于其未来的发展方向进行讨论，希望可以为相关企业的发展和研究提供参考。 标签：含油污泥；无害化处理；发展方向 引言 当前在石油开采以及炼制的过程中，会产生大量的含油污泥，含油污泥中含有取得原有材料，还有污泥中也有许多有害的物质，例如重金属，以及一些有毒性的阴离子和放射物性质，长期存放在空气中，会对环境产生重要的影响和危害，同时也会造成石油资源的浪费，当前国内外含油污泥处理技术方法比较多，但是实用性比较强，难以真正实现无害化处理，因此，对于当前含油污泥无害化处理技术进行分析，对于帮助人们，对于现有含有工具无害化处理技术进行更好的掌握，有着重要的作用，同时能够推动进行无害化深度处理技术的开发。 一、机械分离技术分析 机械分离技术作为一种传统的处理方法，相对来说是比较有效的，这种方法主要就是通过加入调制剂，对于污泥的稳定状态进行破坏，提高脱水效率，也可以使用加热处理和冷冻熔处理法进行调质。开展污泥调质，主要的目的就是通过一定的手段，对于固体粒子群的性状和排列状态进行有效的调整，使其能够适合不同的脱水条件。机械分离技术处理污泥油的回收率比较高，国外这一技术相对来说已经很成熟。当前，国内含油污泥系统中，使用絮凝剂进行调质的比较多。由于不同地区含油污泥成分有一定的差异，需要药剂和脱水机械更好的适应，保证通用优化。如果其中的化学试剂不能够有效处理，容易造成二次污染。机械分离，主要就是把含油污泥中的游离水进行脱离，脱除一些有害杂质，当然，污泥处理后还需要进一步的处理。 二、萃取处理技术分析 含油污泥经过加药和机械脱水后，含油量一般在百分之八十左右，并且其中的水主要是以游离水为主，更多的是以间隙水和内部结合水以及附着水的形式存在。在常规操作中难以实现油、水和固体物的彻底分离。萃取法主要就是把含油污泥溶解，把含油污泥中的原油回收利用。萃取法中包含了相似相溶的原理，选择比较适合的有机溶剂作为萃取剂把含油污泥进行溶解，把含油污泥中的原油进行回收和利用。

含油污泥处理工艺综述

含油污泥处理工艺综述 摘要：含油污泥是油田生产过程中产生的固体废物，已被列入《国家危险废物 名录》，它的处理时国内外一个难攻克的难题。通过介绍不同的处理工艺技术， 对目前的技术路线进行对比。 关键词：含油污泥技术对比 原油在开采、炼制、储存等过程中会产生大量的油泥，其特点是含有多种有毒有害物质，含水率高，固体颗粒小，油水固乳化严重，脱水困难，其处置问题一直是石化企业的老大难 问题。2008年国家对危险废物名录进行修改，将含油污泥划为危险废物。随着国家危废名录 的出台，地方环保部门也加大了石化企业油泥外委处置的监管力度，有些地方环保部门严格 限制油泥出厂。由此可见，油泥处理已成为各级环保部门的一大关注热点。随着国家环保法 规的日益严格和公众环保意识的增强，环保执法力度的增大，油泥无害化处置将是石化企业 继污水达标处理和废气达标处理之后的又一项环保要求【1】。 一、油泥类型 根据成因不同，油泥通常分为：落地油泥、罐底油泥、地面溢油、炼油厂含油污泥等。 （1）落地油泥 在油田采油生产、原油输送、装置检修及拆油井过程中，井喷和防喷后总有后一些石油 无法回收，再加上由于事故、跑冒滴漏等原因会有或多或少的原油流到地上，从而形成落地 油泥。 （2）罐底油泥 油品储罐在储存油品特别是原油时，存放时间一般较长，特别是战略储备时储存时间更长，这时原油及油品中高熔点蜡、沥青质、胶质和所夹带的沙粒、泥土、重金属盐类等无机 杂质因密度差便会和水一起沉降积累在油罐底部，形成又黑又稠的胶状物质，即罐底油泥， 其数量一般高达该储罐容量的1%~2%。 （3）地面溢油 油气田地面溢油中的地面，主要指油气田区域范围农用或可农用的土壤，也包括沿海滩 潦土壤、沙石土壤等；溢油主要指落地原油，也包括落地的煤油、汽油等成品油成品油。油 气田产生地面溢油的环节很多，钻井过程中起下钻作业、试井、井喷、清理钻井设备；采油 过程中抽油管的断裂、采油树机泄漏、抽油机停车进行作业检修；原油集输过程中集输管线 断裂引起的泄漏以及原油炼制加工等环节均能产生地面溢油，从而形成油泥。 （4）炼油厂含油污泥 炼油厂的污水处理系统产生的污泥主要来自隔油池的底泥、浮选池浮渣、剩余活性污泥、统称为“三泥”。炼油厂含油污泥的性质复杂，黏度较大，难以沉降，且浓缩困难，脱水和处 理技术难度大，一直是困扰炼油行业的环保难题【2】。 二、油泥的性质及特征 油泥的成分极其复杂，随地质条件、生产工艺的不同而各异，一般由水、泥土、油类有 机物等组成。油泥主要有以下几个特征：水含量高、体积大；成分复杂、处理难度大；含有 大量污油和可燃物质；有害成分多数超过排放标准。 三、国内外油泥净化处理技术 从80年代中期开始，美国、日本、德国、前苏联等发达国家开始研究高效低耗处理油泥的方法和工艺。现今国内外处理含油污泥的方法一般有：焚烧法、生物处理法、热洗涤法、 溶剂萃取法、化学破乳法、固液分离法等。尽管处理的方法很多，但都因针对性不强、处理 成本高等缺点没有推广。对含油污泥进行无害化、清洁化并回收其中资源的综合处理，成为 国内外环境保护和石油工业的重点之一。 (1）焚烧法 法国、德国的石化企业多采用焚烧的方式，污泥先经过调制和脱水预处理，浓缩后的污 泥再经设备脱水干燥，将泥饼送至焚烧炉进行焚烧，灰渣用于修路或埋入指定的灰渣填埋场，焚烧产生的热能用于供热发电。

