APPLiED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1979, p. 729-7390099-2240/79/04-0729/11$02.00/0

Vol. 37, No. 4

Effect of Environmental Parameters on the Biodegradation ofOil Sludget

J. T. DIBBLEf AND R. BARTHA*

Department ofBiochemistry and Microbiology, Cook College, Rutgers-The State University ofNew Jersey,New Brunswick, New Jersey 08903

Received for publication 24 January 1979

A laboratory study was conducted with the aim of evaluating and optimizingthe environmental parameters of "landfarming", i.e., the disposal by biodegrada-tion in soil of oily sludges generated in the refining of crude oil and relatedoperations. Oil sludge biodegradation was monitored by CO2 evolution and byperiodic analysis of residual hydrocarbons. The parameters studied were soilmoisture, pH, mineral nutrients, micronutrients, organic supplements, treatmentrate, treatment frequency, and incubation temperature. Oil sludge biodegradationwas optimal at a soil water-holding capacity of 30 to 90%, a pH of 7.5 to 7.8, C:Nand C:P ratios of 60:1 and 800:1, respectively, and a temperature of 20°C or above.Addition of micronutrients and organic supplements was not beneficial; sewagesludge interfered with hydrocarbon biodegradation. Breakdown of the saturatedhydrocarbon (alkane and cycloalkane) fraction was the highest at low applicationrates, but higher application rates favored the biodegradation of the aromatic andasphaltic fractions. An application rate of 5% (wt/wt) oil sludge hydrocarbon tothe soil (100,000 liters/hectare) achieved a good compromise between high bio-degradation rates and efficient land use and resulted in the best overall biodeg-radation rate of all hydrocarbon classes. Frequent small applications resulted inhigher biodegradation than single large applications. Two 100,000-liter/hectare(255 barrels per acre) or four 50,000-liter/hectare oil sludge hydrocarbon appli-cations per growing season seem appropriate for most temperate zone disposalsites.

Petroleum refining unavoidably generatesconsiderable volumes of oil sludges. Commonsources of these sludges are storage tank bot-toms, oil-water separators, flotation and biolog-ical wastewater treatment units, cleaning ofprocessing equipment, and soil from occasionalminor spills on refinery grounds. The composi-tion of these sludges varies according to theirorigin, storage, and treatment history. In a typ-ical case hydrocarbons, water, and mineral solidsare present in roughly equal proportions. Oilysludges constitute a disposal problem and,among other options, biodegradation in soil or"landfarming" offers a cost-effective yet environ-mentally acceptable alternative. In contrast toburial in anaerobic landfills, this mode of dis-posal leads to the relatively rapid biodegradationof the hydrocarbons by soil microorganisms,thus reducing the danger of groundwater con-tamination.

t Paper of the Journal Series of the New Jersey Agricul-tural Experiment Station.

t Present address: Research and Technical Department,Texaco, Inc., Port Arthur, TX 77640.

The biodegradation of accidentally spilled orwaste oils in aquatic and terrestrial environ-ments has been the subject of several recentreviews (3, 5, 7). A careful and extensive studyof oil biodegradation in soil by Raymond et al.(20), as well as previous work reviewed in thispaper, were all conducted in the field under localweather and precipitation conditions. To com-plement this work, we conducted a laboratorystudy on the effects of controlled environmentalparameters on oil hydrocarbon biodegradationunder simulated landfarming conditions. We ex-pected this study to clarify the feasibility andefficiency of the landfarming practice under avariety of climatic and soil conditions. We werealso seeking to optimize those parameters of thelandfarming practice that can be readily con-trolled. Using an actual refinery sludge and soilfrom a prospective disposal site, we evaluatedthe effects of soil moisture content, soil pH,levels of inorganic fertilizer, micronutrients, andorganic supplements, sludge loading rates, ap-plication frequencies, and temperature on hy-drocarbon degradation in the landfarming proc-ess.

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730 DIBBLE AND BARTHA

MATERIALS AND METHODS

Soil. The soil for these studies was collected duringthe summer season from a prospective landfarmingsite on the grounds of a petroleum refinery in centralNew Jersey. After removal of surface litter, soil wascollected to a 20-cm depth, passed through a 3-mmsieve, and stored at 20°C in plastic bags. The site hasa history of industrial use; its soil was highly com-pacted and had poor drainage and aeration character-istics. The plans for site preparation included theadmixture of sand to improve the above characteristicsof the soil, and in our laboratory experiments we alsomixed this soil with an equal weight of distilled wa-ter-washed sharp gardeners' sand. This sand passedthrough a 0.5-mm sieve but was retained on a 0.16-mmsieve. Out of concern that the poor quality of this soilmay have resulted in a low microbial diversity, beforeuse in experiments the soil was inoculated with 0.1%(wt/wt) good-quality agricultural soil preincubatedwith 0.5% (wt/wt) oil sludge hydrocarbons.

Oil sludge. An oil sludge sample was collected froman open storage pit at the refinery. The sources of thematerial were oil-water separators, storage tank bot-toms, and small spills on the refinery property. Toeliminate the water content and to facilitate handling,the sludge was air dried until sufficiently friable topass through a 3-mm sieve. The air-dried oil sludgewas stored at 5°C in sealed glass containers.

