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Municipal solid waste compost dose effects on soil microbial biomass determined by chloroform fumigation-extraction and DNA methods

Municipal solid waste compost dose effects on soil microbial biomass determined by chloroform... Annals of Microbiology, 57 (4) 681-686 (2007) Municipal solid waste compost dose effects on soil microbial biomass determined by chloroform fumigation-extraction and DNA methods Olfa BOUZAIANE*, Hanene CHERIF, Fethia AYARI, Naceur JEDIDI, Abdennaceur HASSEN Centre de Recherches et Technologies des Eaux (CERTE), Laboratoire de Traitement et Recyclage, B.P. 95 - 2050, Hammam Lif Tunis, Tunisia Received 25 June 2007 / Accepted 8 October 2007 Abstract - We evaluated the relationship between microbial biomass C and N (B and B ) as estimated by the chloroform fumiga- C N tion-extraction (CFE) method and microbial biomass DNA concentration in a loam-clayey wheat cultivated soil. The soil received munic- -1 -1 ipal solid waste compost at rates of 40 or 80 t ha and farmyard manure at 40 t ha . Microbial biomasses C and N and DNA con- -1 centration showed the highest values for microorganisms counts with compost and farmyard manure at 40 t ha . Compost applica- -1 tions at 40 t ha-1 improve the micro-organisms growth than that of 80 t ha . Moreover a significant decrease of soil microbial bio- mass was noted after fertilisation for three years. The presence of humic acid and proteins impurities in DNA extracts; even in impor- tant level as in F-treated soil; did not affect the microbial biomass. The decrease of microbial biomass was due to heavy metals con- -1 tent elevation in compost at 80 t ha treated soil. Thus the highest rate of municipal solid waste compost induced the lowest ratio of biomass C to soil organic carbon and the lowest ratio of biomass N to soil organic nitrogen. There was a positive relationship between B , B and DNA concentration. DNA concentration was significantly and positively correlated with B and with B . However there was C N C N a negative correlation between either micro-organisms numbers and DNA concentration, or B and B . The comparison of the two C N used methods DNA extraction and CFE showed the lowest coefficient of variation (cv %) with DNA extraction method. This last method can be used as an alternative method to measure the microbial biomass in amended soils. Key words: municipal solid waste compost, microbial biomass C, microbial biomass N, chloroform fumigation extraction, DNA extraction. INTRODUCTION soils (Wu and Brookes, 2005). The DNA quantification method (DNA) has been compared to the CFE method in dif- The soil microbial biomass (SMB) conducts biochemical ferent soils (Marstorp et al., 2000; Bailey et al., 2002; transformations in soil (Breland and Eltun, 1999). The Leckie et al., 2004) and has been proposed as an alterna- potential influence of the SMB in a soil sample may be tive method of CFE to measure SMB (Marstorp et al., 2000). assessed by its amount (Anderson and Domsch, 1989). Direct extraction of total DNA from the soil, often results in Several studies showed that the SMB varies with soil man- coextraction of DNA and other soil components mainly agement including the farming system (Hu et al., 1997), humic substances, which negatively interfere with DNA fertilisation (Salinas-Garcia et al., 1997), organic amend- dosage (Steffan et al., 1988; Trevors et al., 1992). ments (Jedidi et al., 2004) and heavy metals (Garcia-Gil et The objectives of this study were: (i) to observe the al., 2000). Soil microbial biomass may be used as a sensi- effects of municipal solid waste compost dose on the tive and as an indicator of environmental changes of soil progress of microbial biomass determined by fumigation- management (Bouzaiane et al., 2007). extraction and DNA methods (ii) and to observe whether Assessment of SMB can be achieved by direct methods, purified, extracted DNA may be an appropriate measure of such as the micro-organisms counts (Paul and Johnson, total microbial biomass and an alternative method to the 1977), or by indirect methods, such as the chloroform fumi- fumigation-extraction in municipal solid waste compost gation-extraction method (CFE) (Vance et al., 1987; Tate et treated soil. al., 1988). The CFE (Vance et al., 1987; Brookes, 1995) has been widely used to estimate microbial biomass under dif- ferent field and laboratory conditions. The microbial biomass MATERIALS AND METHODS C and N have been estimated, using CFE in both cultivated and uncultivated soils (Vong et al., 1990), in forest soils Experimental site and soil sampling. The study was (Gallardo and Schlesinger, 1990), and in seasonnally dried conducted in the north of Tunisia, in a wheat (Var. Karim) cultivated plot. The climate is semi arid with annual mean temperature of 18.6 °C and of 119.15 mm precipitation. * Corresponding author. Phone: +21671788436; For the wheat cultivated plot, the experiment was set up in Fax: +21671410740, E-mail: olfa_bz2004@yahoo.fr 682 O. Bouzaiane et al. a random block design with four replications and an ele- MBC = CE/0.35 mentary plot size of 2.25 m . where CE was the difference between organic C extracted Total N was determined by the Kjeldahl method as rec- from fumigated and non-fumigated treated soils. ommended by Brookes et al. (1985), while the organic C Total N in the extracts was determined according to the content was determined by dry combustion (Walkley and Kjeldahl methods as described by Brookes et al. (1985). Black 1934). The total heavy metals (Cu, Zn, Ni, Pb, Cr and The microbial biomass N was estimated using the following Cd) in soils were determined by an atomic absorption spec- equation: trophotometer after acid digestion (nitric acid and chloridric MBN = NE/0.68 acid, 3/1, v/v). where NE was the difference between total N extracted Treatments used were: non-treated soil (S); a livestock from fumigated and non-fumigated soils. Amounts of -1 farmyard manure of cow is used in this experiment at rate microbial biomass C or N were expressed (mg C or N kg -1 of 40 t ha (F); mature municipal solid waste compost dry weight) on air-dry soil basis and represent the average (MSWC) was obtained from sorted municipal solid wastes of three