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Detection of microbial groups metabolizing a substratein soil based on the [l4C] quinone profileKatsuaki Saitou a , Koichi Fujie b & Arata Katayama aa Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japanb Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi,441-8580, JapanPublished online: 04 Jan 2012.

To cite this article: Katsuaki Saitou , Koichi Fujie & Arata Katayama (1999): Detection of microbial groups metabolizing asubstrate in soil based on the [l4C] quinone profile, Soil Science and Plant Nutrition, 45:3, 669-679

To link to this article: http://dx.doi.org/10.1080/00380768.1999.10415830

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Soil Sci. Plant Nutr., 45 (3), 669-679, 1999 669

Detection of Microbial Groups Metabolizing a Substrate in Soil Based on the [ l4e] Quinone Profile

Katsuaki Saitou, Koichi Fujie*, and Arata Katayama l

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan; • Department of Ecological Engineering, Toyohashi University of Technology,

Toyohash~ 441-8580 Japan

Received January 21, 1999; accepted in revised form June 17, 1999

Analysis of [l4C]respiratory Quinones synthesized in soil for 6 h after spiking with [U -14C]glucose, [U -l4C]glycine, and [1,2- l4C]acetate enabled to finger­print the microorganisms metabolizing each substrate in soil and to determine the whole structure of the microbial communities at the same time. The [l4C]_ Quinones synthesized from [U _l4C] glucose were the same as those from [U­l4C]gIycinc in soil, suggesting that the same microbial groups metabolized glucose and glycine under the given conditions. No [l4C]quinones from [1,2-14C] acetate were detected in soil, indicating that the metabolism of acetate by microorganisms is negligible. The profiles of [l4C]quinones from [U- l4C]_ glucose were compared between Nagoya University Farm soils subjected to 4 different fertilizing practices. The soils receiving farmyard manure contained [l4C]menaquinones with highly hydrated isoprenoid units, which indicated the presence of Actinobacteria metabolizing glucose. The soil receiving only chemical fertilizers contained [14C]ubiquinone with 8 isoprenoid units (Q-8), indicating the presence of beta and gamma subdivisions of Proteobacteria. All the 4 soils were characterized by the high proportions of [14C] MK-6 and a mixture of [l4C]MK-8(H4) and [l4C]MK-9. The Q-9 and Q-10(H2)' indicators of fungi, were not labeled under most of the conditions.

Key Words: [l4C] -labeling of quinones, metabolizing activity, soil microbial community, [U -14C]glucose, [D _l4C] glycine.

Microbial activities determine the rate of nutrient cycling and the degradation rates of environmental pollutants in soil (Alexander 1994). Therefore, identification of the active microbial groups should contribute to supplying information on the role of microorganisms in the degradation of organic materials in soil. Classically, microorganisms with such a specific ability (e.g., cellulose decomposition) have been isolated by enrichment culture techniques (Brock et al. 1994). However, this isolation procedure enables to select only the culturable microorganisms that can express a specific ability under given culture conditions. Many microorganisms in soil are not culturable and conditions in a pure culture are completely different from those in actual soil (Torsvik et al. 1996). Even when an isolate is the major microorganism involved in the phenomenon in question, other unculturable microorganisms may also be involved in actual soil.

1 To whom correspondence should be addressed.

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670 K. SAITOU, K. FUJIE, and A. KATAYAMA

To identify the microorganisms responsible for the degradation of organic materials in soil, the degrading microorganisms should be detected without culture in laboratory media. Immunofluorescence staining techniques using probes targeting specific enzymes and ONAs have been developed for the detection (Carter and Lynch 1993). However, these methods enable to detect only the microorganisms with the same characteristics for the enzymes or the same sequences of ON As as those of the culturable isolates. All the microorganisms degrading a compound in soil are not always detected. Grouping of bacteria and fungi has been successfully carried out through selective inhibition by antibiotics (Mori et al. 1998). However, the selective inhibition method does not provide any information on the tax­onomic subsets of bacteria and fungi.

