Emon Technolgy, Vl 2. pp 10181027 © Selper Ltd, 2000 A NOVEL METHOD FOR MEASURING BIOMASS ACTIVITY APPLIED TO SOILS CONTAMINATED WITH OIL REFINERY TAR S.R. MARTIN, A. J. GUWY", B. ELLIS?, P. HAROLD®, F. R. HAWKES! AND D. L. HAWKES School of Technology and "School of Applied Sciences, University of Glamorgan, Pontypridd, Mid Glamorgan CF37 1DL, UK Celtic Technologies Ltd., Cambrian Buildings, Mount Stuart Square, Cardiff CF10 5QQ, UK (Received 20 July 1999; Accepted 10 March 2000) ‘ABSTRACT Soil biomass activity may be wsed as an indicator of pollutant degradation in sol, but methods for estimating sol biomass ‘can be time-consuming. Activity in soll contaminated with ol refinery tar has been assayed sing a low flow gas meter tO ‘ensure oxygen evolved by the decomposition of hydrogen peroxide by catalase inthe soil microflora. Each analysis takes 15 minutes, Samples from contaminated land undergoing bioremediation were analysed through a 14 week treatment period and biomass activity compared with pollitant concentrations. An increase in microbial activity corresponded #0 ‘Secrease in pollutant concentrations Keywords: INTRODUCTION Soil biomass is an important parameter in studies of ecology and fertility in soils and the quantity and activity of the microorganisms in the soil are useful factors for assessing sil quality. Classical methods of soil biomass estimation involve isolation and enumeration of soil bacteria, for example by plate counts [1,2]. Viable counts are an inadequate method of cell enumeration, due to significant difficulties in fully representing the bacterial population in the soil [3]. More common assays of soil biomass include the fumigation {incubation technique [4], the fumigation extraction method 5], bacterial respiration (61, ATP content (71, ‘microcalorimetry [8] and carbon dioxide evolution [9]. These methods have drawbacks either because of the complexity of the experiment, the length of time an analysis takes, use of hazardous chemicals or large margins of experimental error. Commonly used methods for estimating microbial biomass content of soils using enzyme activity are the activities of dehydrogenase [10,11], urease [12] and phosphatase (9]. Klein etal, (13] described the use of soll enzymes as a fast response indicator of changes in soils, specifically in reclamation processes. Most enzymes perform specific reactions and only a small fraction of the soil population may possess a particular enzyme at any one time. ‘Catalase is another enzyme used to measure soil biomass activity [14-17]. This enzyme is present in all aerobic micro- organisms. It catalyses the decomposition of hydrogen Catalase, bioremediation peroxide into oxygen and water. 2H,0,-° 2H,0+ 0, ® Catalase is present in cells to protect against damage from hydrogen peroxide, endogenously via the respiratory chain of active aerobically respiring cells, or through the action of superoxide dismutase {18}, Its particularly suitable for activity measurements as it is highly active over a wide pH range (pH 3-9), requires no cofactors {171 and H,O, freely enters the cel. ‘A method for continuous measurement of catalase activity in activated sludge has previously been described {19]. The method depends on the use of a low-flow gas meter which may be produced [201 to measure the gas evolved from H,O, decomposition. In activated sludge samples it was found that catalase activity correlated with respirometric measurements, and as expected, not with total or volatile solids measurements which include inert organic matter. It was concluded that catalase activity in a heterogeneous population might reflect the active aerobic microbial community. A biomass monitor using this method. described in a patent [21] may be applied to soil samples. Guwy et al, [22] showed that catalase activity was linearly related to the dry weight of garden soil. The catalase activity ‘measurement should be more rapid than existing methods for ‘measuring active biomass in soils, and uses no toxic reagents, Here we apply this method by which soil biomass activity can be quantified to a soil contaminated with oil refinery tar for the duration of a contaminated site remediation process. 