determinations. by aerobic fermentation of 120 days in the composting plant. The annual application rates given as ton dry matter DNA extraction method. DNA was extracted and purified -1 used in our case study were MSWC at rates of 40 t ha from equivalent dry weights of each soil sample (500 mg -1 (C1) and at 80 t ha (C2). Some physico-chemical charac- fresh soil), using the Bio 101 Fast DNA Kit for Soil (Biogène, teristics of the soil and organic amendments used in this France), according to the manufacturer instructions. study were summarised in Table 1. Purified DNA was quantified by spectrophotometer (Bio- TM The soil sampling was performed three years after RAD Smart Spec Plus, France) (Leckie et al., 2004). The organic matter application; in June at the end of culture spectrophotometric A260 /A280 and A260 /A230 ratios and at a depth of 0-20 cm. All samples were stored at 4 °C were determined to evaluate levels of protein and humic prior to analyses. acid impurities, respectively, in the extracted DNA (Ogram et al., 1987; Steffan et al., 1988). TABLE 1 - Soil and organic amendments characteristics used Microorganisms counts. Aliquots of 5 g of soil were used Soil Compost Farmyard manure to determine the number of culturable microorganisms. Samples were plated onto 10-fold-diluted tryptic soy agar pH (in water) 8.5 (0.2) 7.9 (0.2) 7.8 (0.30) -1 (BIO RAD, France) containing 100 µg cycloheximide ml to C (%) 0.87 (0.01) 17.50 (1.30) 29.20 (2.40) inhibit fungal growth. Plates were incubated at 25 °C for three days and then the numbers of colonies forming units N (%) 0.095 (0.002) 1.800 (0.030) 2.600 (0.090) (CFU) were counted. C/N 9.15 9.8 11.4 HR (%) 8.2 25.8 7.1 Statistical analysis. The ANOVA analysis was carried out using the SPSS statistical program for Windows (SPSS Inc., Clay (%) 27 (0.8) Na Na Chicago, IL). The means were compared according to the Silt (%) 62 (1.4) Na Na Newman and Keuls multiple range-test. Pearson’s correlation Sand (%) 11 (0.5) Na Na coefficients were calculated for selected parameters. All sta- -1 tistical analyses were performed at P ≤ 0.05 or at P ≤ 0.01. Cd (mg kg ) 1.1 (0.03) 2.3 (0.30) 2.1 (0.05) -1 Pb (mg kg ) 49.5 (2.3) 80.1 (3.6) 8.9 (0.9) -1 Cr (mg kg ) 22.5 (1.1) 78.9 (2.9) 25.9 (2.5) RESULTS AND DISCUSSION -1 Ni (mg kg ) 21.9 (1.8) 90.8 (4.1) 22.4 (1.8) pH, total organic C and organic N -1 Cu (mg kg ) 42.5 (0.3) 337 (6.8) 25.5 (1.3) There were no significant differences (P < 0.05) between pH -1 Zn (mg kg ) 115.7 (2.2) 290.2 (11.7) 117.1 (3.1) values of all soil treatments (Table 2), reflecting the buffer capacity of the soil. Similar results were obtained by C: carbon; N: nitrogen; HR: moisture; Cd: cadmium; Pb: lead; Duchaufour (1997) in a clayey soil with high CaCO content. Cr: chrome; Ni: nickel; Cu: copper; Zn: zinc. 3 The total organic C (TOC) investigation showed a posi- n = 3; (In brackets): standard deviation; NA: not applicable. tive effect of organic matter amendment with either com- post or farmyard manure after three years application. In fact a significant increase of 22, 48 and 245% for F-treat- The chloroform-fumigation method. Microbial biomass ed soil, C1-treated soil and C2-treated soil, respectively C and N were analysed by the CFE method, according to (Table 2). Similar results were obtained by Kaschl et al. Vance et al. (1987) and Brookes (1995), respectively. (2002) who reported an increase in the organic matter in Duplicate samples (20 g) of treated soils and non-treat- compost amended soil. Also Bouzaiane et al. (2007) ed were fumigated with ethanol-free CHCl 24 h. noticed that the MSWC used during this study appeared 3for Fumigated and non-fumigated soil samples were extracted loaded with organic matter and micro-organisms than the with 0.5 M K SO (1/4, w/v). Organic C was quantified by farmyard manure. Application of compost at rates of 40 t 2 4 -1 -1 the potassium dichromate oxidation method (Jenkinson ha and 80 t ha increased the TOC and this increase is and Powlson, 1976) and subsequent back-titration of the higher that with F-treated soil. Similar results were unreduced dichromate. The soil microbial biomass C (MBC) obtained by Eghball (2002); after 4 years of compost appli- was estimated using the following equation (Jenkinson and cation higher organic matter content than with farmyard Powlson, 1976): manure application were obtained. Ann. Microbiol., 57 (4), 681-686 (2007) 683 TABLE 2 - pH, total organic C, microbial C ratio, organic N and microbial N ratios Treatments pH TOC (%) B (%) B /TOC N (%) B (%) B /N C C org N N org S 8.57 (0.06)a 1.10 (0.1)a 6.51 (0.62)a 5.9 0.17 (0.03)a 4.52 (0.4)a 26.6 F 8.63 (0.14)a 1.35 (0.2)a 14.30 (2.10)b 10.5 0.32 (0.1)a 12.70 (3.6)c 39.7 C1 8.49 (0.05)a 1.63 (0.1)b 22.10 (3.12)d 13.5 0.33 (0.09)a 15.70 (2.7)d 47.6 C2 8.60 (0.05)a 3.88 (0.3)c 18.20 (1.11)c 4.77 0.58 (0.11)b 9.90 (1.6)b 17.0 -1 -1 -1 S: non-treated soil; F: farmyard manure at 40 t ha treated soil; C1: compost at 40 t ha treated soil; C2: compost at 80 t ha treated soil; TOC: total organic C; N : organic N. org n = 4; (In brackets): standard deviation; within a column different letter after bracket means that the value is significantly different according to Student-Newmann-Keuls test at P < 0.05. The soil organic nitrogen was also increased by fertili- ments that contained microbial biomass in the organic sation. Subsequent values of organic nitrogen were 88 and residues and the addition of substrate-C, which stimulates -1 94%, after farmyard manure and compost at 40 t ha the indigenous soil microbiota. This could be explained by application, respectively (Table 2). The higher value of the soil enrichment in humus instead of microbial C when organic nitrogen of 241% was obtained after with compost compost is applied at elevated rates. -1 at 80 t ha application. Results showed that organic nitro- gen did not differ significantly for non-treated soil, F-treat- ed soil and C1-treated soil. However there is a significant difference between C2-treated soil and F-treated soil or between C1-treated soil and C2-treated soil. Amendment dose and type effects on soil microbial biomass and DNA concentration Microbial biomass C and N in the non-treated soil were sig- nificantly different from those of treated soils (Fig. 