Analytical methods. Soil analyses were per-formed by the New Jersey Soils Testing Laboratory,Cook College, Rutgers University. Soil pH was deter-mined for a soil-distilled water slurry (1:1, wt/vol)with a pH meter. Soil reaction was adjusted by theaddition of CaCO3. Water-holding capacity was deter-mined by the Hilgard cup technique. Moisture con-tents of the soil and oil sludge were measured gravi-metrically after drying at 105°C for 12 h. Soil moisturewas adjusted by the addition of distilled water.The ash content of the oil sludge was determined

gravimetrically after dry ashing 10-g samples in amuffle furnace at 850'C for 12 h. The hydrocarboncontents of the oil sludge and soil were extracted bythe following method. A 25-g sample was mixed with25 g of anhydrous Na2SO4 and extracted with diethylether in a Soxhlet extractor for 12 h. The ether extractwas concentrated over steam in a Kuderna-Danishconcentrator with a Synder column to a volume of lessthan 10 ml. The extract was dried with an additional0.1 g of anhydrous Na2SO4 and transferred, with rins-ings, to a tared vial. After evaporation of the solventat 25°C, the extractable hydrocarbon was quantifiedgravimetrically. Extraction efficiency of the hydrocar-bons from the sludge-soil-sand mixture was identicalto that from the oil sludge alone.The extracted hydrocarbons were separated further

into class fractions by silica gel column chromatogra-phy. The silica gel was activated at 1050C for 12 h.The glass column (outer dimensions, 2 by 28 cm; bedvolume, 65 ml) was packed with silica gel (Woelm,Eschwege, West Germany) suspended in hexane. Hy-drocarbon samples (0.2 to 0.5 g) were absorbed on 3 gof silica gel and placed on the column. A 3-g layer ofanhydrous Na2SO4 was placed over the sample to

prevent mixing of the sample with solvents. The sat-urated, aromatic, and asphaltic classes were succes-sively eluted by 120-ml volumes of hexane, benzene,and chloroform-methanol (1:1, vol/vol), respectively.The solvents were dried with anhydrous Na2SO4 foruse. South Louisiana crude oil of known composition(2, 18) was used for calibrating column separationefficiency. The fractions were collected in tared 250-ml Erlenmeyer flasks and weighed after solvent evap-oration. The n-alkanes in the saturated hydrocarbonclass were analyzed by gas-liquid chromatography asdescribed earlier (8). Carbon dioxide evolution wasmonitored in Biometer flasks (6) and plotted cumula-tively.

Biodegradation experiments. Unless specifiedotherwise, the Biometer flasks were prepared andincubated in the following manner. Each flask wascharged with 10 g of soil, 10 g of sand, and 2.08 g ofair-dried oil sludge containing 500 mg of extractablehydrocarbons. Water was added to 60% of the holdingcapacity, and the soil pH was adjusted to 7.5 by using7.5 mg of CaCO3 per flask. To avoid any interferencewith the CO2 evolution measurements from the oilsludge, CaCO3 treatment of the soil was performed atleast 10 days before the oil sludge addition. Nitrogenfertilizer was added as NH4NO3 in a C:N ratio of 60:1.Phosphorus and potassium were added as K2HPO4 inC:P and C:K ratios of 800:1 and 400:1, respectively.Incubation was at 28°C. The replacement of trappingalkali and a flushing of the Biometer flasks with C02-free air were performed as dictated by the CO2 evolu-tion activity in 1- to 10-day intervals.

Individual environmental parameters were variedas specified below. All parameters not otherwise spec-ified below were maintained in the following experi-ments at the standard conditions described above.

Soil moisture. Soil moisture was adjusted withdistilled water to 30, 60, and 90% of the holding capac-ity of the soil. Oil sludge was added at 0.5 g/flask (120mg of extractable hydrocarbons).

Soil pH. Soil pH was adjusted to 5.0, 6.0, 7.0, and7.8 with varying amounts of CaCO3. The bufferingcharacteristics of this soil prevented its adjustment toa higher pH without the use of unrealistic amounts ofCaCO3. Oil sludge was applied at 0.5 g/flask. Controlflasks received CaCO3 but no sludge. After 103 days ofincubation, the active flasks received a second 0.5 g ofoil sludge.

Mineral nutrients. Mineral nutrients were addedas NH4NO3 and K2HPO4 to give a range of initialcarbon-mineral nutrient ratios. In respective flasks,these were as follows: C:N ratios, 15:1, 60:1, and 300:1,C:P ratios, 200:1, 800:1, and 4,000:1; and C:K ratios,100:1, 400:1, and 2,000:1. Control flasks received nomineral nutrient supplementation. On day 131, allflasks were reloaded with the initial amount (2.08 g)of oil sludge.