To characterize the microbial communities in soil, methods analyzing biomarkers have been developed, such as the analysis of phospholipid fatty acids and the various analyses of ONAs using polymerase chain reactions (Morgan and Winstanley 1997). In previous papers, we reported that the respiratory quinones were useful biomarkers for characterizing the soil microbial communities (Fujie et al. 1998a, b; Katayama et al. 1998; Uchida et al. 1999). Most of the microorganisms contain a major respiratory quinone although some minor quinones are also present (Collins and Jones 1981). Each quinone has been assigned to the corre­sponding microbial groups in soil (Fujie et al. 1998a; Iwasaki and Hiraishi 1998).

In this report, we developed a new method for detecting the microbial groups metaboliz­ing a substrate in soil, which is based on the analysis of the [I4C] respiratory quinones synthesized from a [14C] substrate through the metabolism of microorganisms in soil.

MATERIALS AND METHODS

The soils used were sampled from the plough layer of fields in Nagoya University Farm (a Palehumult) subjected to four different fertilizing practices: unfertilized soil (NF-soil), soil amended with chemical fertilizers (CF-soil, 370 kg-N ha- I y-I of nitrogen, 420 kg-P20 5

ha- I y-I of phosphorus, and 370 kg-K20 ha-I y-I of potassium), soil amended with chemical fertilizers and 40 t ha- I y-I of farmyard manure (CF + FYM-soil), and soil amended with 400 t ha- I y-I of farmyard manure (FYM-soil). The properties of the farmyard manure and soils were reported previously (Ghani Nugroho and Kuwatsuka 1990; Katayama et al. 1998).

The fresh soil samples were sieved through a 2-mm mesh sieve just after sampling and used. For the labeling of microbial biomass, 20 g of soil (on a dry weight basis) was placed in a 50 mL-volume beaker, and for the labeling of quinones, 1 g of soil (on a dry weight basis) was placed in a 10 mL-volume test tube. Moisture content of the soil was adjusted to pF l.8, and the moistened soil was pre-incubated at 30°C for 7 d in the dark. The preincubat­ed soils were spiked with a [I4C] substrate and the replicated soil samples were placed in a 1 L-volume of sealed container, and incubated at 30"C in the dark. The l4C02 was trapped by 10 mL of 10% N aOH solution placed in the container. After 6 or 8 h of incubation, the soil was frozen using liquid nitrogen to stop the reaction. The [I4C] substrates added included 19.8 to 182 nmol g-I dry soil of [U-I4C]glucose (8.2 GBq mmol- I, 98.9% radio­active purity, Amersham, Buckinghamshire), 15.2 nmol g-I dry soil of [U-I4C]glycine (3.52 GBq mmol- I, 99.2% radioactive purity, Amersham, Buckinghamshire), and 134 nmol g-I dry soil of [1,2- I4 C]acetic acid sodium salt (4.0 GBq mmol- I, 99.6% radioactive purity, Mora­veck, Brea, CA).

The amount of [l4C] microbial biomass carbon was measured by a fumigation-extrac­tion method (Brookes et al. 1985; Vance et al. 1987) in CF + FYM-soils after 8 h of spiking

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[l4e] Quinone Profile 671

with 19.2 nmol g-l dry soil of [U_14C] glucose. The radioactivity of the extracts was measured with a liquid scintillation counter (LSC-5100, Aloka, Tokyo) after mixing with a scintillation cocktail (Scintisol EX-H, Dojindo, Tokyo). The radioactivity of the soil residue after extraction was determined as 14C02 by combustion of the residue using a sample oxidizer (ASC-113, Aloka, Tokyo). Since the non-extractable radioactivity is consid­ered to have been incorporated into the microbial biomass (Sugai and Schimel 1993), the amount ofradioactive biomass carbon was calculated as the sum of the radioactivity of flush carbon and non extractable carbon. The amount of total microbial biomass carbon was also measured using a TOC analyzer (Shimadzu TOC-500, Shimadzu, Kyoto) in the soil sample spiked with the same amount of [U_14C] glucose.

