Soil Sci. Plant Nutr., 29 (4), 48~97, 1983

TIC REDUCTION IN SUBMERGED SOILS

Masanori OKAZAKI, Eiichi HIRATA, and Kiyoshi TENSHO·

Faculty of Agricultur~. Tokyo Univ~rsity of Agriculture and T~chnoloKY. Tokyo. /83 Japan

• Radioisoto~ and Nucl~ar Engin~erinK School. Japan Atomic Energy R~search Institute. Tokyo. 113 Japan

Received March 22. 1983

The reduction of TIC (2.3.S-triphenyltetrazolium chloride) to TPF (triphenylformazan) in submerged soils was investigated. In terms of nmol TPF per g of dry soil per 30 min, the amounts of TIC reduction in soils after 1 to 28 day incubation under submerged condi­tions at 30°C in the dark were 0-749 for the Nagano sample and 0-216 for the Nyuzen sam­ple, indicating an increase with the time of incubation. {-Ray irradiation with cobalt 60 at a dose of 3 Mrad which almost completely sterilized soil microorganisms decreased TPF formation in the Nagano and Nyuzen samples by 62 and S7%, respectively. After {-ray irradiation a linear correlation between the assay time and the amount of TPF was observed. The rate of enzymatic TPF formation in the Nagano sample after ,,-ray irradiation was the same as that of the Nyuzen sample. The estimated percentages of TIC reduction in sub­merged soils were 68-76% for dehydrogenase activities and 24-32% for highly reducing substances. K~y Words: TIC reduction, ,,-ray irradiation, dehydrogenase activity, highly reducing substances.

The reduction of TIC (2,3,5-triphenyltetrazolium chloride) to TPF (triphenyl­formazan) has been used for the determination of dehydrogenase activities in soils (4, 7, 8, 10, Jl, /5, 20, 23, 27). STEVENSON (27) studied dehydrogenase activities as a reliable index of microbial activities. He found positive correlations between de­hydrogenase activities and respiration of soil. Ross and ROBERTS (22), however, failed to observe a relationship between dehydrogenase activities and oxygen uptake. MOORE and RUSSELL (15) concluded that dehydrogenase activity was not an appro­priate index of soil fertility. Despite the lack of useful correlation between dehydro­genase activities and soil fertility, dehydrogenase activities have been considered to reflect the level of biological activities in soil, because dehydrogenase activities involve many different enzymes transferring hydrogen or electron from substrates to acceptors. Some factors that influence dehydrogenase activities in soil have been studied by many researchers (1,9, /5,20). CASIDA et 01. (7) developed a method for the determination of dehydrogenase activities and observed that the dehydrogenase activities in soil with added nutrients paralleled only the rapid multiplication of gram-positive bacteria.

Generally, the enzyme activities in soil are estimated by determining the amount 489

490 M. OKAZAKI, E. HIRATA, and K. TENSHO

of endproducts or the elimination of substrates added to soil samples after treatment with bacteriostatic compounds or )I-rays. Since abiogenetic dehydrogenases are un­likely to be present in soil (24), as far as dehydrogenase assay is concerned, both toluene addition and )I-ray irradiation have not been adopted to suppress microbial activities, although Ross (23) showed that toluene significantly decreased dehydrogenase activities by 44 to 66% under aerobic conditions.

MACLAREN et al. (/3, /4) studied the sterilization of soil by )I-ray irradiation. They indicated that the number of surviving organisms approached zero at a dose of 2 Mrep and that survival curves for facultative anaerobes and microaerophiles were similar to those for aerobic organisms. Phosphatase and urease activities, however, could still be detected in soils sterilized by )I-ray irradiation. The sterilization of soil by )I-ray irradiation may have mild and often negligible effects on the biochemical properties of soil as compared with toluene (25).

Meanwhile, TIC is one of the redox indicators, with a redox potential of -0.081 V. Accordingly, succinate dehydrogenase and glutamate dehydrogenase, of which the redox potentials are -0.00 and -0.030 V, are unable to reduce TIC. However, substances which have lower redox potential than -0.081 V would enable TIC reduc­tion in an assay system based on non-biochemical methods. W ADA et al. (30) used the activities of chemical reduction of TIC for the estimation of easily decomposable organic matter in paddy soils.