Micronutrients and organic supplements.Trace elements were added to a series of flasks as 2.6ml of a 1:1,000 dilution of Hoagland trace elementsolution (21). The tested organic supplements included7.5 mg of Difco yeast extract per flask. Controls re-ceived yeast extract but no oil sludge. Another seriesof flasks received dried domestic sewage sludge (31.4%

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BIODEGRADATION OF OIL SLUDGE 731

organic matter) at a 1% (wt/wt) concentration. Con-trols received sewage sludge but no oil sludge, or oilsludge only.Treatment rate. Treatment rate was tested at

increasing sludge loading rates, corresponding to 0.25,0.5, 1.0, 2.0, and 3.0 g of extractable hydrocarbons perflask. After 131 days of incubation, flasks received asecond charge corresponding to the initial one.Treatment frequency. Treatment frequency was

tested at an overall sludge loading rate correspondingto 2.0 g of extractable hydrocarbons per flask. Thisamount of sludge was added as a single charge on day0, as two 1.0-g charges on days 0 and 87, or as fourcharges of 0.5 g each on days 0, 57, 87, and 124. Aproportional amount of fertilizer accompanied each oilsludge application.Temperature effects. Temperature effects were

tested by incubation of oil sludge treated and un-treated control flasks at 5, 13, 20, 28, and 30°C. Thesoil used in these experiments was preincubated for 40days at corresponding temperatures.

Treatments were made in duplicate. In experimentsthat involved reloading, initial treatment was in quad-ruplicate. From the four flasks, two were reloaded andtwo were sacrificed for analysis. All data points rep-resent the mean of duplicate or quadruplicate samples.Variation between replicates was within ±7%.

RESULTSSoil analysis. The soil from the prospective

landfarming site had a loam texture (42% sand,34% silt, 24% clay). Its organic matter contentwas 3.2%, total nitrogen was 0.03%, the cationexchange capacity was 8.1 mg/100 g of soil, andthe pH was 3.7. Major mineral nutrient concen-trations were found to be as follows (in milli-grams per kilogram): Mg, 371; P, 12; K, 13; NOJ3-,6; NH4', 8. Because of the poor soil qualityindicated by the above data, the soil was condi-tioned before use in our experiments as de-scribed above. Similar conditioning was recom-mended for the disposal site.

Oil sludge analysis. The composition of theair-dried oil sludge used in our experiments wasas follows: extractable hydrocarbon, 24%; ash,72%; water, 4%. By column chromatography, thehydrocarbon composition was found to be: sat-urated hydrocarbons, 57%; aromatic hydrocar-bons, 29%: asphaltic hydrocarbons, 14%. Thehigh ash content of the sludge was consistentwith its origin and storage history. The absenceof alkanes shorter than C14 reflected the effectsof weathering during open pit storage. The rel-atively high proportion of saturated hydrocar-bons was expected to favor microbial utilizationof the sludge hydrocarbons. As sludges fromvarious sources vary in their oil content, forcomparison purposes we found it useful to ex-press our loading rates in terms of extractablehydrocarbons rather than as total sludge weight.

Soil moisture. Carbon dioxide evolution ofoil sludge-treated soil was monitored for 60 daysat 30, 60, and 90% of the water-holding capacity.Identical CO2 evolution rates were recorded atall three moisture levels (data not shown).

Soil pH. Raising the soil pH by the additionof CaCO3 had a marked positive effect on oilsludge biodegradation (Fig. 1). The CO2 evolu-tion of controls not receiving oil sludge showeda similar trend, but the increases were smaller.The CO2 evolution of untreated controls wassubtracted from the CO2 evolution of the treatedsamples to show the net effect of pH on oilsludge biodegradation. The highest pH tested(pH 7.8) resulted in the highest rate of CO2evolution. Although with this particular soil itwas not practical to test a higher pH, we believethis value approaches the optimum.

After 60 days of incubation, CO2 evolutionfrom the oil sludge stopped for all practicalpurposes. This could have been due to the ex-haustion of metabolically accessible hydrocar-bon or to the accumulation of toxic metabolites.The reapplication of oil sludge on day 103showed that the former was the case. CO2 evo-lution was more rapid upon the second applica-tion than it was at the beginning of the experi-ment. The positive effect of increased soil pHwas more marked after the second sludge appli-cation, with pH 7.0 and pH 7.8 giving the great-est rate and extent of CO2 evolution.

Information gained from residual hydrocar-bon extraction is shown in Fig. 2. In this histo-gram, percent hydrocarbon degraded gave ahigher conversion percentage than did CO2 ev-olution. This is to be expected, since some hy-drocarbons are converted to cell material anddegradation products other than CO2.

7EFFECT OF SOIL REACTION

6-

5-

pH 5.0

c t@2[ > ]~~~~~~~~N

0 50 00 50TOE (Days)

FIG. 1. Effect of soil reaction on carbon dioxideevolution.

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732 DIBBLE AND BARTHA

The greatest extent of hydrocarbon conver-

sion occurred at the highest pH tested (pH 7.8).Subtracting the values for days 0 to 103 from thevalues for days 0 to 180 shows a greater biodeg-radation activity during the last 77 days thanduring the first 103 days. The residual hydrocar-bon from the first oil sludge application was notinhibitory to the biodegradation of the secondcharge. On the contrary, the apparent enrich-ment of a hydrocarbon-degrading population re-

sulted in a more rapid rate and a greater extentof hydrocarbon biodegradation. Residual recal-citrant hydrocarbons from the first applicationmay have been degraded by cometabolism dur-ing the biodegradation of the second charge. Theoverall decrease in extractable hydrocarbons forthe 180-day period was 57%.

o lo

Ix

pH

FIG. 2. Effect ofsoil reaction on oil sludge biodeg-radation. The total height of the histograms repre-

sents hydrocarbon degradation; the white portion ofthe histograms represents C02 evolution as a per-centage of the theoretical total. The C02 evolutionand hydrocarbon degradation at 100 days and pH5.0 were identical and are represented by the cross-

hatched bar. The group of histograms on the right ison a different scale because of the reloading on day103.