The contents of [14C] quinones in soil were determined 6 h after spiking with a [14C]_ substrate by the modified method described in the previous paper (Fujie et al. 1998a). The soil was extracted with a solution of chloroform and methanol (2: I) and the extract was partitioned between n-hexane and saline water. The radioactivity of the saline water fraction was measured as the [14C] water soluble fraction. From the n-hexane fraction, the [14C]_ quinones were fractionated using Sep-Pak Plus Silicailll cartridges (Waters Chromatography Division, Milford, Massachusetts) and analyzed with a high performance liquid chromato­graph (HPLC) equipped with a reverse-phase column and a photodiode array detector (Fujie et al. 1998a). In the analysis ofHPLC, the [14C]quinones extracted from I g (on a dry weight basis) of soil were combined with non-labeled quinones from 19 g (on a dry weight basis) of soil treated in the same manner with non-labeled substrate. This procedure enabled to detect the quinones by using a photodiode array detector in the HPLC. Since the menaquinone with 8 isoprenoid units tetrahydrated (MK-8(H4)) and menaquinone with 9 isoprenoid units (MK-9) were not resolved well on the chromatogram, the two mena­quinones were measured as a mixture (MK-8(H4) + 9). The resolution between MK-9(H4) and MK-lO and between MK-7(H4) and MK-8 also became poor often, and these mena­quinones were measured as a mixture. Since the menaquinones with highly hydrogenated isoprenoid chains were not identified, they were determined as MK-others. The eluate of the HPLC was collected with a fraction collector (LKB Redifrac, Pharmacia, Tokyo). The radioactivity of each fraction was determined by the liquid scintillation counter. Recovery was examined with a measuring the radioactivity of 14C02, the [14C] water-soluble fraction, the soil residue and the residue in Sep-Pak Plus Silicailll cartridges. The radioactivity of the residue in Sep-Pak Plus Silicailll cartridges was eluted with diethyl ether and determined using the scintillation counter.

In an experiment to determine the labeling time of quinones, the extract was fractionat­ed into ubiquinones and·menaquinones by thin layer chromatography (TLC) (Silica Gel 70FM Plate-wako, Wako, Osaka). The silica-gel TLC was developed with n-hexane and diethyl ether (90: 10, v Iv) according to the method described by Hiraishi (Hiraishi et al. 1992). The radioactivity of the quinones was measured by liquid scintillation spectrometry after scraping off the spots.

The quinone profiles of the soils were analyzed based on a statistical index of dissimilar­ity. The dissimilarity index (D) was calculated by the equation (Hiraishi et al. 1991):

n

DU. j)=1/2~lpki-PkjIX100 k=l

where Pki and Pkj are the mol fractions of the k quinone species for the i and j samples, respectively. The unit of dissimilarity here was expressed as percentage. The fluctuations of the dissimilarity index of the [14C] quinone profiles were examined empirically. Using the

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672 K. SAITOU, K. FUJIE, and A. KATAYAMA

differences in duplicate determinations, the confidence intervals were calculated based on the normal distribution for the differences in dissimilarity.

RESULTS

Figure 1 shows the changes in the amounts of 14C02, the [14C] water-soluble fraction and the [14C] quinones in CF + FYM-soil after spiking with 19.8 nmol g~l dry soil of [U-14C] glucose and 15.2 nmol g~l dry soil of [U_14C] glycine. The [14C] water-soluble fraction, an indicator of [14C] glucose and [14C] glycine, decreased to less than the detection limit (0.1 nmol g~l dry soil) in 2 h. In the case of [14C] glucose, the radioactivity of the [14C] mena­quinones was saturated after 2 h and that of the [14C] ubiquinones after 6 h. The distribution of the radioactivity from [U-14C]glucose into the microbial biomass in soil accounted for 86.6±3.7% of the total radioactivity after 8 h of spiking and corresponded to 1.58 llg C g~l dry soil, comprising 0.7% of the total microbial biomass carbon (228 llg C g~l dry soil). In the case of [14C] glycine, the radioactivity of both [14C] menaquinones and [14C] ubi­quinones was saturated after 6 h. These results indicated that the [14C] quinones were synthesized in soil within 6 h.