We studied TIC reduction in submerged soils and evaluated the contribution of dehydrogenase activities and highly reducing substances in submerged soils to TIC reduction.

MATERIALS AND METHOD

Soil samples. Soil samples used were taken from the surface horizon of paddy soils. They consisted of a gray brown clayey paddy soil (Eutric Fluvisol, Nagano soil) collected at Nagano City, Nagano Prefecture, Japan, and an excessively eluviated sandy paddy soil (Dystric Fluvisol, Nyuzen soil) collected at Nyuzen-Cho, Toyama Prefecture, Japan. Soil samples were air-dried and passed through a 2 mm sieve.

Incubation method. Approximately 30 ml of distilled water was added to 5 g of an air-dried soil sample in a SO ml centrifuge tube. The contents were thoroughly mixed and the tube was filled up with additional distilled water. Immediately after the centrifuge tube was stoppered with a double gum cap equipped with a needle, the needle was removed so that no air space remained at the top of the centrifuge tube (/7). Soil samples were incubated under submerged conditions at 30°C in the dark for I to 28 days.

Measurement of Eh. Eh was measured with a Hitachi-Horiba M-7 pH meter. Platinum electrodes were inserted in the soil samples before submerged incubation was performed and a saturated calomel cell was used as a reference electrode accord­ing to Y AMAIIo'E and SA TO (3/).

TIC Reduction by Submerged Soils 491

t--U04JDII lum cap

Water

Fig. 1. Method of incubation under submerged conditions and injection of TIC-tris buffer solution.

Determination of organic carbon in soil solution. Organic carbon in the soil solu­ion of incubated samples was determined by the wet combustion method of MENZEL and VACCARO (16), using the infrared carbon dioxide absorption technique.

Determination of Fe( 1/). After incubation, Fe(U) in the soil samples was ex­tracted with 0.6% aluminum chloride solution. The amount of Fe(lI) extracted was determined colorimetrically by the o-phenanthroline method.

Determination of TPF. Upon the completion of incubation,S ml of soil solution in the centrifuge tube was removed with a syringe, and 5 m1 of 0.2% TIC-O.5 M tris (hydroxy methyl) amino methane buffer solution (pH 7.6) was added conversely using a syringe (Fig. I). The centrifuge tube was shaken and allowed to stand for 3~120 min at 30°C in the dark. Thereafter, TPF produced was extracted with acetone and measured spectrophotometrically at a wave length of 485 nm according to the method of W ADA et al. (30). The blank sample consisted of a mixture of acetone and 0.5 M

tris buffer solution without TIC. 'I-Ray irradiation. Soil samples in the 50 ml centrifuge tube after 14 and 28 days

incubation under submerged conditions were irradiated with 3.0 Mrad dose of ,,-rays at a rate of 0.15 Mrad per hour using an irradiator, AECL gamma cell 200.

Bacterial counts. Mter the submerged soil samples were incubated for 14 days, counts of total bacteria with or without 'I-ray irradiation were performed by the dilu­tion plate method using an albumin agar culture. The colonies were allowed to grow for three days at 30°C in the dark. All colonies grown on the medium were counted (29).

RESULTS AND DISCUSSION

Chemical properties of soil sample.f Some of the chemical properties of the soil samples are briefly presented in Table

492 M. OKAZAKI. E. HIRATA. and K. TENSHO

1. Free iron and organic carbon contents were slightly higher in the Nagano sample than in the Nyuzen sample, while the C/Fe ratio was smaller in the Nagano sample than in the Nyuzen sample. This may depend on the difference of native free iron content and the eluviation of iron on account of the utilization of the soils for paddy cultivation (18).

Dehydrogenase activities are thought to be related to the soil organic matter content. Nevertheless, the relation between dehydrogenase activities and soil organic matter content is not clear, and varies with the season (22). As the dehydrogenase activities are considered to reflect the total range of oxidative and reductive activities of soil microorganisms, the changes in the compounds responsible for the redox reac­tion in submerged soils were determined.