1). The application of either compost or farmyard manure showed an increase in B , B and DNA concentration. The microbial C N biomass C and N, and DNA concentration in the C1-treated soil were significantly different from those in C2-treated soil (P < 0.05). It is notable that DNA concentration in C1- treated soil was higher than that in C2-treated soil (Fig. -1 1C). So that, the application of compost at 40 t ha -1 enhance microorganisms growth than that of 80 t ha , this result support the suggestion made by Jedidi et al. (2004), -1 who used compost at 40 t ha but in laboratory studies, and recommended this level for field. Our results revealed -1 that when compost dose increased from 40 to 80 t ha , the microbial B and B and DNA concentration decreased sig- C N nificantly. -1 Microbial biomass C and N in compost at 40 t ha treat- ed soil were significantly different from those obtained in F- treated soil. However, no significant differences between the microbial biomass C and N in compost-treated soil or in -1 farmyard manure-treated soil at 40 t ha in laboratory study was found by Jedidi et al. (2004). Moreover, DNA concentration in compost treated soil was higher than that in the F-treated soil. These findings could be explained by the enhancement of C-retaining microbial activities in soil with the compost application than with the farmyard manure use. This could be also resulted from more C-sta- bilizing (or composting) micro-organisms that may be introduced into the soil with the compost application. FIG. 1 - Influence of compost rate on the biomass C (A), bio- Carbon and nitrogen microbial ratios mass N (B) and DNA concentration (C). S: soil without The microbial C ratio (B / TOC) increased after fertilisation -1 amendment; F: farmyard manure at 40 t ha treated (Table 2). In non-treated soil, the microbial ratio was 5.9, -1 soil; C1: fompost at 40 t ha treated soil; C2: fompost this ratio increased with farmyard manure (10.5) and with -1 at 80 t ha treated soil. Means followed by the same let- -1 compost at 40 t ha (13.5) while decreased with compost ter are not significantly different according to the -1 at 80 t ha (4.77). Also Garcia-Gil et al. (2000) showed Newmann and Keuls test at P < 0.05; bars are standard that microbial biomass increased with the organic amend- deviation. 684 O. Bouzaiane et al. The microbial N ratio (B / Norg) increased in C1-treat- Heavy metals effect on microbial biomass ed soil and F-treated soil and this increase was of 39.7 and Heavy metals content in soil under different treatments 47.6, respectively (Table 2). However, the application of C2 (Table 4) showed an increase in Cd, Ni, Cr, Zn, Cu and Pb decreased the microbial N ratio wich is of 17.0. The results with compost addition. The ratio of soil microbial C to soil showed that the high level of compost enriched the soil in organic C has been proposed as a useful measure of soil organic N instead of microbial N. pollution by heavy metals (Brookes, 1995) and a reduction in this ratio as a result of metal has been reported from Humic acid and protein impurities in soil and organic other studies (Chander and Brookes, 1991; Fliessbach and treatments Reber, 1992). In semiarid conditions, soil biomass is sub- Soil DNA was often contaminated with humic acid or pro- ject to seasonal variations and has an influence on this teins that interfered with accurate quantification of DNA by ratio. Our data showed that the highest rate of MSW com- -1 UV absorbance at 260 nm (Tebbe and Vahjen, 1993; Kuske post (80 t ha ) had the lowest ratio of biomass C to soil C et al., 1998). The A260 /A230 and A260 /A280 ratios for (Table 2), indicating a low biomass C content in compari- soil DNA were significantly lower than the ratios for DNA son with the organic C in soil. Similar result was obtained solutions from pure cultures (Zhou et al., 1996) showing for the ratio of biomass N to soil nitrogen. This low ratio that soil DNA was coextracted with humic compounds could be attributed not only to heavy metal that had been (Table 3). added with the elevated rates of fertilisers (compost at 80 -1 DNA extracts from the C1-treated soil showed higher t ha ), but also to a high condensation and humification of A260/A280 and A260/A230 ratios than those obtained with organic matter that is resistant to microbial attack (Tate, other treatments. The F-treated soil showed the lowest 1987). This may account for the results, particularly the ratio which may due to the high proportion of humic acids low microbial biomass content in the soils amended with and proteins added to soil after farmyard manure applica- MSW compost, compared to the farmyard manure treat- tion. Accordingly, the decrease in the microbial biomass C ment, which is a labile source of organic C for soil biota. and N and DNA concentration in the C2-treated soil could not be explained by the inhibition effects of proteins or Relationship between DNA and microbial biomass C humic acid. So these results may be explained by the pres- and N ence of other inhibitory source such as the cumulative There was a linear relationship between microbial biomass effect of heavy metals after three years of compost appli- C and microbial biomass N (Fig. 2). Franzluebbers et al. cation. (1995) found similar relationship between the microbial biomass C and N. On the other hand, Jedidi et al. (2004) found linear relationship in compost treated soil after 2, 4 TABLE 3 - Comparison of soil DNA yields and purity and 8 weeks of laboratory incubation. A linear relationship between biomass C and DNA concentration was found (Fig. Treatments DNA yield A /A ratio A /A ratio 260 280 260 230 -1 3B). DNA concentrations and B in the soil were highly cor- (µg DNA g C related (Fig. 3B). Nevertheless, the DNA concentration was dry wt soil) generally proportional to the B and both methods seemed S 0.54 (0.06) 1.23 (0.05)b 0.84 (0.02)b to give reliable values of soil microbial biomass. Similar F 0.81 (0.05) 1.05 (0.05)a 0.71 (0.03)a results were obtained by Marstorp et al. (2000), who found a strong relationship between B , estimated by CFE, and C1 1.52 (0.04) 1.38 (0.02)c 0.98 (0.04)c extracted DNA in a mineral soil. They suggested that DNA C2 1.04 (0.04) 1.2 (0.03)b 0.86 (0.03)b could be used as a measure of microbial biomass in agri- Pure culture 1.89 1.57 cultural soils with low organic matter content. These find- ings are, however, different from those of Griffiths et al. -1 S: non-treated soil; F: farmyard manure at 40 t ha treated soil; (1997) who found no relationship between B and DNA in -1 -1 C1: compost at 40 t ha treated soil; C2: compost at 80 t ha mineral soils incubated with heavy metals under laborato- treated soil; Pure culture: DNA from Gram positive bacteria. ry conditions. Furthermore, Leckie et al. (2004) reported n = 3 determined by spectrophotometry at 260 nm (A ), 280 nm (A ) and 230 nm (A ); (In brackets): standard deviation; no relationship between DNA yield and B in forest humus. 