Mineral nutrients. The CO2 evolution dataof this 285-day experiment with reloading at day131 are summarized in Table 1. The kinetics ofCO2 evolution resembled those in Fig. 1. Be-tween days 0 and 131, the two lower levels offertilization gave almost identical CO2 evolutionvalues (24.0 and 23.3%, respectively), but for theperiod from day 131 to day 285 and for the wholeduration of the experiment the intermediatetreatment level was clearly superior. The highestfertilization level resulted in only a slight CO2evolution increase as compared with the unfer-tilized control.

Determination of residual hydrocarbons con-

firmed that percent biodegradation was greatestat the intermediate fertilization level. At thehighest level of fertilization, biodegradation was

only slightly greater than in the control, whichreceived no mineral nutrients. The greateramount of fertilizer itself did not inhibit biodeg-radation. In other experiments discussed below,flasks were prepared with identical amounts ofmineral nutrients, but amounts of hydrocarbon(2 g/20 g of soil) were added to bring the ratiosto the following values: C:N, 60:1; C:P, 800:1; andC:K, 400:1. No loss of activity was observed inthese treatments. Apparently, conditions ofoverly low ratios of carbon to mineral nutrientsdo not favor oil sludge biodegradation.

It should be pointed out that here and in otherexperiments, unless specifically noted otherwise,no new fertilizer was added upon reloading withoil sludge. Little or no fertilizer loss was expectedin the aerobic closed-flask system, and it was

anticipated that the bulk of the fertilizer ele-ments, after exhaustion of the readily utilizablehydrocarbons and decline of the responsible mi-crobial population, would be available for use inbiodegradation of the subsequent oil sludge load.Micronutrients and organic supplements.

Hoagland trace element solution slightly stimu-lated CO2 evolution (Table 2) but not biodegra-

TABLE 1. Effect of mineral nutrients on oil sludge biodegradation% Biodegradation as determined by:a

Mineral nutrient ra-

CO2 evolution for days: Residual hydrocarbon for days:

0-131 131-285 0-285 0-131 131-285 0-285

No nutrients added 13.5 22.1 17.8 26.1 35.1 30.0C:N, 300:1; C:P, 24.0 30.0 27.0 28.3 53.7 39.9

4,000:1; C:K,2,000:1

C:N, 60:1; C:P, 800: 23.3 38.5 30.9 33.2 59.6 45.21; C:K, 400:1

C:N, 15:1; C:P, 200: 13.9 28.9 21.4 26.5 48.9 36.71; C:K, 100:1a Loadings were 0.5 g of oil sludge hydrocarbon each on days 0 and 131. The total biodegradation period was

285 days.

CJ C02 Evolved 0 - 160 doys_DOersa in Hydrocorbon Estracted

0100-doys 100 160I1d

5 6 7 ZS 5 6 7 .Z 6 7

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BIODEGRADATION OF OIL SLUDGE 733

dation, as measured by residual hydrocarbon.Yeast extract acted in a similar manner. Thelatter test was prompted by a report (15) thataddition of brewers' waste yeast, at the sameconcentration as the yeast extract added in thisexperiment, stimulated the biodegradation oftwo oils in soil, perhaps by supporting cometa-bolic hydrocarbon transformations. In our tests,neither trace element nor yeast extract supple-mentation proved to be of substantial value. Anobserved slight stimulation, which was neverthe-less greater than the excess C02 produced bythe yeast extract treatment alone, must be as-cribed to an enhanced mineralization of hydro-carbon biodegradation intermediates. However,the overall hydrocarbon biodegradation did notincrease.

In past years, domestic sewage sludge hasreceived much attention as a soil conditioner (1).Since some organic components ofsewage sludgemay support cometabolic transformations of themore recalcitrant hydrocarbons, we tested theeffects of this readily available and inexpensiveadditive. Although sewage sludge at 1% (wt/wt)increased C02 evolution of soil without oilsludge, it depressed both C02 evolution and soil

TABLE 2. Effect of trace elements, yeast extract,and sewage sludge on oil sludge biodegradation

% Biodegradation as determined by:'Treatment C02 Residual

evolution hydrocarbon

Control 19.0 34.4Hoagland trace 22.1 33.2

elementsYeast extract 22.6 33.7Sewage sludge 13.6 26.1

a Loading was 0.5 g of oil sludge hydrocarbon. Thebiodegradation period was 83 days. The control re-ceived oil sludge but no other additives. The data arecorrected for CO2 evolution from soil alone, from soilwith yeast extract but no oil sludge, and from soil withsewage sludge but no oil sludge, respectively. Conse-quently, the CO2 evolution data represent net CO2evolution from the oil sludge.

biodegradation in treated samples (Table 2).Consequently, this material does not appear tobe a desirable conditioner for landfarming sites.