The changes in Eh and Fe(II) in soil and of organic carbon in the soil solution after incubation under submerged conditions are shown in Fig. 2. The decrease of the Eh values was closely related with the increase of Fe(U) in the submerged soils. The contents of Fe(ll) produced during the incubation ranged from 2 to 459 mg Fe per 100 g of dry soil in the Nagano sample and 9 to 57 mg Fe per 100 g of dry soil in the Nyuzen sample. Throughout the incubation period these values were higher in the former sample than in the latter one, which is compatible with their free iron con­tents. As microbial activities in submerged soil were stimulated by the addition of free iron (3), the microbial activities in the Nagano sample would be higher than in the Nyuzen sample. BREMNER and TABATABAI (4) observed that the addition of Fe20 a

Table 1. Chemical properties of soil samples. ~-- - -~-----

Soil Free iron- Org-C T-N C/N C/Fe b

Name FAO/UNESCO (%) (%) (%) ----------------------~- ------ ----------

Nagano Eutric Fluvisol 1. 14 1. 87

Nyuzen Dystric Fluvisol 0.30 1.34

_ ASoUn and KUMADA (1). b Org-C/Free iron.

lOO

~ 500

-b 400

~ lOO

~200

't 'OO

041~l~7~~1~4::=2~,==~28 01 3 7 14 21 28

Incubation time in days Incubation time in days

o. 18 10.4 1.6

o. 12 11. 2 4. S

01 3 7 14 21 28

Incubation time in days

Fig. 2. Olanges in Eh, and Fe(lI) in soil samples and organic carbon in soil solution after incu­bation under submerged conditions. •• Nagano soil; O. Nyuzen soil. Eh is indicated at pH 7; Eh value of 0.06 V corresponds to pH one.

TIC Reduction by Submerged Soils 493

and MnO. stimulated dehydrogenase activity. Figure 2 also shows that the concen­tration of organic carbon in the soil solution was higher in the Nagano sample than in the Nyuzen sample. The decrease of organic carbon in the soil solution at the later stage of incubation may be caused by the consumption by microorganisms as a carbon source. These results corresponded with the data presented in the previous papers (18, 19).

TTC reduction in submerged soils The changes of TIC reduction in the Nagano and Nyuzen samples after incu­

bation under submerged conditions are shown in Fig. 3. In both the Nagano and Nyuzen samples the amounts of TPF produced increased with the incubation period except for the later stage of incubation in the Nagano sample. In all cases the amounts of TPF produced was much larger in the Nagano sample than in the Nyuzen sample. The largest amount of TPF produced was 749 nmol TPF per g of dry soil per 30 min, in the case of the Nagano sample after 21 day incubation. This value was about 12~~ that of the total amount of TIC added. Meanwhile, the maximum value in the Nyuzen sample was 216 nmol TPF per g dry soil per 30 min. These maximum values were considerably higher than the values ranging from 12.4 to 23.9 nmol TPF per g of dry soil per 30 min reported by CASIDA et aJ. (7) and those ranging from 104 to 216 nmol TPF per g of dry soil per 30 min reported by Ross (23). The total amount of TPF produced in the Nagano sample was about 3 times as high as that of the Nyuzen sample.

There were no remarkable changes in the Fe(II) concentration in the submerged soils following the injection of TIC-tris buffer solution. This finding indicates that TIC reduction in submerged soils did not depend on the oxydation of Fe(II). TIC reduction in this study would be due to dehydrogenase activities and to non-enzymatic

800

Incubation time in days

Fig. 3. Changes in TPF produced in soil samples incubated under submerged conditions. Five I of soil samples were assayed with S ml of O.2~/~ TIC-tris butTer in the dark for 30 min. ., Nagano soil; O. Nyuzen soil.

494 M. OKAZAKI, E. HIRATA, and K. TENSHO

substances such as highly reducing substances which could function as electron ac­ceptors (12). Preliminary experiments with these samples showed that TIC reduction could not be detected in the submerged soils after autoclaving, resulting in the inac­tivation of dehydrogenases and in the destruction of highly reducing substances.

ITC reduction in submerged soils sterilized by y-ray irradiation The sterilization of submerged soils by y-ray irradiation was adopted to exclude

the influence of microorganisms on 30 min' assay, since y-ray irradiation exerts milder effects on the biochemical properties of soils than other bacteriostatic or sterilizing agents (6, 13,21,26, 28). The y-ray irradiation of dose 3 Mrad at a rate of 0.15 Mrad per hour resulted in sterilizing almost completely microorganisms in the submerged soil, as shown in Table 2. In the case of the Nyuzen sample, after sterilization by y-ray irradiation the average number of colonies growing on the albumin agar culture for the triplicate petri dishes was 0.7 colonies per g of dry soil, while the values rang­ing from 4.8 x 101 to 6.0 x 105 colonies per g of dry soil were obtained in the non­sterilized sample, thus supporting the findings of BOWEN and CA WSE (5).