1019MATERIALS AND METHODS. Soils and sampling procedures, CELTIC Technologies Lid (Cardiff, UK) undertook bioremediation of ground contaminated with waste tars, as part of the reclamation of a former shale oil refinery site in Scotland. The treatment program was designed to mediate a total of approximately 10 000 tonnes (7000m") ‘of waste tars over an 18 month period commencing in August 1997, ‘The waste tars, produced as a by-product from oll shale refining activities, comprised viscous sludge, with high concentrations of diesel range hydrocarbons (DRO) and elevated poly aromatic hydrocarbons (PAH). Additional contaminants included volatile organic compounds (BTEX), and phenols, together with certain heavy metals. ‘The remediation goals for the site were set in agreement with the Scottish Environment Protection Agency (SEPA), and were calculated using assumptions based upon treated ‘material being used as a soil within the final site development asa golf course. Bioremediation works comprised a program of, physical waste processing, and aerobic biological treatment of, wastes, together with treatment monitoring and management. Waste processing involved combining tar sludge with a soil conditioning agent, nutrients and other amendments inthe first three or four weeks of treatment in order to produce a matrix suitable to promote aerobic microbial activity. Aerobic treatment within sol bed systems was then facilitated, employing conventional agricultural equipment to mix and aerate soils, together with regular amendment to provide inorganic and organic macro- and _micro-nutrients. Each treatment process lasted approximately 12 to 18 weeks, comprising 3-4 weeks for construction and processing of sol beds (approximately 0.5 m deep, 60 m long and 15 m wide), followed by 8 to 14 weeks of aerobic treatment. In order to accommodate the volume of wastes for treatment, the process was undertaken in 3 consecutive phases with a total of 14 to 16 soil beds in each phase. Preliminary tests for catalase activity were carried out coma sample T, taken from freshly remediated soil. The sample was taken from the centre of a 2m high stock pile, measuring approximately 50 m in diameter, ata depth of 05 to Im. The full experiment measuring catalase activity and pollutant levels followed the time course of remediation with samples taken from each of two treatment soil beds, numbers 2 and 4 Six composite samples were taken from each bed in weeks 3 (when bed construction was completed), 6, 9, and 14 of treatment (dates 27 August, 14% September, 6® October and 10" November 1998 respectively). Samples were not taken during week 12 of the treatment due to a two-week period of heavy rainfall. Due to these adverse weather conditions, the beds had not been worked and were considered to be anaerobic. Samples taken in week 14 were still very wet. To collect the six composite samples the bed was segregated into 6 sectors (designated 1-6), approximately 10 m in length, and manually retrieved 0.5m cores were collected, from at least three separate locations within each sector. Cores were homogenised manually to give a composite sample (1 kg). Following this procedure, all samples for catalase activity testing were sent in a cool box via overnight delivery and analysis was carried out within three days of sampling Method for assaying soll catalase activity Between 1-5g of sol sample was buffered to pH 7 with 50 cm? of 0.1M sodium dihydrogen orthophosphate buffer and placed in a 260 em’ total volume plastic reaction chamber connected to a LFM300 low flow gas meter (G. H. Zeal Lid., London, UK) operating on the principle described by Guwy et 41, [20]. The chamber was maintained at 25°C by placing into a temperature controlled cabinet (Fisher Scientific UK) and, stirred using a magnetic stirrer and follower bar (Fisher Scientific, UK), A 3%w/v solution of hydrogen peroxide Fisher Scientific UK) was added at a rate of 22 em? min" using a Watson Marlow pump (Poole UK) fitted with a 304MC/A pumphead. The cumulative gas production was measured over 15 minutes. ‘The sample T (taken from the treated stockpile) was air dried overnight and subsequently sieved through a 2 mm sieve. Aliquots assayed (1-5g) were weighed from the sieved sample. The total volume