280 230 within a column different letter after bracket means that the value The ratios of DNA concentration and B and DNA concen- is significantly different according to Student-Newmann-Keuls tration and B in the non-treated soil (Fig. 3 A and B) did test at P < 0.05. not differ significantly from the other treated soils. TABLE 4 - Heavy metals following soil fertilisation Treatments Cd (ppm) Pb (ppm) Cr (ppm) Ni (ppm) Cu (ppm) Zn (ppm) S 1.11 (0.14)a 70.46 (13.0)a 37.58 (4.63)a 31.88 (3.83)a 53.14 (2.74)a 96.97 (3.84)a F 1.68 (0.21)b 111.94 (6.24)b 48.70 (3.58)b 47.33 (4.07)b 72.91 (4.51)b 116.12 (2.19)b C1 2.33 (0.21)c 135.40 (6.61)c 77.02 (10.11)c 53.47 (3.16)c 93.74 (4.57)c 190.53 (19.01)c C2 2.98 (0.35)d 158.24 (12.11)d 88.31 (10.69)d 71.69 (5.76)d 111.81 (10.23)d 216.60 (12.65)d -1 -1 -1 S: non-treted soil; F: farmyard manure at 40 t ha treated soil; C1: compost at 40 t ha treated soil; C2: compost at 80 t ha treat- ed soil. Cd: Cadmium; Pb: Lead; Cr: Chrome; Ni: Nickel; Cu: Copper; Zn: Zinc. n = 4; (In brackets): standard deviation; within a column different letter after bracket means that the value is significantly different according to Student-Newmann-Keuls test at P < 0.05. Ann. Microbiol., 57 (4), 681-686 (2007) 685 TABLE 5 - Coefficients of variation (%) of microbial C biomass (B ), microbial N biomass (B ) and DNA concentration Y = 20.2x + 4.6 C N in treated soil r = 0.94 0.8 P <0.05 Treatments Coefficients of variation (%) 0.6 BC BN DNA concentration 0.4 S 9.7 3.9 1.8 F 14.8 12.6 6.2 0.2 C1 11.2 7.6 2.6 C2 6.1 4.9 2.8 0 10 20 30 40 -1 biomass N ( g g soil) N -1 S: non-treated soil; F: farmyard manure at 40 t ha treated soil; -1 -1 C1: compost at 40 t ha treated soil; C2: compost at 80 t ha FIG. 2 - Relationship between biomass N and biomass C. treated soil. TABLE 6 - Pearson’s correlation coefficients between the micro- Y = 22.9x + 4.15 bial C biomass (B ), microbial N biomass (B ), DNA C N r = 0.72 concentration, microorganisms counts, TOC and N org P < 0.05 B B DNA Microbe TOC N C N org counts B 1 0.91** 0.92** –0.41 0.44 0.52* B 1 0.83** –0.46 0.46 0.58* DNA 1 –0.32 0.28 0.37 Microbe 1–0.23–0.31 Y = 536.3x + 17.4 0.8 counts r = 0.90 P < 0.05 0.6 TOC 1 0.90** N 1 org 0.4 TOC: total organic carbon; N : organic nitrogen; * correlation is org 0.2 significant at the 0.05 level; ** correlation is significant at the 0.01 level. 0.5 11.5 2 -1 (g g soil) DNA concentration N CONCLUSION FIG. 3 - Relationship between DNA concentration and biomass N (A) and biomass C (B) in soil. The application of mature municipal solid waste compost at -1 40 t ha was the best rate which improves soil microbial biomass and DNA extracts in wheat cultivated soil. However the application of municipal solid waste compost Moreover the coefficient of variation in the DNA extrac- -1 at 80 t ha enriched the soil on organic C and N. And this tion method was lower than the one of the fumigation rate included a higher content of heavy metals have a neg- extraction method (Table 5). These results indicated that ative effect on soil microbial biomass growth. Besides, it the quantification of DNA yields could be used as an alter- exists a significant correlation between microbial biomass native and a reliable method than chloroform fumigation C, microbial biomass N and DNA extracts. Moreover the extraction method to estimate microbial biomass in culti- coefficient of variation in the DNA extraction method was vated-amended soils. lower than the one of the fumigation extraction method. A correlation matrix (Table 6) shows some significant These results indicated that the quantification of DNA yields relationships between the biomass C and N, DNA concen- could be used as an alternative and a reliable method than tration, micro-organisms counts, total organic C and organ- chloroform fumigation extraction method to estimate ic N. There was a strong positive correlation between B microbial biomass in cultivated-amended soils. and B or between B and DNA concentration. B or B N N C N showed a positive correlation with organic N. However B , Acknowledgements B and DNA concentration showed a negative correlation We wish to thank Dr Hafedh Nasr, National Research with micro-organisms counts. These results could be Institute for Rural Engineering Water and Forest, Tunisia, explained by the fact that viable and culturable micro- Dr Vanessa Bailey, Pacific Northwest National Laboratory organisms represent only 1 to 10% of the total soil micro- Richland, USA and Dr Claudio Mondini, Instituto organisms. However the extraction of DNA involved the Sperimentale per la Nutrizione delle Piante, Italy, for help- total soil micro-organisms, including culturable and non ful comments on the manuscript. The present study is a culturable ones. 686 O. Bouzaiane et al. part of the 1999-2002 research programme “Municipal Jenkinson D.S., Powlson D.S. (1976). The effects of biocidal treatments on metabolism in soil - I. Fumigation with chlo- solid waste treatment and compost agriculture application“ roform. Soil Biol. Biochem., 8: 167-177. which is supported jointly by the Tunisian State Secretariat Kaschl A., Romheld V., Chen Y. (2002). 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DNA recovery from soils soils. FEMS Microbiol. Ecol., 24: 103-112. of diverse composition. Appl. Environ. Microbiol., 62: 316- Hu S., Grunwald N.J., Van Bruggen A.H.C., Gamble G.R., Drinkwater L.E., Shennan C., Demment M.H. (1997). Short term effects of cover crop incorporation on soil carbon pools and nitrogen availability. Soil Sci. Soc. Am. J., 61: 901-911. Jedidi N., Hassen A., Van Cleemput O., M’hiri A. (2004). Microbial biomass in soil amended with different types of organic wastes. Waste Manage. Res., 22: 93-99. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Municipal solid waste compost dose effects on soil microbial biomass determined by chloroform fumigation-extraction and DNA methods