By spiking CF + FYM-soi1 with 182 nmol g~l dry soil of [14C]glucose, the recovery of the radioactivity was examined. After 6 h, 9.3± l.7% of the radioactivity was distributed to 14C02, 2.3± l.8% to the [14C]water-soluble fraction, and 101.0±8.8% to the soil residue. About 0.1% of the radioactivity was distributed to respiratory quinones. With the radioactiv-

1.2 '6 rn Cl rn 0.8 o E c:

8' 0.4 u

v

[14Cj_glucose 0.8

[14Cj-glycine

0.6

0.4

0.2

2 4 6 8 0----~--~4--~6--~8

2 4 6 B 4 6 B

2 4 6 8

Incubation time (hours)

Fig. 1. Release of 14C02, the disap­pearanceof [ 14 C] water soluble fraction and the increase in the contents of [l4C]_ quinones in CF + FYM-soil after the addition of [U-14C] glucose (19.8 nmol g-l soil) and [U-l4C] glycine (15.2 nmol g-l soil). Vertical bars for the [14C]_ qui nones denote the standard deviation. The standard deviation of the [l4C]_ water soluble fraction was smaller than the size of the symbols. The standard deviation l4C02 was not measured be­cause l'C02 released from the replicated samples was trapped in one sealed bot­tle.

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[14C] Quinone Profile 673

ity of the residue in Sep-Pak Plus SilicalRi cartridges, the recovery of radioactivity from [U­I4C]glucose was 113±8%.

Figure 2 shows the chromatograms obtained to determine the amounts of total and radioactive quinones in soil spiked with [U _l4C] glucose. The radioactivity of each fraction was calculated by subtraction of the radioactivity at the baseline just before the peak. However, since the baseline of the radioactivity fluctuated, especially in the ubiquinone analysis the detection limit of the ubiquinone with 8 isoprenoid units (Q-8) reached 800 dpm. For the other quinones, the minimum detection level corresponded to a difference of 50 dpm between the baseline and the peak, which was equivalent to 0.l0 pmol of quinone in the labeling by [U-l4C]glucose, 0.23 pmol in that by [U-14C]glycine, and 0.22 pmol in that by [1,2-I4C]sodium acetate.

Table I shows the [l4C] quinones and total quinones in CF + FYM-soil spiked with [U-14C]glucose and [U-l4C] glycine. The [14C]quinone species synthesized from [U_14C]_ glycine were the same as those labeled by [U-l4C] glucose (Table 1). The dissimilarity between the two [I'C] quinone profiles amounted to 15.4%. The confidence interval (95%) in the dissimilarity of the [ l4C] quinone profiles from one soil was 20.5% in this study. Therefore, the two [ l4C] quinone profiles did not show any significant differences, suggesting

70

60

E 50 Co :s 40 i!' ~ 30

'" ~ 20

'" a:: 1 0

500

i400 :s i!'300 ·5

~ 200 o =g 100 a::

o

Ubiquinones

Menaquinones

MK·l0(HB)

I

40 50 60 70 Fraction number

Fig. 2. Chromatograms of respiratory quinones in CF + FYM-soil with radio­activity of quinone frac­tions. The soil was spiked with [U-14C] glucose. Each fraction consisted of I mL. Q stands for ubiquinones and MK for menaqui­nones, then follows a hy­phenation and the num­bers of isoprenoid units and of hydrogenated groups of the double bonds in the side chain.

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674 K. SAITOU, K. FUJIE, and A. KATAYAMA

Table 1. Respiratory quinones in CF + FYM-soil labeled with [U-l4C]glucose and [U-l4C] glycine.

Glucose" Glycineb

Quinones Total Labeled Incorporation Total Labeled Incorporation (nmol/ g-soil) (pmol/ g-soil) activityc (X 10-3 ) (nmol/ g-soil) (pmol/ g-soil) activityC (X 10-3

)

Q-8 0.08 NDd 0 0.09 ND 0 Q-9 0.03 ND 0 0.03 ND 0 Q-IO 0.08 0.12 1.5 0.08 0.24 3.0 Q-IO(H2) 0.02 ND 0 0.02 ND 0 MK-6 0.05 0.77 15 0.08 0.99 12 MK-7 0.09 0.14 1.6 0.21 0.38 1.8 MK-7(H2) ND ND 0 0.02 ND 0 MK-7(H.)+8 0.25 0.18 0.73 0.49 0.71 1.4 MK-8(H2) 0.11 0.10 0.92 0.22 Trace Trace MK-8(H.)+9 0.12 0.71 5.9 0.24 2.27 9.5 MK-9(H2) 0.02 0.18 9.1 0.05 0.52 10 MK-9(H.)+ 10 0.04 0.51 13 0.08 1.23 15 MK-IO(H2) 0.03 ND 0 0.08 ND 0 MK-IO(H.) 0.11 ND 0 0.20 ND 0 MK-IO(Ha) 0.01 ND 0 0.02 ND 0 MK-IO(H.) 0.01 ND 0 0.03 ND 0 MK-Il ND ND 0 ND ND 0 MK-others ND ND 0 ND ND 0