was then made up to 10 cm? with water in a measuring cylinder and emptied into the reaction chamber. The measuring cylinder was washed out with a further 5 em! of water into the reaction chamber. For every soil sample assayed control samples were autoclaved at 15 psi for 30 minutes. The dry weight of the samples was determined by Standard Methods [23] A calibration plot was, constructed in order to establish the relationship between soil dry weight and activity Samples from treatment beds 2 and 4 were air dried toa workable consistency, and sieved through a 4 mm sieve, Four aliquots were taken from each sieved sample for catalase measurement, allowing for two to three tests and one autoclaved control. Three 5g aliquots were also taken from the sieved sample for dry weight determination by standard methods. Analyses were carried out within 36 hours of being, sieved, as described above. ‘Chemical analysis during the treatment period The soll samples taken for catalase activity ‘measurements were also analysed for target contaminants, specifically n-alkanes, total hydrocarbons and the 16 EPA priority pollutant PAHs, [24] while a range of parameters such as nutrients, pH, moisture and temperature were used to assess ongoing treatment requirements. Samples were despatched in cool boxes by overnight courier to Robertson's Laboratories, (Llandudno, UK), for analysis. On receipt of samples, target contaminants were analysed using the following methods. Soil (10g), as rece from the field with large stones 1020removed, was spiked with surrogate standards and extracted with dichloromethane (250 cm?) for 4 hours using Soxhlet extraction apparatus. Sub-samples of this extract were then used to determine both oil hydrocarbons and PAHs. The amount of extract used was adjusted depending upon the level of contamination within the soil samples received. Analysis of oll hydrocarbons (n-alkanes and total hydrocarbons) involved analysis of the extract by gs chromatography (Fisons 8000 series, 30m DBS capillary column) using a flame ionisation detector. Compounds were {quantified using a 5-point diesel oil calibration standard, with squaline used as an internal standard to assess recovery. Poly aromatic hydrocarbons (PAHs) were determined, after passage of the extract through an activated alumina ‘column and subsequent elution of the retained PAHs. This fraction was then analysed using gas chromatography (Hewlett Packard 5890 with 30m DBSms capillary column) ‘and mass spectroscopy detection (Hewlett Packard 5970). Individual PAHs were quantified against a5 point calibration standard of the 16 EPA priority pollutant PAHs, while contaminant recoveries were determined using deuterated internal standards of biphenyl d-10 and deuterated semi volatile standard (Alldrich, Poole, UK). Total organic carbon (TOC) was determined by the combustion of a known weight of dry powdered sample in oxygen, using a high temperature LECO CS125 induction furnace carbon sulphur determinator (LECO Instruments Ltd, Cheshire, UK) Kjeldah! digestion with sulphuric acid in the presence of a copper catalyst, Following digestion the solution was distilled under alkaline conditions and the liberated ammonia absorbed in boric acid was then titrated with standard hydrochloric acid [25], Potassium and phosphorus were extracted from the wil using a mixture of nitric and hydrochloric acids. The extracted metals were then analysed using a Perkin Elmer Total nitrogen was determined by "EB 180 % 160 3 140 = 120 2 100 3 80 2 0 40 § 20 % gO 6 0 1 2 Dry Figure 1 102 3300DV (Llantrisant, Mid Glamorgan UK) inductively coupled plasma-atomic emission spectrometer. In addition to the laboratory analyses, parameters were measured on-site including volatile organic compounds determined using a HNU photo-ionisation (Geotechnical Instruments Leamington Spa, ‘Warwickshire UK) calibrated against toluene and pH using a Coming pH meter calibrated with pH 7 and 10 buffers. Percentage moisture was determined gravimetrically in treatment beds 1, 5,9 and 13 weekly in accordance with the British Standard [26]. Temperature measurements were performed weekly at four different depths across all treatment beds using an electronic thermometer with internal