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Springer Journals
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Annals of Microbiology, 57 (4) 681-686 (2007) Municipal solid waste compost dose effects on soil microbial biomass determined by chloroform fumigation-extraction and DNA methods Olfa BOUZAIANE*, Hanene CHERIF, Fethia AYARI, Naceur JEDIDI, Abdennaceur HASSEN Centre de Recherches et Technologies des Eaux (CERTE), Laboratoire de Traitement et Recyclage, B.P. 95 - 2050, Hammam Lif Tunis, Tunisia Received 25 June 2007 / Accepted 8 October 2007 Abstract - We evaluated the relationship between microbial biomass C and N (B and B ) as estimated by the chloroform fumiga- C N tion-extraction (CFE) method and microbial biomass DNA concentration in a loam-clayey wheat cultivated soil. The soil received munic- -1 -1 ipal solid waste compost at rates of 40 or 80 t ha and farmyard manure at 40 t ha . Microbial biomasses C and N and DNA con- -1 centration showed the highest values for microorganisms counts with compost and farmyard manure at 40 t ha . Compost applica- -1 tions at 40 t ha-1 improve the micro-organisms growth than that of 80 t ha . Moreover a significant decrease of soil microbial bio- mass was noted after fertilisation for three years. The presence of humic acid and proteins impurities in DNA extracts; even in impor- tant level as in F-treated soil; did not affect the microbial biomass. The decrease of microbial biomass was due to heavy metals con- -1 tent elevation in compost at 80 t ha treated soil. Thus the highest rate of municipal solid waste compost induced the lowest ratio of biomass C to soil organic carbon and the lowest ratio of biomass N to soil organic nitrogen. There was a positive relationship between B , B and DNA concentration. DNA concentration was significantly and positively correlated with B and with B . However there was C N C N a negative correlation between either micro-organisms numbers and DNA concentration, or B and B . The comparison of the two C N used methods DNA extraction and CFE showed the lowest coefficient of variation (cv %) with DNA extraction method. This last method can be used as an alternative method to measure the microbial biomass in amended soils. Key words: municipal solid waste compost, microbial biomass C, microbial biomass N, chloroform fumigation extraction, DNA extraction. INTRODUCTION soils (Wu and Brookes, 2005). The DNA quantification method (DNA) has been compared to the CFE method in dif- The soil microbial biomass (SMB) conducts biochemical ferent soils (Marstorp et al., 2000; Bailey et al., 2002; transformations in soil (Breland and Eltun, 1999). The Leckie et al., 2004) and has been proposed as an alterna- potential influence of the SMB in a soil sample may be tive method of CFE to measure SMB (Marstorp et al., 2000). assessed by its amount (Anderson and Domsch, 1989). Direct extraction of total DNA from the soil, often results in Several studies showed that the SMB varies with soil man- coextraction of DNA and other soil components mainly agement including the farming system (Hu et al., 1997), humic substances, which negatively interfere with DNA fertilisation (Salinas-Garcia et al., 1997), organic amend- dosage (Steffan et al., 1988; Trevors et al., 1992). ments (Jedidi et al., 2004) and heavy metals (Garcia-Gil et The objectives of this study were: (i) to observe the al., 2000). Soil microbial biomass may be used as a sensi- effects of municipal solid waste compost dose on the tive and as an indicator of environmental changes of soil progress of microbial biomass determined by fumigation- management (Bouzaiane et al., 2007). extraction and DNA methods (ii) and to observe whether Assessment of SMB can be achieved by direct methods, purified, extracted DNA may be an appropriate measure of such as the micro-organisms counts (Paul and Johnson, total microbial biomass and an alternative method to the 1977), or by indirect methods, such as the chloroform fumi- fumigation-extraction in municipal solid waste compost gation-extraction method (CFE) (Vance et al., 1987; Tate et treated soil. al., 1988). The CFE (Vance et al., 1987; Brookes, 1995) has been widely used to estimate microbial biomass under dif- ferent field and laboratory conditions. The microbial biomass MATERIALS AND METHODS C and N have been estimated, using CFE in both cultivated and uncultivated soils (Vong et al., 1990), in forest soils Experimental site and soil sampling. The study was (Gallardo and Schlesinger, 1990), and in seasonnally dried conducted in the north of Tunisia, in a wheat (Var. Karim) cultivated plot. The climate is semi arid with annual mean temperature of 18.6 °C and of 119.15 mm precipitation. * Corresponding author. Phone: +21671788436; For the wheat cultivated plot, the experiment was set up in Fax: +21671410740, E-mail: olfa_bz2004@yahoo.fr 682 O. Bouzaiane et al. a random block design with four replications and an ele- MBC = CE/0.35 mentary plot size of 2.25 m . where CE was the difference between organic C extracted Total N was determined by the Kjeldahl method as rec- from fumigated and non-fumigated treated soils. ommended by Brookes et al. (1985), while the organic C Total N in the extracts was determined according to the content was determined by dry combustion (Walkley and Kjeldahl methods as described by Brookes et al. (1985). Black 1934). The total heavy metals (Cu, Zn, Ni, Pb, Cr and The microbial biomass N was estimated using the following Cd) in soils were determined by an atomic absorption spec- equation: trophotometer after acid digestion (nitric acid and chloridric MBN = NE/0.68 acid, 3/1, v/v). where NE was the difference between total N extracted Treatments used were: non-treated soil (S); a livestock from fumigated and non-fumigated soils. Amounts of -1 farmyard manure of cow is used in this experiment at rate microbial biomass C or