Oil sludge loading rate. To make the mostefficient use of the limited land area availablefor disposal, a high rate of oil sludge applicationis desired. However, oil sludge concentration inthe soil should not be so high as to depress therate of biodegradation.With an increase in the oil sludge loading rate,

there was an increase in the absolute amount ofCO2 evolution up to 1 g of hydrocarbons per

flask containing 20 g of soil-sand mixture (datanot shown). A loading rate of 2 g of hydrocarbonper flask did not result in a further increase inC02 evolution, and at 3 g/flask a 12% decreasein C02 evolution was observed as compared withthe 1- and 2-g/flask loading rates. Table 3 showsthe percent biodegradation of the hydrocarbonsin the oil sludge as measured by C02 evolutionand by residual hydrocarbon analysis. As ex-

pected, the greatest percentage of degradationoccurred at the lowest application level. A re-

duction in percent biodegradation occurredabove the 1-g/flask application rate. Of course,

the lowest loading rate would not make optimaluse of the biodegradation capacity of the avail-able land area. Table 4 shows the absoluteamounts of hydrocarbon degradation and theresulting residue levels. The best compromisebetween high degradation rates and relativelylow residue levels was achieved with two addi-tons of 1.0 g of hydrocarbon per flask, and we

consider this the maximum useful loading rate.With the usual conversion factors to the ploughlayer of field soil, this translates to approxi-mately 100,000 liters of hydrocarbon per hectare(255 barrels per acre). This amount can be ap-plied twice during a 9-month growing season.

The above application rate refers to the hydro-carbon content of the sludge rather than to thetotal bulk of the sludge, since its water andinorganic solid content is not expected to influ-ence greatly the maximum useful loading rate.The above projection should not be considered

TABLE 3. Effect of loading rate on oil sludge biodegradation% Biodegradation as determined by:

Loading ratea (g) CO2 evolution for days: Residual hydrocarbon for days:

0-131 131-285 0-285 0-131 131-285 0-285

3.00 6.3 8.7 7.5 12.0 20.5 14.52.00 11.7 14.5 13.1 20.4 26.0 22.41.00 23.7 26.3 25.0 34.5 48.8 40.40.50 23.3 38.5 30.9 36.0 56.9 45.20.25 32.0 48.0 40.0 36.0 60.7 48.7

aGrams of extractable hydrocarbon per 20 g of soil-sand mixture. The flasks were reloaded with equalquantities on day 131, and the total biodegradation period was 285 days.

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734 DIBBLE AND BARTHA

valid for so-called "biosludges" from units forthe biological treatment of oily effluents. Thesesludges usually have a low hydrocarbon contentcombined with a high amount of nonhydrocar-bon organic material, and their maximum usefulloading rates remain to be determined.

Analysis of the residual hydrocarbons by com-ponent classes revealed that the loading rate hadan effect on the types of hydrocarbons degraded.This heretofore unrecognized effect deserves ad-ditional consideration in decisions on treatmentrates. Of interest is both the absolute mass andalso the percentage of each hydrocarbon classdegraded in relation to the amount applied. Bi-odegradation ofthe saturated hydrocarbon class,expressed as mass degraded, closely followed thepattern established by CO2 evolution (Fig. 3A).The mass degraded after one and two loadingsincreased with increasing loading rates up to 1g of hydrocarbon per flask. The mass degradedat a loading rate of 2 g/flask was only slightlyhigher, and a loading rate of 3 g of hydrocarbon

TABLE 4. Comparison of hydrocarbonbiodegradation and residue at various loading

rates

Lang No. of load- Amt of hydro- Amt of residualLaiag No. of load- carbon biode-Ato rsdarate ings graded hydrocarbon(g) ~~~ ~~~~~(g)g

3.00 2 0.88 5.122.00 2 0.90 3.101.00 2 0.81 1.190.50 2 0.45 0.550.25 2 0.24 0.26

Grams of extractable hydrocarbon per 20 g of soil-sand mixture. The flasks were reloaded with equalquantities on day 131, and the total biodegradationperiod was 285 days.

per flask resulted in a lesser mass degraded. Thepercent biodegradation of the saturated hydro-carbon class (Fig. 3B) decreased with increasingcharge size, leaving a larger residue of saturatedhydrocarbons at higher loading rates.During the biodegradation of the first load of

oil sludge, the mass of aromatic hydrocarbonsdegraded was highest at a loading rate of 1 g ofhydrocarbon per flask; at higher loading ratesbiodegradation decreased. The mass of aromatichydrocarbons degraded after two oil sludge load-ings increased with increasing loading rates.However, the percentage of aromatics degradedafter two loadings decreased at rates above 1 gof hydrocarbon per flask. Enrichment of orga-nisms that can degrade aromatics and/or theirenhanced cometabolic activity in the presenceof saturated hydrocarbons from the secondsludge application are the most plausible expla-nations for the improved utilization of the aro-

matics at the higher application rates. Somehighly condensed polynuclear aromatic hydro-carbons are considered to be potentially carci-nogenic. Loading rates of up to 1 g of hydrocar-bon per flask (100,000 liters/hectare) resulted inthe highest percentage of biodegradation of thearomatic hydrocarbon residue.The asphaltic hydrocarbons are considered to

be the least susceptible to biodegradation, theircomplex structure rendering them relatively re-

calcitrant to microbial attack. Some asphaticmaterial is considered analogous to the humicmatter in soil. The asphaltic class also has anonhydrocarbon component consisting of com-

pounds that contain nitrogen, sulfur, and oxy-gen. During the metabolism of hydrocarboncompounds, the accumulation of oxygenated in-termediates contributes to the asphaltic materialin the soil (12).