calibration, supplied by Fisher Scientific UK (Loughborough, Leicestershire UK). several detector RESULTS AND DISCUSSION ry test for catalase activity Figure 1 shows the relation of dry weight to sol catalase activity (measured as oxygen evolution) for sample T at five different weights. There was a linear relationship between sample weight and catalase activity (r? = 0.998) with an activity of 37.42 1 0,/kg dry weight/15mins. The value for a control autoclaved sample was always zero. Catalase activity during the treatment period ‘Samples were initially heterogeneous in appearance, 35, the beds were still relatively unmixed. As the treatment progressed the samples became visually more homogenous, ‘The catalase activities for beds 2 and 4 are shown in Tables 1 and 2 respectively. ‘The value for control autoclaved samples taken from beds? and 4 was always zero. Each composite Weight (g) Relationship of catalase activity to dry weight in sample T.Table 1. Catalase activity, hydrocarbon and n-alkane values for each sample in bed 2 Date Sectornumberof mn Mean catalase activity SD SD Hydrocarbon alkane composite sample (/kg in 15 min) concentrations (g kg")_concentrations (g kg) 24/8/98 1 3 20.81 182 73 263, 79 (week 3) 2 3 49.47 an 63 45 97 3 3 4456 2 22 285 8a 4 3 3138 246 78 4 102 5 3 2221 421 100 302 84 6 3 54.02 084 16 362 ua 14/9/98 1 3 56.72 737 130 174 34 (week 6) 2 2 10992 mm 65 198 38 3 2 10173 3133 25. 52 4 2 66.75 an 62 259 68 5 2 10780 498139 269 62 6 3 1631, 33028 344 57, 06/10/98 1 3 47.80 956 200 92 16 (week 9) 2 3 36.28 130 36 108 20 3 3 37.60 an 99 61 10 4 3 32.82 266° 81 no 19 5 3 2757 363 132 86 18 6 2 88.02 48855 187 43 10/11/98 1 3 4655 06 15 37 13 (week 14 2 3 4194 159 38 72 12 3 3 22.76 ost 12 28 16 4 3 12 21335 55 12 5 3 m2 am 45 80 4 6 3 45.46 669 147 95 4s. Tenner sap Table2. Catalase activity, hydrocarbon and n-alkane values for each sample in bed 4. Date Sectornumber of An Mean catalase activity SD 98D Fydrocarbon walkane composite sample kg in 15 min) concentrations (g,kg.")_ concentrations (g kg) TA7BTIR 1 z 6256 bar 106 178 47 (week 3) 2 2 79.15 46859) 21 83 3 2 38.29 03706 821 241 4 2 6456 278 43, 685, 98 5 2 85.50, 233 25 751 218 6 2 6541 39060 862 267, 1479758 i 3 126.13 56345 303 39 (week 6) 2 3 10907 204 110 160 27 3 3 138.36 51437, 151 23 4 3 95.08 1067 112 uy 15 5 2 1201 94878 199 29 6 2 116.93 1403 120 150 23 D67T0798 T 3 63.32 7.05 111 36 33 (week 9) 2 3 2523 237 9a 76 14 3 3 4931 44490 107 V7 4 3 301 15640 103 14 5 3 5592 18633, 107 14 6 3 4911 29760 4 Ww TOT 798 T 3 3278 20462 64 5 (week 14) 2 3 49.32 66135 162 26 3 3 80.70 355 69) 72 12 4 3 98.64 14715. 58 cet 5 3 88:30 05306 66 1 6 3 58.27 2136 127 23 Tanumber or sample 1022sample representing sectors 1-6 for each date was a mixture of, cores sampled from three locations with each sector (10m length of bed). The standard deviation of triplicate analysis for catalase activity on each composite sample was on average 8% for bed 2 and 6.5 % for bed 4 (minimum and maximum, values of 1.6% and 25.2% for bed 2 and 0.6% and 13.5% for bed 4). However, when the mean catalase activies shown in Tables 1 and 2 for each week were averaged, the percentage standard deviation between the sectors of each bed during, the entire treatment period was between 24-49% in bed 2 and 12.37% in bed 4, It is thus evident that the major source of variation in obtaining values for catalase activity was sampling, The problems with the lack of heterogeneity between soil samples has been identified as a major disadvantage in the analysis and assessment of pollutants in soil (27) ‘The same problem of heterogeneity affected the hydrocarbon and n-alkane determinations. In Table 1 the standard deviation of the hydrocarbon concentrations for the ‘composite samples (representing the 6 sectors) in weeks 3, 6, 9 ‘and 14 varied from 12 to 40% in bed 2 and 18 to 50% in bed 4. Similarly the standard deviation of rvalkane concentration varied between 12 to 54% in bed 2and between 31 to 51% in bed 4. However, despite the variability due to sampling, certain trends are evident. The averages of the mean catalase activity in sectors 1 to 6 for beds 2 and 4 are plotted against time in Figures 2 and 3 respectively. Figures 2 and 3 also show the mean hydrocarbon and n-alkane concentrations in sectors 120 60 e 3 100 0 g 22 g_ 80 wo Ze c2 2 8 ZF 60 30 8 ¢ Pe be g° 40 20 3 3 4 20 i = 3 OT 0 o ° 5 10 1s Time (weeks) Figure2. Mean catalase activity compared with mean pollutant concentrations in bed 2 over the treatment period (Mean. catalase activi 140 2 120 z 3 100 og 80 2 z5 & 40 8 a a 20 & 0 o 2 4 6 ‘@; mean total hydrocarbon concentrations (); mean n-alkane concentrations A). 