Total 1.05 2.72 2.57 1.94 6.44 3.24

"The soil was spiked with 19.8 nmol g-l dry soil [U- l4C] glucose. bThe soil was spiked with 15.2 nmol g-l dry soil [U-14C] glycine. cIncorporation activity= [l4C]-labeled amount of a quinone/Total amount of a quinone. dND: not detected. The minimum detection levels (MDL) were 0.10 pmol g-l soil for [U-14C]­glucose and 0.23 pmol g-l soil for [U- l4C] glycine, respectively. The MDL of quinones was 0.01 nmol g-l soil. Trace indicates that the radioactivity was detected but not determined because the level was lower than MDL.

that the same microbial groups may metabolize glucose and glycine. The [14C] quinone species labeled consisted mainly of menaquinones, especially MK-6, MK-8(H4)+9, and MK-9 (H4)+ 10. When the spiking amount of [U_14C] glucose was increased 9 times almost the same [14C] quinone profile was obtained except for the detection of low levels of [14C]_ MK-1O(H2) and [14C]MK-1O(H4)' The [1,2-14C] acetic acid did not yield [14C]quinones in the CF + FYM-soil in spite of the larger amount added compared with the other two compounds. The 14C02 production was negligible (0.013% of the initial amount). These results indicated that the acetate-metabolizing activity of microorganisms in the CF + FYM­soil was not appreciable.

Figure 3 shows the profiles of total and [14C]quinones obtained by spiking with [U-14C] glucose in the soils subjected to four different fertilizing practices. The [14C] quinone profiles were significantly different between any of two soils with a dissimilarity index ranging from 24% to 39%. The FYM- and CF + FYM-soils differed from the CF - and NF-soils by the detection of [14C]MK-1O(H4) and [14C]MK-others. In the FYM-soil, [14C]_ Q-9 was also detected, and [14C]Q-I0 accounted for a large proportion. The CF-soil differed from the other soils by the detection of [14C]Q_8 and a large fraction of [14C]MK-8(H2)' The [14C] quinone profiles of the individual soils were significantly different from the corre­sponding total quinone profiles with a dissimilarity index ranging from 45% to 48%. The high dissimilarity was caused by the high proportions of [14C] MK-6 and [14C] MK-8(H4) + 9 in all the soils, reflecting the high metabolic activities of the corresponding microbial

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Q-8 Q-9

Q-10 Q-10(H2)

MK-6 MK-7

gj MK-7(H2) 5 MK-7(H4) c MK-8 "g. MK-8(H2) '5 MK-8(H41+9 ;J MK-9(H2) "ll MK-9(H41+10 l'l. MK-10(H2) rn MK-10(H4)

MK-10(HsJ MK-10(H8i

MK-ll

[ l4C) Quinone Profile

A) Total quinones FYM-soil CF+FYM-soil CF-soil NF-soil

MK-Others _>=--::-'---::-=--::-: ""'-----'-_--'--_ "---':--:::-'::----=-' ~--::'---::-'::--=--0.1 0.2 0.3

FYM-soil Q-8 Q-9

Q-10 ..... Q-10(H2i

MK-6 MK-7

;J MK-7(H2i 5 MK-7(H4i r:: MK-8

~ MK-8(H2i •••• '0 MK-8{H4i+9 ;J MK-9(H2) "ll MK-9(H4)+ 10 l'l. MK-10(H2i rn MK-10(H4i

MK-10(Hs) MK-l0(Hs)

MK-11

Mole fractions of quinones

B) [14C]-labeled quinones CF+FYM-soil CF-soil NF-soil

MK-Others "-------'--_'--------.J L----,,-',-~,---,,-J o-----coc':---::-':---co-' o----,,-L----::-'::--,,-l o 0.1 0.2 0.30 0.1 0.2 0.30 0.1 0.2 0.30 0.1 0.2 0.3