0.

0-

_0

0-

EDCOOU3

i.4A1.2.~z~ZfI

0-B D

10-~ Jod2od

LOADING RATE

FIG. 3. Effect of loading rates on the biodegradation of hydrocarbon classes. (A) Absolute amount (mass)

of a class removed afer one loading; (B) percentage ofa class removed after one loading; (C) absolute amountof a class removed after two loadings; (D) percentage of a class removed after two loadings. Loadings were

at 0 and 131 days. The total biodegradation period was 285 days. Symbols: G, saturated hydrocarbons;@,aromatic hydrocarbons; A, asphaltic hydrocarbons.

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VOL. 37, 1979

The level of asphaltic residues is the net resultof the degradation of hydrocarbon and nonhy-drocarbon components of the asphaltic class andthe accumulation of oxygenated intermediatesfrom the metabolism of hydrocarbons from allof the hydrocarbon classes. The results of thisexperiment (Fig. 3A and C) show that the netremoval in mass of asphaltic material increaseswith increasing rates of oil sludge application.The percentage of net asphaltic material re-

moval was at a maximum at a loading rate of 2.0g of hydrocarbon per flask after the first loadingand at a loading rate of between 2.0 and 3.0 g/flask after the second loading. Apparently, thenet removal of asphaltic material was greatestunder the conditions where saturated hydrocar-bons are least biodegraded. It seems possiblethat the rapid utilization of saturated hydrocar-bons at low loading rates results in the accu-mulation of metabolites that contribute to theasphaltic material.

Considering the above data on hydrocarbonclass removal as a function of loading rate, the1.0-g/flask (100,000 liters/hectare) rate still re-

mains the loading rate of choice since it resultsin an efficient removal of potentially carcino-genic aromatics and at least strongly limits ac-cumulation of the recalcitrant asphaltic mate-rial.Treatment frequency. An identical load of

2 g of oil sludge hydrocarbons per flask was

applied as a single charge or divided into incre-ments of 0.5 or 1 g and applied, with correspond-ing amounts of fertilizer, over the same timeperiod. The incremental loading of small chargesresulted in a higher rate and a higher total ofhydrocarbon biodegradation than did the single-charge loading (Fig. 4). The percent biodegra-dation data (Table 5) confirmed that the extentofbiodegradation was greatest in flasks receivingfour loadings of 0.5 g of hydrocarbon per flask.Small multiple loadings apparently kept the hy-drocarbonoclastic microbial population in an ac-

tive growth phase with metabolic processes attheir peak. After four consecutive loadings, theresidual material was not inhibitory to biodeg-radation.

Separation of the residual hydrocarbons intocomponent classes revealed that the saturated

BIODEGRADATION OF OIL SLUDGE 735

hydrocarbon class was preferentially utilized ata smaller charge size (Table 5). The percentageof aromatic hydrocarbons biodegraded was alsoslightly higher at a charge size of 0.5 g. The netremoval of asphaltic material, as in the loadingrate experiment, was greatest at the highercharge size of 2.0 g.The results of this and the preceeding experi-

ment were in good agreement and suggested thatthe pattern of biodegradation of the hydrocar-bon classes may be controlled by regulation ofthe oil sludge loading rate and frequency. Smallcharge size loadings resulted in a preferentialutilization of the saturated hydrocarbon classand in a maximum overall rate and extent ofbiodegradation. Larger charge size loadings re-

sulted in slightly lower overall biodegradation,but they favored the net removal of asphalticmaterial.Temperature. Temperature profoundly in-

fluences all biological processes and can be ex-

pected to have a significant effect on oil sludgebiodegradation. The CO2 evolution curves (Fig.5) indicate negligible activity at 5°C, a 2-week

Days

FIG. 4. Effect of reloading on carbon dioxide evo-lution. Increment charge size, expressed as grams ofhydrocarbon per loading, appears at the right. CO2evolution was determined at 1- to 10-day intervals asdictated by the rate of evolution. Because of theirdense spacing, the data points were omitted.

TABLE 5. Effect of loading frequency on the biodegradation of oil sludge hydrocarbons% Biodegradation as determined % of the following hydrocarbon clases biodegraded:

Charge size No. of load- by:

CO2 evolution Reiduahydro- Saturated Aromatic Asphaltic

2.0 1 17.9 24.5 17.7 34.8 31.91.0 2 25.8 33.1 35.1 33.8 24.50.5 4 30.8 45.5 54.0 40.7 22.0

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736 DIBBLE AND BARTHA

20'C

>3w

82139C

5"C0 0 20 30 , 50

TIME (Days)FIG. 5. Effect of temperature on CO2 evolution of oil sludge-treated soil. All curves were corrected for CO2

evolution of untreated soil at the respective temperature; the curves represent net C02 evolution (A C02) fromoil sludge.

lag phase at 13°C followed by a fair rate of C02release, almost identical rates at 20 and 280C,and only a slightly higher rate at 370C.Hydrocarbon biodegradation, calculated from

the residue levels at the conclusion of a 47-dayexperiment, was plotted as a function of incu-bation temperature (Fig. 6). It verifies the un-expected pattern indicated by the C02 data, i.e.,little or no increase of the biodegradation activ-ity above 200C.