The results of TIC reduction in the soil samples exposed to a y-ray dose of 3 Mrad after 28 day incubation under submerged conditions are shown in Fig. 4. y­

Ray irradiation decreased considerably TIC reduction by 62 and 57% for the Nagano and Nyuzen samples, respectively. This decrease in TIC reduction was due to the elimination of the activity of soil microorganisms. Therefore, it is assumed that TIC reduction in the submerged soils immediately after irradiation with y-rays was caused by the activity of dehydrogenases derived from irradiated microorganisms and by highly reducing substances (12, 19). Further, it is evident from Fig. 4 that a linear relationship was observed between the assay time and the amount of TPF produced, with typical regression curves. This clear relation, compared with the findings of Ross (23), suggests that TIC reduction after y-ray irradiation is not mediated by microbial growth, but by dehydrogenase activities. After irradiation the rate of en­zymatic TIC reduction in the Nagano sample was very similar to that in the Nyuzen sample. These rates of dehydrogenase activities were 1.17 and 1.37 nmol TPF per g dry soil per min for the Nagano and for the Nyuzen samples, respectively, which showed smaller values as compared with the findings of Ross and ROBERTS (22) and of Ross (23). In this study TTC reduction based on the rapid reduction by highly reducing substances was estimated at zero time of assay, although highly reducing substances might show different levels of reducing ability.

Table 2. Number or colonies grown on the albumin agar after 3-day culture in Nyuzen soil. --~ --~-----------

Treatment

y-Irradiation Non-irradiation A

B

Number or colonies

O. 7 4. 8x loa 6. Ox loa

-----~---~-----

A, 10' dilution; B, 10' dilution. Each value represents an average or triplicate samples.

TIC Reduction by Submerged Soils 49'

400

o~----~----~--------~ o 30 60 120

ASsay time(min)

Fig. 4. TIC reduction in soil samples irradiated with y-rays after 28-day incubation under sub­merged conditions. Five g of soil sample was assayed with S ml of 0.2% TIC-tris buffer in the dark .• 0 Nagano soil Y=1.l7X+207; 0, Nyuzen soil Y=1.37X-rS1.7. A and B show the amount of non-enzymatic TPF formatioll.

Table J. Contribution of dehydrogenase activities and highly reducing substances to TPF formation (nmol TPF per g dry soil per 30 min).

Dehydrogenase activities

Soil Without Highly Total With reducing substances

microorganisms microorganisms

Nagano 403 (62) 36 ( 6) 207 (32) 646 (100)

Nyuzen 123 (.57) 41 (19) .52 (24) 216 (100) -~------ ----.-~------

Parentheses represent %.

According to the regression curves, the amounts of TPF at zero time, which are represented as A and D in Fig. 4, were 207 and 51.7 nmol TPF per g of dry soil for the Nagano and for the Nyuzen samples, respectively. These values are equivalent to 32 and 24~1o of TPF produced in the assay for 30 min (Table 3), which may be due to the presence of highly reducing substances such as aldehydes (19) formed in soil incubated under submerged conditions. Therefore, it is possible to consider that dehydrogenase activities in submerged soils have been overestimated, because of TPF formation by highly reducing substances.

With regard to the reducing capacity of submerged soil, OKAZAKI et 01. (19) showed that the iron-reducing capacity of leachate which depends on the amount of reducing organic substances was obviously higher in the Nyuzen sample than in the Nagano sample. The amount of highly reducing substances responsible for TIC reduction in the present experiment, however, was apparently larger in the Nagano

496 M. OKAZAKI, E. HIRATA, and K. TENSHO

sample than in the Nyuzen sample. The discrepancy in findings may be ascribed to the difference in redox potentials between Fe(III) and TTC as reductive reagents and the occurrence of highly reducing substances with different reducing abilities.

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TIC Reduction by Submerged Soils 497

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