N were expressed (mg C or N kg -1 of 40 t ha (F); mature municipal solid waste compost dry weight) on air-dry soil basis and represent the average (MSWC) was obtained from sorted municipal solid wastes of three determinations. by aerobic fermentation of 120 days in the composting plant. The annual application rates given as ton dry matter DNA extraction method. DNA was extracted and purified -1 used in our case study were MSWC at rates of 40 t ha from equivalent dry weights of each soil sample (500 mg -1 (C1) and at 80 t ha (C2). Some physico-chemical charac- fresh soil), using the Bio 101 Fast DNA Kit for Soil (Biogène, teristics of the soil and organic amendments used in this France), according to the manufacturer instructions. study were summarised in Table 1. Purified DNA was quantified by spectrophotometer (Bio- TM The soil sampling was performed three years after RAD Smart Spec Plus, France) (Leckie et al., 2004). The organic matter application; in June at the end of culture spectrophotometric A260 /A280 and A260 /A230 ratios and at a depth of 0-20 cm. All samples were stored at 4 °C were determined to evaluate levels of protein and humic prior to analyses. acid impurities, respectively, in the extracted DNA (Ogram et al., 1987; Steffan et al., 1988). TABLE 1 - Soil and organic amendments characteristics used Microorganisms counts. Aliquots of 5 g of soil were used Soil Compost Farmyard manure to determine the number of culturable microorganisms. Samples were plated onto 10-fold-diluted tryptic soy agar pH (in water) 8.5 (0.2) 7.9 (0.2) 7.8 (0.30) -1 (BIO RAD, France) containing 100 µg cycloheximide ml to C (%) 0.87 (0.01) 17.50 (1.30) 29.20 (2.40) inhibit fungal growth. Plates were incubated at 25 °C for three days and then the numbers of colonies forming units N (%) 0.095 (0.002) 1.800 (0.030) 2.600 (0.090) (CFU) were counted. C/N 9.15 9.8 11.4 HR (%) 8.2 25.8 7.1 Statistical analysis. The ANOVA analysis was carried out using the SPSS statistical program for Windows (SPSS Inc., Clay (%) 27 (0.8) Na Na Chicago, IL). The means were compared according to the Silt (%) 62 (1.4) Na Na Newman and Keuls multiple range-test. Pearson’s correlation Sand (%) 11 (0.5) Na Na coefficients were calculated for selected parameters. All sta- -1 tistical analyses were performed at P ≤ 0.05 or at P ≤ 0.01. Cd (mg kg ) 1.1 (0.03) 2.3 (0.30) 2.1 (0.05) -1 Pb (mg kg ) 49.5 (2.3) 80.1 (3.6) 8.9 (0.9) -1 Cr (mg kg ) 22.5 (1.1) 78.9 (2.9) 25.9 (2.5) RESULTS AND DISCUSSION -1 Ni (mg kg ) 21.9 (1.8) 90.8 (4.1) 22.4 (1.8) pH, total organic C and organic N -1 Cu (mg kg ) 42.5 (0.3) 337 (6.8) 25.5 (1.3) There were no significant differences (P < 0.05) between pH -1 Zn (mg kg ) 115.7 (2.2) 290.2 (11.7) 117.1 (3.1) values of all soil treatments (Table 2), reflecting the buffer capacity of the soil. Similar results were obtained by C: carbon; N: nitrogen; HR: moisture; Cd: cadmium; Pb: lead; Duchaufour (1997) in a clayey soil with high CaCO content. Cr: chrome; Ni: nickel; Cu: copper; Zn: zinc. 3 The total organic C (TOC) investigation showed a posi- n = 3; (In brackets): standard deviation; NA: not applicable. tive effect of organic matter amendment with either com- post or farmyard manure after three years application. In fact a significant increase of 22, 48 and 245% for F-treat- The chloroform-fumigation method. Microbial biomass ed soil, C1-treated soil and C2-treated soil, respectively C and N were analysed by the CFE method, according to (Table 2). Similar results were obtained by Kaschl et al. Vance et al. (1987) and Brookes (1995), respectively. (2002) who reported an increase in the organic matter in Duplicate samples (20 g) of treated soils and non-treat- compost amended soil. Also Bouzaiane et al. (2007) ed were fumigated with ethanol-free CHCl 24 h. noticed that the MSWC used during this study appeared 3for Fumigated and non-fumigated soil samples were extracted loaded with organic matter and micro-organisms than the with 0.5 M K SO (1/4, w/v). Organic C was quantified by farmyard manure. Application of compost at rates of 40 t 2 4 -1 -1 the potassium dichromate oxidation method (Jenkinson ha and 80 t ha increased the TOC and this increase is and Powlson, 1976) and subsequent back-titration of the higher that with F-treated soil. Similar results were unreduced dichromate. The soil microbial biomass C (MBC) obtained by Eghball (2002); after 4 years of compost appli- was estimated using the following equation (Jenkinson and cation higher organic matter content than with farmyard Powlson, 1976): manure application were obtained. Ann. Microbiol., 57 (4), 681-686 (2007) 683 TABLE 2 - pH, total organic C, microbial C ratio, organic N and microbial N ratios Treatments pH TOC (%) B (%) B /TOC N (%) B (%) B /N C C org N N org S 8.57 (0.06)a 1.10 (0.1)a 6.51 (0.62)a 5.9 0.17 (0.03)a 4.52 (0.4)a 26.6 F 8.63 (0.14)a 1.35 (0.2)a 14.30 (2.10)b 10.5 0.32 (0.1)a 12.70 (3.6)c 39.7 C1 8.49 (0.05)a 1.63 (0.1)b 22.10 (3.12)d 13.5 0.33 (0.09)a 15.70 (2.7)d 47.6 C2 8.60 (0.05)a 3.88 (0.3)c 18.20 (1.11)c 4.77 0.58 (0.11)b 9.90 (1.6)b 17.0 -1 -1 -1 S: non-treated soil; F: farmyard manure at 40 t ha treated soil; C1: compost at 40 t ha treated soil; C2: compost at 80 t ha treated soil; TOC: total organic C; N : organic N. org n = 4; (In brackets): standard deviation; within a column different letter after bracket means that the value is significantly different according to Student-Newmann-Keuls test at P < 0.05. The soil organic nitrogen was also increased by fertili- ments that contained microbial biomass in the organic sation. Subsequent values of organic nitrogen were 88 and residues and the addition of substrate-C, which stimulates -1 94%, after farmyard manure and compost at 40 t ha the indigenous soil microbiota. This could be explained by application, respectively (Table 2). The higher value of the soil enrichment in humus instead of microbial C when organic nitrogen of 241% was obtained after with compost compost is applied at elevated rates. -1 at 80 t ha application. Results showed that organic nitro- gen did not differ significantly for non-treated soil, F-treat- ed soil and C1-treated soil. However there is a significant difference between C2-treated soil and F-treated soil or between C1-treated soil