DISCUSSIONSoil moisture. The aerobic biodegradation

of simple or complex organic material in soil iscommonly greatest at 50 to 70% of the soil water-holding capacity (19). Inhibition at lower valuesis due to inadequate water activity, and highervalues interfere with soil aeration. The thin soillayer (1 cm) in our experimental design mini-mized the effects of waterlogging on soil aera-tion, and, therefore, the extrapolation of thisresult to the field may not be valid. The lack ofinhibition at 30% of the water-holding capacity,although unexpected, cannot be regarded as anexperimental artifact. It suggests that the mois-ture requirements for maximum activity on hy-drophobic petroleum may be different than theoptimal moisture levels for the biodegradationof hydrophilic substrates. It is likely that thehydrocarbons, by rendering some surfaces hy-drophobic, reduce the water-holding capacity ofthe soil and thus increase the availability of thewater that is present.

Soil pH. Extremes ofpH are inhibitory to thegreat majority of microbial degradation proc-esses. Most fungi are less adversely affected by

30-

o20-00

10-

5 10 15 20 25 30 35 40

TEMPERATURE (*C)

FIG. 6. Effect of temperature on hydrocarbon bio-degradation in oil sludge-treated soil.

low pH values than are bacteria. Although therelative role of bacteria versus fungi in the bio-degradation of hydrocarbons in soil has not beendetermined, liming in our experiments favoredthe biodegradation of oil. Our results are inagreement with previous fundings (15, 23). Withour test soil, liming to an over-optimal pH wasnot practical, but considering that Verstraete etal. (23) found optimal activity at a pH of 7.4 anda considerable inhibition at pH 8.5, our highestpH tested (pH 7.8) should be close to the opti-mum.Mineral nutrients. The limitation of petro-

leum biodegradation due to a nutritional imbal-ance between the substrate carbon supplied bypetroleum and the nitrogen and phosphorus re-quired for microbial growth has been extensivelyreviewed in relation to the marine environment(3, 5). As the nitrogen and phosphorus reservesof most soils are low, a similar situation can beexpected to exist in soil. Several previous reports

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BIODEGRADATION OF OIL SLUDGE 737

(13, 14, 16, 20, 23) on the stimulation of oilbiodegradation by mineral fertilizers have ap-peared, but no systematic effort was made todetermine the optimal fertilizer levels.The quantity of nitrogen and phosphorus re-

quired to convert 100% of the petroleum carbonto biomass may be calculated from the C:N andC:P ratios found in cellular material. Acceptedvalues for a mixed microbial population in thesoil are: C:N, 10:1 (24); and C:P, 100:1 (22). Inreality, a complete assimilation of petroleumcarbon into biomass is not achievable undernatural conditions. Some of the petroleum com-pounds are recalcitrant or are metabolizedslowly over long periods. From petroleum com-pounds that are readily metabolized, some car-bon will be mineralized to C02. Thus, efficiencyof conversion of substrate (petroleum) carbon tocellular material is less than 100%. Populationturnover allows for the recycling of nutrients.The optimal C:N and C:P ratios for petroleumbiodegradation were expected to be wider thanthe theoretical values of 10:1 and 100:1, respec-tively, but they could not be predicted accu-rately and required experimental determination.The mineral nutrient ratios resulting in the

greatest biodegradation were similar to the ra-tios found to be optimal for stimulating thebiodegradation of crude oil in seawater (4). Al-though the seawater ratios were rather broadlybracketed in the soil work, i.e., four times higherand five times lower, the results do suggest asimilarity in petroleum biodegradation in thetwo systems.

Potassium, a standard ingredient in agricul-tural fertilizer formulations, was present in ex-cess of its actual microbial requirement, and noeffort was made to determine the exact require-ment level for this element.Our experiment design eliminated leaching

and minimized denitrification losses. These fac-tors are likely to remove at least some of theapplied nitrogen fertilizer in a landforming op-eration, and it is expected that fertilizer in theoptimal C:N and C:P ratios will have to bereapplied with every new oil sludge charge. Theuse of slow-release fertilizers is beneficial interms of fertilizer savings and minimized leach-ing. These aspects of mineral fertilization aredealt with in a separate publication (Dibble andBartha, Soil Sci., in press).Micronutrients and organic supplements.