含油污泥处理技术

含油污泥处理技术及资源化利用途径* 匡少平1,2,3宋峰2 (1.清华大学环境工程博士后流动站，北京100084； 2.青岛科技大学化学与分子工程学院，山东青岛266042； 3. 中原油田博士后工作站，河南濮阳457001） 摘要石油开采和加工过程中产生的大量含油污泥，对生产和生态环境产生极大危害，同时又是一种宝贵的二次资源，对其既必须进行无害化处理又可以进行资源化利用。国内外主要的含油污泥处理技术有：调质－机械脱水、生物处理、固化处理、焚烧、填埋与干化和综合利用等。介绍了各技术的原理、特点和研究应用进展。目前较受重视的技术是调质－机械脱水、生物处理、固化处理和综合利用。调质－机械脱水可有效的分离油-水-泥，但产生的泥饼需进一步处理；生物处理可以将含油污泥中的有机物彻底降解为CO2和H2O，但降解效率有待提高；固化可以大大降低有害物质的渗出率；资源化利用将成为含油污泥处理技术的发展趋势。 关键词含油污泥调质－机械分离生物处理固化资源化 Treatment and comprehensive utilization of oily sludge Kuang Shaoping1, 2, 3, Song Feng2. (1. Post-Doctor Station, Tsinghua University, Beijing 100084；2. College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao Shandong 266042；3. Post-Doctor Work Station, Zhongyuan Oilfield Branch, Puyang Henan 457001) Abstract:Oily sludge produced in the course of exploitation and processing does great harms to the production and the environment. Oily sludge must be treated harmlessly and be utilized comprehensively. Oily sludge treating techniques include sludge quality adjustment and mechanical dewatering, biological treatment, solidification, combustion, natural drying and burial method, and comprehensive utilization, and so on. This paper expatiated on the principles, characteristics and applications of different methods. Quality adjustment and mechanical dewatering, biological treatment, solidification and comprehensive utilization are regarded as important treating techniques. Oily sludge can be divided into oil, water and mud through quality adjustment and mechanical dewatering, but the sludge cake must be treated finally; Microorganism can degrade the organic substance of oily sludge into CO2 and H2O, but efficiency of biodegradation need to be enhanced; Solidification can decrease the exudation of injurant. Comprehensive utilization will be the dominant technique for oily sludge treatment in the future. Keywords:Oily sludge Quality adjustment and mechanical dewatering Biological treatment Solidification Comprehensive utilization 油田和炼油厂的污水处理系统以及原油生产储运系统会产生大量含油污泥。目前我国每年产生的含油污泥总量达500余万t[1]。随着大多数油田进入中后期开采阶段，采出油中含水率越来越高，含油污泥量还会继续增加。人们对含油污泥的处理进行了大量的研究，但至今没有一种成熟有效的处理方法。笔者对目前国内外含油污泥处理技术和资源化利用途径进行了探讨。 1 含油污泥的性质与危害 含油污泥成分极其复杂，主要由乳化油、水、固体悬浮物等混合组成, 其成分与地质 第一作者：匡少平，男，1966年生，教授，主要从事环境科学与工程的教学与研究工作，研究方向为固体废物资源化处理、工业废水处理。 *山东省教育厅科技发展项目（J05D51）；中原油田博士后基金资助项目（2005418）。