and C2-treated soil. Amendment dose and type effects on soil microbial biomass and DNA concentration Microbial biomass C and N in the non-treated soil were sig- nificantly different from those of treated soils (Fig. 1). The application of either compost or farmyard manure showed an increase in B , B and DNA concentration. The microbial C N biomass C and N, and DNA concentration in the C1-treated soil were significantly different from those in C2-treated soil (P < 0.05). It is notable that DNA concentration in C1- treated soil was higher than that in C2-treated soil (Fig. -1 1C). So that, the application of compost at 40 t ha -1 enhance microorganisms growth than that of 80 t ha , this result support the suggestion made by Jedidi et al. (2004), -1 who used compost at 40 t ha but in laboratory studies, and recommended this level for field. Our results revealed -1 that when compost dose increased from 40 to 80 t ha , the microbial B and B and DNA concentration decreased sig- C N nificantly. -1 Microbial biomass C and N in compost at 40 t ha treat- ed soil were significantly different from those obtained in F- treated soil. However, no significant differences between the microbial biomass C and N in compost-treated soil or in -1 farmyard manure-treated soil at 40 t ha in laboratory study was found by Jedidi et al. (2004). Moreover, DNA concentration in compost treated soil was higher than that in the F-treated soil. These findings could be explained by the enhancement of C-retaining microbial activities in soil with the compost application than with the farmyard manure use. This could be also resulted from more C-sta- bilizing (or composting) micro-organisms that may be introduced into the soil with the compost application. FIG. 1 - Influence of compost rate on the biomass C (A), bio- Carbon and nitrogen microbial ratios mass N (B) and DNA concentration (C). S: soil without The microbial C ratio (B / TOC) increased after fertilisation -1 amendment; F: farmyard manure at 40 t ha treated (Table 2). In non-treated soil, the microbial ratio was 5.9, -1 soil; C1: fompost at 40 t ha treated soil; C2: fompost this ratio increased with farmyard manure (10.5) and with -1 at 80 t ha treated soil. Means followed by the same let- -1 compost at 40 t ha (13.5) while decreased with compost ter are not significantly different according to the -1 at 80 t ha (4.77). Also Garcia-Gil et al. (2000) showed Newmann and Keuls test at P < 0.05; bars are standard that microbial biomass increased with the organic amend- deviation. 684 O. Bouzaiane et al. The microbial N ratio (B / Norg) increased in C1-treat- Heavy metals effect on microbial biomass ed soil and F-treated soil and this increase was of 39.7 and Heavy metals content in soil under different treatments 47.6, respectively (Table 2). However, the application of C2 (Table 4) showed an increase in Cd, Ni, Cr, Zn, Cu and Pb decreased the microbial N ratio wich is of 17.0. The results with compost addition. The ratio of soil microbial C to soil showed that the high level of compost enriched the soil in organic C has been proposed as a useful measure of soil organic N instead of microbial N. pollution by heavy metals (Brookes, 1995) and a reduction in this ratio as a result of metal has been reported from Humic acid and protein impurities in soil and organic other studies (Chander and Brookes, 1991; Fliessbach and treatments Reber, 1992). In semiarid conditions, soil biomass is sub- Soil DNA was often contaminated with humic acid or pro- ject to seasonal variations and has an influence on this teins that interfered with accurate quantification of DNA by ratio. Our data showed that the highest rate of MSW com- -1 UV absorbance at 260 nm (Tebbe and Vahjen, 1993; Kuske post (80 t ha ) had the lowest ratio of biomass C to soil C et al., 1998). The A260 /A230 and A260 /A280 ratios for (Table 2), indicating a low biomass C content in compari- soil DNA were significantly lower than the ratios for DNA son with the organic C in soil. Similar result was obtained solutions from pure cultures (Zhou et al., 1996) showing for the ratio of biomass N to soil nitrogen. This low ratio that soil DNA was coextracted with humic compounds could be attributed not only to heavy metal that had been (Table 3). added with the elevated rates of fertilisers (compost at 80 -1 DNA extracts from the C1-treated soil showed higher t ha ), but also to a high condensation and humification of A260/A280 and A260/A230 ratios than those obtained with organic matter that is resistant to microbial attack (Tate, other treatments. The F-treated soil showed the lowest 1987). This may account for the results, particularly the ratio which may due to the high proportion of humic acids low microbial biomass content in the soils amended with and proteins added to soil after farmyard manure applica- MSW compost, compared to the farmyard manure treat- tion. Accordingly, the decrease in the microbial biomass C ment, which is a labile source of organic C for soil biota. and N and DNA concentration in the C2-treated soil could not be explained by the inhibition effects of proteins or Relationship between DNA and microbial biomass C humic acid. So these results may be explained by the pres- and N ence of other inhibitory source such as the cumulative There was a linear relationship between microbial biomass effect of heavy metals after three years of compost appli- C and microbial biomass N (Fig. 2). Franzluebbers et al. cation. (1995) found similar relationship between the microbial biomass C and N. On the other hand, Jedidi et al. (2004) found linear relationship in compost treated soil after 2, 4 TABLE 3 - Comparison of soil DNA yields and purity and 8 weeks of laboratory incubation. A linear relationship between biomass C and DNA concentration was found (Fig. Treatments DNA yield A /A ratio A /A ratio 260 280 260 230 -1 3B). DNA concentrations and B in the soil were highly cor- (µg DNA g C related (Fig. 3B). Nevertheless, the DNA concentration was dry wt soil) generally proportional to the B and both methods seemed S 0.54 (0.06) 1.23 (0.05)b 0.84 (0.02)b to give reliable values of soil microbial biomass. Similar F 0.81 (0.05) 1.05 (0.05)a 0.71 (0.03)a results were obtained by Marstorp et al. (2000), who found a strong relationship between B , estimated by CFE, and C1 1.52 (0.04) 1.38 (0.02)c 0.98 (0.04)c extracted DNA in a mineral soil. They suggested that DNA C2 1.04 (0.04) 1.2 (0.03)b 0.86 (0.03)b could be used as a measure of microbial biomass in agri- Pure culture 1.89 1.57 cultural soils with low organic matter content. These find- ings are, however, different from those of Griffiths et al. -1 S: non-treated soil; F: farmyard manure at 40 t ha treated soil; (1997) who found no relationship between B and DNA in -1 -1 C1: compost at 40 t ha treated soil; C2: compost at 80 t ha mineral soils incubated with heavy metals under laborato- treated soil; Pure culture: DNA from Gram positive bacteria. ry conditions. Furthermore, Leckie et al. (2004) reported n = 3 determined by spectrophotometry at 260 nm (A ), 280 nm (A ) and 230 nm (A ); (In brackets): standard deviation; no relationship between DNA yield and B in forest humus. 