Trace element deficiency in soils is rarely en-countered, and the marginal response to thetrace element solution was not unexpected. Ourfinding that yeast extract failed to stimulatehydrocarbon biodegradation contrasts with thereport of Lehtomiike and Niemela (15) on the

stimulatory effect of brewers' waste yeast. Thediscrepancy may be due to differences in mate-rials, soils, and test conditions. The fact thatLehtomake and Niemela did not use mineralfertilizer in addition to the waste yeast, whereaswe did, is particularly relevant. In the formercase, waste yeast may have supplied merelynitrogen and phosphorus for hydrocarbon-de-grading microorganisms.The reduced hydrocarbon biodegradation in

the presence of sei age sludge, evident from boththe C02 and the residual hydrocarbon data (Ta-ble 2), is explainable by either a diauxic effect(17) or by population selection unfavorable forhydrocarbon degradation. The positive effect ofthe sewage sludge on C02 production of controlsoil excludes a general antimicrobial effect bythe sewage sludge.Treatment rate. To date, oil sludge applica-

tion rates in landfarming operations were chosenin an arbitrary manner, the criteria being landavailability and the appearance and surface sta-bility of the land after application. Jensen (11)studied the influence of oily waste concentrationon biodegradation activity as monitored by ox-ygen uptake. Oily waste was incorporated intosoil at concentrations varying from 0 to 30% (wt/wt). The highest oxygen uptake rate was at aconcentration of 5%, but only small positive ornegative differences were detected at other con-centrations. Total microbial counts increasedwith oil application to the 5% level but declinedthereafter, supporting the 02 uptake data. Noother systematic effort for the optimization ofapplication rates could be located in the litera-ture. On a hydrocarbon basis, the 5% optimalapplication rate shows a surprisingly good agree-ment with our own results (1.0 g of hydrocarbonper 20 g of soil-sand mixture).To date, the effect of the loading rate on the

absolute and relative removal of the three hy-drocarbon fractions (saturated, aromatic, andasphaltic) has gone unrecognized. Our resultsshow that application rates that are over optimalfor a rapid removal of saturated hydrocarbonsfavor removal of the aromatic and asphalticclasses. Our interpretation of this effect is at thistime, by necessity, speculative. We believe thatthe biodegradation of the higher aromatics andof the asphaltic compounds, few of which canserve as growth substrates by themselves, isdependent upon a continued presence of satu-rated hydrocarbons to support the cometabolicbiodegradation of the former classes. Additionalstudies will be required for the verification orrejection of this hypothesis.Treatment frequency. Results showing that

smaller and more frequent applications yield

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738 DIBBLE AND BARTHA

higher overall biodegradation rates than doesthe infrequent application of large batches arenot unexpected, but this parameter has not beenevaluated previously specifically with oilywastes. Small, frequent applications minimizethe adverse effects of toxic oil sludge compo-nents and, at the same time, keep the hydrocar-bon-degrading microbial population in a contin-uous state of high activity. In a practical situa-tion a compromise has to be reached betweenefficient site use through high biodegradationrates and an avoidance of excessive labor costsfor frequent applications. At most temperatezone landfarming sites two 100,000-liter/hectare(255 barrels per acre) or four 50,000-liter/hectareoil sludge hydrocarbon applications per growingseason seem appropriate. In the latter case91,000 liters of oil sludge hydrocarbon per hec-tare (232 barrels per acre) was degraded perseason. This is slightly less than one-half of theapplied amount, and longer-term experimentswill be necessary to determine whether eventualaccumulation of hydrocarbon residues might af-fect site performance. In a 250-day experiment(Fig. 4) involving four reapplications, we ob-served a steady increase instead of a decline inhydrocarbon biodegradation, suggesting that asteady state between the described applicationrates and biodegradation may be reached withno further residue accumulation. Nevertheless,this assumption will need to be verified by ex-periments and/or long-term field experience.Temperature effects. A rise in metabolic

activity of microbial communities is commonthroughout the mesophilic range, and the level-ling-off of the hydrocarbon biodegradation ratein soil above 20°C was unexpected. Preacclima-tion of the soil samples at the respective incu-bation temperatures seems to exclude the pos-sibility of an experimental artifact. As a reason-able but nevertheless speculative explanation wesuggest that an increased toxicity of hydrocar-bons at higher temperatures may counteract thestimulation of metabolic processes.

Direct temperature regulation of the disposalsites is not technically or economically feasible.Gudin and Syratt (10) suggested the use of blackplastic film mulch as a means of stimulatingwaste oil biodegradation, thus increasing soiltemperatures during the winter. The use of filmmulch would preclude tilling of the soil andwould decrease its aeration. For these reasons,we do not believe this measure would have apositive effect. It is also likely to be unnecessary,since Freedman and Hutchinson (9) reportedthat the albedo decrease due to oil contamina-tion raised the temperature in the upper 10 to20 cm of tundra soils by as much as 5°C. Similar

effects would be expected in temperate zone soilsduring the winter. 4

General remarks. A laboratory approachallowed the testing of a large number of con-trolled environment parameters within a rela-tively short time. We are, however, acutelyaware of the limitations of such studies in repro-ducing field situations. Important parameters,such as tillage and soil texture, could not bemeaningfully tested in our laboratory system.Poorly drained, waterlogged conditions in thefield may drastically reduce biodegradation ratesprojected from laboratory studies, although thiseffect was not observed in our laboratory testsystem. It also remains to be determinedwhether temperatures that fluctuate around adaily mean during a 24-h cycle can be equatedwith a steady incubation temperature at thesame mean. Consequently, our findings will needvalidation and possible adjustments in the field.Nevertheless, we feel that our laboratory studywill greatly reduce and simplify field experimen-tation by providing pilot data that can be brack-eted in future field tests.

ACKNOWLEDGMENTSThis work was supported by Chevron, USA., Inc.

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bunker C, Kuwait crude oil and South Lousiana crudeoil. Report no. AID. IBA. 74. Esso Research, Linden,N.J.

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