60 50 40 30 20 n-Alkane conc (g kg") Hydrocarbon cone (g kg") 10 ° 8 10 12 14 16 Time (weeks) Figure 3, Mean catalase activity compared with mean pollutant concentrations in bed 4 over the treatment period (Mean catalase activity ‘mean total hydrocarbon concentrations C1; mean n-alkane concentrations ‘A. 10231 to 6 for beds 2 and 4 plotted against time. Reproducibility between triplicates inthis test were good. However fora large sampling area, more samples may need to be taken in order to decrease variation between samples. Alternatively, a large ‘composite sample may be formed for the entire bed from ‘which subsamples may be taken for analysis, which reduces ‘error caused by topographical variation. Taking a composite sample of each of six sectors in the bed as performed here provided more detail on the bioremediation process. At the start of treatment, (week 3) catalase activity in bed 2 was lower than in bed 4 (Figures 2 and 3). In week 3 the ‘mean n-alkane and hydrocarbon concentrations in bed 2 were also lower than bed 4. For catalase activity beds 2 and 4 showed similar trends with time, seen in Figures 2 and 3 respectively. Catalase activity in bed 2 showed an increase of 131% from week three to week six (from 4041 litres O, evolved/kg in 15 minutes in week 3 to 93.21 litres O; evolved/kg in 15 minutes in week 6 of treatment). An increase of about 70% was observed in bed 4 from week three to week six. Between weeks 6 and 9 the activity in bed 2 dropped by just under 51.7% while in bed 4 the activity dropped by 60%. By week 9 the mean catalase activities for both beds 2 and 4 were similar. In both beds the catalase activity increased between weeks 9 and 14. A similar study {28] using microbial activity measured by fluorescein diacetate hydrolysis and microbial numbers also reported a corresponding sharp decrease in pollutant (et fuel) concentration in the first weeks of the treatment period. Figures 2 and 3 show that the decrease in pollutant concentrations was greatest during the first weeks of treatment. In bed 2 mean n-alkane concentrations dropped. by 44%, and mean total hydrocarbon concentrations dropped. by 21.2%. In bed 4 mean n-alkane concentrations dropped by 85, and mean total hydrocarbon concentrations dropped 70%. ‘Mean n-alkane concentrations in week 3 were higher in bed 4 than in bed 2, as was the catalase activity. The n-alkane activity dropped more sharply in bed 4 than in bed 2 between, ‘weeks 3 and 6, possibly related to higher catalase activity. Table 3 shows that between weeks 3 and 9 the mean VOC, phenol, and TOC concentrations all decreased, corresponding to the reduction in pollution shown in Figures 2 and 3. This dramatic reduction in pollutant concentration also observed in Tables 5 and 6. These tables also show the reduction of total PAH's and individual polynuclear aromatics (PNA’s) concentration for treatment beds 2 and 4 respectively. In weeks 3 to 8 the temperature in the beds was 20- 25°C higher than the ambient temperature (Figure 4, Table 4). ‘After week & the temperature in the treatment beds dropped; Figures 2 and 3 indicate that at this point the catalase activity Table3. Mean chemical analysis of treatment beds during weeks 3 and 9, Determinant “Total concentration Week 3 (24/8/98) Week 9 (6/10/98) PH 7.46 7.46 moisture % 237 27 VOC's (mg kg?) 10-500 s Phenols (mgkg') 425 24 TOC kg") 70.89 57.03 Nitrogen (gkg") 372 219 Phosphorus (gk) 1.16 a7 Potassium (g kg") 231 1.60 Table 4 ‘Temperature °C profiles of the treatment beds. “Ambient Depth (m ‘Surface 02 04 06 Week5 11 25.4(286,17.1) 793.8399,182) | 28G57,193) 25.405, 18.6) Weeks 14 25.2(278,213) 33(363,299) 33.2(363,30:1) 32.1 (36,283) Week7 16 29.9(324,283) 38.49.1,306) 34072,289) 33.2666, 285) Weeks 9 20.203,199) 30(965,21.2 323(37.7,24) 31.7365, 24.1) Week9 9 234(24,222) 34.1G7,325) 35.839, 38) 31.4.5, 30) Week 10 9 228 244,21.7) 38.4(40,31,7) 38.2(42.7,33) 37.5423, 32.1) Week 11 4 329(142,111) 24.4803,208) 317.1,27) 31.7677, 266) Week 12 4 16.4(182, 15:1) 23.709.1,202) 278G44,237) 29.1656, 247) Week 13. 