280 230 within a column different letter after bracket means that the value The ratios of DNA concentration and B and DNA concen- is significantly different according to Student-Newmann-Keuls tration and B in the non-treated soil (Fig. 3 A and B) did test at P < 0.05. not differ significantly from the other treated soils. TABLE 4 - Heavy metals following soil fertilisation Treatments Cd (ppm) Pb (ppm) Cr (ppm) Ni (ppm) Cu (ppm) Zn (ppm) S 1.11 (0.14)a 70.46 (13.0)a 37.58 (4.63)a 31.88 (3.83)a 53.14 (2.74)a 96.97 (3.84)a F 1.68 (0.21)b 111.94 (6.24)b 48.70 (3.58)b 47.33 (4.07)b 72.91 (4.51)b 116.12 (2.19)b C1 2.33 (0.21)c 135.40 (6.61)c 77.02 (10.11)c 53.47 (3.16)c 93.74 (4.57)c 190.53 (19.01)c C2 2.98 (0.35)d 158.24 (12.11)d 88.31 (10.69)d 71.69 (5.76)d 111.81 (10.23)d 216.60 (12.65)d -1 -1 -1 S: non-treted soil; F: farmyard manure at 40 t ha treated soil; C1: compost at 40 t ha treated soil; C2: compost at 80 t ha treat- ed soil. Cd: Cadmium; Pb: Lead; Cr: Chrome; Ni: Nickel; Cu: Copper; Zn: Zinc. n = 4; (In brackets): standard deviation; within a column different letter after bracket means that the value is significantly different according to Student-Newmann-Keuls test at P < 0.05. Ann. Microbiol., 57 (4), 681-686 (2007) 685 TABLE 5 - Coefficients of variation (%) of microbial C biomass (B ), microbial N biomass (B ) and DNA concentration Y = 20.2x + 4.6 C N in treated soil r = 0.94 0.8 P <0.05 Treatments Coefficients of variation (%) 0.6 BC BN DNA concentration 0.4 S 9.7 3.9 1.8 F 14.8 12.6 6.2 0.2 C1 11.2 7.6 2.6 C2 6.1 4.9 2.8 0 10 20 30 40 -1 biomass N ( g g soil) N -1 S: non-treated soil; F: farmyard manure at 40 t ha treated soil; -1 -1 C1: compost at 40 t ha treated soil; C2: compost at 80 t ha FIG. 2 - Relationship between biomass N and biomass C. treated soil. TABLE 6 - Pearson’s correlation coefficients between the micro- Y = 22.9x + 4.15 bial C biomass (B ), microbial N biomass (B ), DNA C N r = 0.72 concentration, microorganisms counts, TOC and N org P < 0.05 B B DNA Microbe TOC N C N org counts B 1 0.91** 0.92** –0.41 0.44 0.52* B 1 0.83** –0.46 0.46 0.58* DNA 1 –0.32 0.28 0.37 Microbe 1–0.23–0.31 Y = 536.3x + 17.4 0.8 counts r = 0.90 P < 0.05 0.6 TOC 1 0.90** N 1 org 0.4 TOC: total organic carbon; N : organic nitrogen; * correlation is org 0.2 significant at the 0.05 level; ** correlation is significant at the 0.01 level. 0.5 11.5 2 -1 (g g soil) DNA concentration N CONCLUSION FIG. 3 - Relationship between DNA concentration and biomass N (A) and biomass C (B) in soil. The application of mature municipal solid waste compost at -1 40 t ha was the best rate which improves soil microbial biomass and DNA extracts in wheat cultivated soil. However the application of municipal solid waste compost Moreover the coefficient of variation in the DNA extrac- -1 at 80 t ha enriched the soil on organic C and N. And this tion method was lower than the one of the fumigation rate included a higher content of heavy metals have a neg- extraction method (Table 5). These results indicated that ative effect on soil microbial biomass growth. Besides, it the quantification of DNA yields could be used as an alter- exists a significant correlation between microbial biomass native and a reliable method than chloroform fumigation C, microbial biomass N and DNA extracts. Moreover the extraction method to estimate microbial biomass in culti- coefficient of variation in the DNA extraction method was vated-amended soils. lower than the one of the fumigation extraction method. A correlation matrix (Table 6) shows some significant These results indicated that the quantification of DNA yields relationships between the biomass C and N, DNA concen- could be used as an alternative and a reliable method than tration, micro-organisms counts, total organic C and organ- chloroform fumigation extraction method to estimate ic N. There was a strong positive correlation between B microbial biomass in cultivated-amended soils. and B or between B and DNA concentration. B or B N N C N showed a positive correlation with organic N. However B , Acknowledgements B and DNA concentration showed a negative correlation We wish to thank Dr Hafedh Nasr, National Research with micro-organisms counts. These results could be Institute for Rural Engineering Water and Forest, Tunisia, explained by the fact that viable and culturable micro- Dr Vanessa Bailey, Pacific Northwest National Laboratory organisms represent only 1 to 10% of the total soil micro- Richland, USA and Dr Claudio Mondini, Instituto organisms. However the extraction of DNA involved the Sperimentale per la Nutrizione delle Piante, Italy, for help- total soil micro-organisms, including culturable and non ful comments on the manuscript. The present study is a culturable ones. 686 O. Bouzaiane et al. part of the 1999-2002 research programme “Municipal Jenkinson D.S., Powlson D.S. (1976). The effects of biocidal treatments on metabolism in soil - I. Fumigation with chlo- solid waste treatment and compost agriculture application“ roform. Soil Biol. Biochem., 8: 167-177. which is supported jointly by the Tunisian State Secretariat Kaschl A., Romheld V., Chen Y. (2002). 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Annals of Microbiology – Springer Journals

Published: Nov 21, 2009

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