Mole fractions of quinones

675

Fig. 3. Profiles of total quinones (A) and [l4C)-labeled qui nones (B) obtained by the addition of [U-l4C)­glucose in four soils subjected to different fertilizing practices: FYM­soil, CF + FYM-soil, CF-soil, and NF-soil. Details of fertilization practices are described in the text. The spiking amounts of [U-l4C]­glucose were 132 nmol g-l dry soil in FYM-soil, 88 nmol g-l dry soil in CF + FYM-soil and in CF-soil, and 66 nmol g-l dry soil in NF-soil. Abbreviations of quinone species are the same as those in Fig. 2.

groups. Total quinones were also significantly different between any of two soils (values of dissimilarity index> 14%) because the confidence intervals (95%) in dissimilarity of total quinones from one soil were 6.7% in this study. Therefore, both the glucose-metabolizing microorganisms and the whole structure of microbial communities were significantly different between the soils subjected to different fertilizing practices.

DISCUSSION

The analysis of [HC] respiratory quinones synthesized in the soils spiked with a [14C]_ substrate allowed the detection of the microbial groups metabolizing the substrate in soil. Most of the microorganisms contain one species of quinone as their major quinones and some minor quinones (Collins and Jones 1981), and the major quinone species do not change with physiological conditions with some exceptions such as in the cases of phototro­phic oxygen non producers (Imhoff 1984), Enterobacteriaceae (Collins and Jones 1981), and Shewanella (Akagawa-Matsushita et al. 1992). Since the populations of these microorgan­isms are small in soil (Akimov 1996), the isotopic labeling of respiratory quinones was considered to be suitable for the detection of microbial groups metabolizing a substrate in soil. However, the microbial resolution was limited to the microbial groups containing the

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676 K. SAITOU, K. FUJIE, and A. KAT A Y AMA

same species of quinone. Fungi contain mainly Q-9 or Q-lO(H2) (Fujie et al. 1998a). Other quinones are indicators of bacteria (Fujie et al. 1998a; Iwasaki and Hiraishi 1998). The [l4C] quinone profile, therefore, may enable to detect the changes in the bacterial groups with a higher sensitivity than those in fungal groups.

The method was characterized by a high sensitivity and enabled to detect 0.1 pmol g-l dry soil of [14C] quinones by using a [l4C]substrate with a high specific radioactivity. The detection limit of [l4C] quinones corresponded to about 10-4 (one to ten thousandth) of total quinones in arable soils. Since the total amounts of quinones had a linear relation with the microbial biomass in soil (Saitou et al. 1999), this detection limit may correspond to about 10-4 of soil microbial biomass. Other methods can be applied to detect the micro­organisms metabolizing a substrate in soil without culture in laboratory media: detection of specific mRNA and selective inhibition by antibiotics. The minimum detection levels of specific mRNA using [32P]-labeled anti-sense RNA were reported to be 106 cfu g-l dry soil (Fleming et al. 1993; Jeffrey et al. 1994; Hoenerlage et al. 1995), which corresponded to about 10-2 of total colony-forming units of soil. Increase of the sensitivity using the reverse­transcriptase-dependent polymerase chain reaction (RT -PCR) has enabled to detect mRNAs of ribulose-l,5-bisphosphate carboxylase in aquatic microorganisms (Xu and Tabita 1996) and peroxidases of a white-rot Basidiomycete, Phanerocheate chrysosporium, in autoclaved soils (Bogan et al. 1996a, b). However, the detection of mRN A of microorganisms in unsterilized soils using RT -PCR has not been reported. Partial inhibition technique using antibiotics was also used to determine the contribution of bacteria and fungi to the degradation of a substrate in soil (Mori et al. 1998). However, this technique did not allow to further resolve taxonomic groups of microorganisms in soil.