14 136 022,142) 166(188,132) 19.1025,147) 20.5 (15.8, 12.3) Week 14_ 6 68 (8,62) 111 133,97) 145 (166,123) __16.2(193, 129) Figures are mean Tigh, ow) 1024Table 5. Mean chemical analysis of treatment bed 2. Date Total concentration (mg kg" dry weight) PAH's Benzo (a) Diberz(ah) Phen- Pyrene Moisture % pyrene __athracene__anthrene Week3 «1023S 20 20 5 AS (24/8/98) Week6 = «NANA NA. NA NA 169 (14/9/98) Week 9 333 40 13 18 8057 (06/10/98) Week12 263-35 03 15 93 A 00/11/98) SN Rota Table 6, Mean chemical analysis of treatment bed 4. Date Total concentration (mg kg" dry weight) PAH’s Benzo(a) Dibenz(a,h) Phen- —Pyrene Moisture % pyrene _athracene __anthrene Week 3 15393) 33 408 200 240 (24/8/98) Week6 = NANA NA. NA NA 138 14/9798) Week9 =» «-54.0— 3.8 10 25 90 Bd (06/10/98) Week 12 27333 os 20 wo | 78 (10/11/98), SN Net a Temperature °C Seen eae a eusSsanSRsunsans $$$ t+ + + +4 0 2 4 6 8 10 12 4 16 Time (weeks) Figure4, Mean temperature profiles of the treatment beds over the treatment period (Surface li 0.2m @; 0.4m A; 0.6m O; ambient O). 1025‘was past its peak and the organic pollution concentrations were much reduced. Possibly there was a reduction in the respiratory activity and hence less heat generation. The ambient temperature, however, was generally lower in weeks 9-12 than weeks 3-8, Also the moisture content of the beds ‘was significantly higher in weeks 9-12 than earlier (Figure 5), due to the increased rainfall. The moisture contents of all beds ‘measured had risen to 25-30% by week 11 and this may have reduced oxygen availability, so decreasing respiration and heat generation. It appears that after the remediation process microbial activity increased, presumably due to growth of the pollutant degrading bacteria. This suggests that biodegradation of pollutants was occurring and not merely loss of pollutants through volatilisation. Following, the rapid decrease of substrate concentration, microbial activity dropped. Figures 2 and 3 showed that although pollutant concentrations kept on falling until week 14, when, treatment stopped, catalase activity rose in week 14 by 14% compared to week 9 in bed 2, and by 45% in week 14 ‘compared to week 9 in bed 4. The final increase in activity may be caused by colonisation of the site by other bacteria ‘when pollutant levels are no longer inhibitory for growth. ‘commencement of ‘CONCLUSIONS Catalase activity may be measured in heavily contaminated soils undergoing bioremediation in an assay that takes only 15 minutes per sample. ‘This method may be useful in monitoring treatment performance as an increase in catalase activity was associated with a decrease of pollutant concentrations and the bioremediation system with higher catalase activity removed rvalkanes and hydrocarbons more rapidly. Reduced variation between samples could be achieved by increasing the number of samples per bed, or creating a ‘composite sample for the entire bed before analysis. ACKNOWLEDGEMENTS, ‘This work was partly funded by the Biotechnology and Biological Sciences Research Council of the UK under the Biological Treatment of Soil and Water LINK grant scheme (BSW05376), We acknowledge the help of our partners in this scheme including Siemens Environmental, Poole Dorset, UK, and thank the holders of patent no. No. WO 96/18896, UGCS, University of Glamorgan, for permission to publish this work. We also acknowledge the intellectual contribution of Dr RM Dinsdale. 40 450 35 400 a 350 g 25 300 = 2 250 & 3 200 € ee 150 2 ty 100 5 50 0 ° o 2 4 6 8 0 2 14 16 Time (weeks) Figure 5. Moisture profiles of treatment beds over the treatment period (Bed no 1 @; Bed no 5M; Bed no 9 &; Bed no 13 O; Rainfall 4; Cumulative rainfall 0). [REFERENCES 1. Frankenberger, W. T. and Dick, W. A., Relationship between enzyme activities and microbial growth and activity indices in soil. Soi. Sci. Amer. ., 47, 945-951. (1989). 102610. UL. 2 13, rn 6 v. 18, ws. 20, a. 2m, RR 2. 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