The labeling time with [U-14C]glucose and [U_14C] glycine was only 6 h. The average doubling time of microorganisms in soil has been estimated at about 9.6 d (Domsch et al. 1978). Therefore, the [14C] quinones are considered to be synthesized directly from the [14C] substrate in the microorganisms but are not produced through indirect synthesis utilizing dead microorganisms that had originally synthesized [l4C] quinones from the [l4C] substrate. It is suggested that the [14C]quinones were detected in the microorganisms which synthesized quinones within 6 h in quick response to the [l4C] substrates or which were characterized by an inherent active metabolism under the experimental conditions. The high incorporation rates of radioactivity from [l4C] substrate into quinones is considered to reflect the high activity of the corresponding microbial groups. In the CF + FYM-soil spiked with [U-14C]glucose and [U-l4C] glycine, MK-8(H4)+9, MK-9(H2)' and MK-9(H4)+10 showed high incorporation rates, which corresponded to Actinobacteria (Yokota 1998) metabolizing the substrates with high activities. On the other hand, [14C] quinones were not detected and 14C02 production was negligible in the CF + FYM-soil by spiking of [1,2_14C]­acetate, indicating that the microbial groups metabolizing acetate were not active or showed lower population levels than the detection limit. Incorporation of labeled acetate into lipids had been observed after 6 to 24 h in soil (Arao 1997) and sediments (Phelps et al. 1994). The longer labeling time may lead to the growth of microorganisms and the incorporation of radioactivity.

The [l4C] quinone profiles from [U_14C] glucose enabled to fingerprint the microbial groups metabolizing glucose in the four soils subjected to different fertilizing practices. The [l4C] quinone profiles of the FYM- and CF + FYM-soils differed from those of the CF- and NF-soils by the presence of [14C]-labeled MK-1O(H4) and MK-others (highly hydrogenated menaquinones), which indicated the presence of Actinomycineae, Micromonosporineae,

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[l4C] Quinone Profile 677

Pseudonocardineae, Streptomycineae, and/or Streptosporangineae in the Class Actinobac­teria (Yokota 1998). The FYM-soil also contained [14C] Q-9, which indicates the presence of the gamma subdivision of Proteobacteria and fungi (Fujie et al. 1998a; Iwasaki and Hiraishi 1998). CF-soil was characterized by [ l4C]Q-8. The microorganisms containing Q-8 were classified into the beta subdivision of Proteobacteria and some into the gamma subdivision (Hiraishi 1996). The common characteristic of the [14C] quinone profiles in all the soils was the large proportion of [l4C]MK-6 and [14C]MK-8(H4 )+9, compared to the total quinone profile. The presence of MK-6 may indicate the presence of microorganisms belonging to Cytophaga-Flavobacterium cluster, the delta and epsilon subdivisions of Proteobacteria and/or Gram positive bacteria with a low G+C content (Iwasaki and Hiraishi 1998). The presence of MK-8(H4) and MK-9 would indicate Corynebacterineae, Micrococcineae, Streptosporangineae, Pseudonocardineae, and/ or Propionibacterineae of the Class Actinobacteria (Yokota 1998). Further studies are required to determine how the microorganisms metabolizing a substrate differ between the soils.

Except for the FYM-soi1, the Q-9 and Q-1O(H2) which are mainly present in fungi (Fujie et al. 1998a) were not labeled under the conditions tested. Law et al. (1971) reported the incorporation of p-hydroxy-[U-14C] benzoic acid into the ubiquinones of Phycomyces during 4 h of incubation of a pure culture. Therefore, it is considered that fungi did not contribute to the degradation of glucose and glycine under such soil conditions.

Thus, a new method to detect the microorganisms metabolizing a single substrate was developed based on the analysis of [ l4C] qui nones in soil. This method enabled to finger­print the microorganisms metabolizing a compound in soil without the bias associated with culture in laboratory media. Although only three substrates, glucose, glycine, and acetate, were examined in this study, the method could be applied to other substrates. It should be noted that some compounds may not act as substrates but as indicators of the rate of biosynthesis of isoprenoid quinones in microorganisms, for example the intermediates in the pathway of isoprenoid biosynthesis such as 4-hydroxybenzoate (Law et al. 1971).

Acknowledgments. We thank Professor M. Kimura and Dr. K. Toyota of Nagoya University for their valuable suggestions. We are also indebted to Professor S. Yoshida, Nagoya University Farm, for his assistance in soil sampling. This study was supported by Nippon Life Insurance Foundation and a Grant­in-Aid for Scientific Research from Ministry of Education, Science, Sports and Culture.

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