003X-0717’81.060455-06102.00,‘0 Pergamon Press Ltd
CHANGES IN MICROBIAL BIOMASS AND ACTIVITY IN SOILS AMENDED WITH PHENOLIC ACIDS G. P. SPARLING, B. G. ORD and D. VAUGHAN Departments of Microbiology and Soil Organic Chemistry. The Macaulay Institute for Soil Research. Craigiebuckler, Aberdeen AB9 205, Scotland (Accepted 28 April 1981) Summary-
The phenolic acids p-hydroxybenzoic. ferulic. caffeic and vanillic acid. were added to soil of the Countesswells series that had been fallow or carried crops of potatoes, peas or barley for two consecutive years. Changes in phenolic acid concentration, the soil biomass, the respiration rate. and soil amylase activity were measured over 28 days. All the phenolic acids were sorbed by the soils which was generally in the order caffeic > feruhc = vanillic > hydroxybenzoic acid. The phenolic acids stimulated soil respiration and increased the biomass as determined by the substrate-induced respiration method. but the fumigation method of biomass assessment gave anomalous results. The soil amylase activity was initially increased by phenolic acid amendments but soon decreased. and after 7 days was less than in non-amended soil although activity had increased again after 28 days. The rates of respiration and the total phenolic acid concentrations were similar to unamended controls after 28 days. The immediate respiration response. measured IL6 h after amendment. Indicated that caffeic acid gave the largest initial response of the phenolic tested. this being 55572”” of that given by glucose. Soil from the potato plot showed the highest immediate response to the phenolic acid amendments measured as a proportion of the respiration response to glucose. The findings suggest that some crops stimulate the growth of phenolic-acid degrading organisms.
INTRODUCTION
MATERIALS AND METHODS Soils and amendments
Soil phenolic acids such as ferulic, p-coumaric, vanillit. p-hydroxybenzoic and syringic acids comprise less than 0.1”” of the total organic matter in soil but they are widespread and both toxic or stimulatory effects on plant and microbial growth have been reported (Whitehead, 1964; Knosel. 1959; Guenzi and McCalla, 1966; Wang rr ul., 1967; Anderson and Domsch, 1974: Glass, 1976; McClure er u/., 1978). Microbial growth in soil is usually limited by low concentrations of available organic substrates and many organisms spend much of their life cycle in resting stages with low metabolic activity (Gray, 1976; Gray and Williams, 1971). The phenolic acids occur as the free acid in the soil solution and hence should be readily available for microbial degradation (Wang ~lr (II., 1967). Although the transformation of phenolic acids to some humic substances has been followed (Martin and Haider. 1979a.b). there is little information on the reaction of the whole soil biomass to phenolic acid additions or the effect of the previous crop on the rate of phenolic acid decomposition. We report the rate of phenolic acid decomposition in soil previously fallow or cropped with potatoes, barley or peas. The soil biomass was estimated, and the respiration and amylase activity also measured because previous studies in unamended soils (Jenkinson and Powlson, 1976; Domsch er al., 1978; Sparling. 1981) had shown these to be reasonable indicators of the biomass metabolism.
0 The Macaulay
Institute
for Soil Research.
The soils (Countesswells series) which were from experimental plots within the grounds of the Macaulay Institute for Soil Research, have been described by Glentworth and Muir (1963). After crop harvest the upper horizons (@200 mm) were collected from plots that had grown potatoes, peas or barley for two consecutive growing seasons or from fallow plots kept free of all plants. The plots differed only slightly in their major characteristics: the soil pH range was between 5.3c5.34, and the percentage loss on ignition between 11.&l 1.2%. Acetic acid (2.5%) extractable between 68-73 PgK g- i, nutrients ranged 14.G14.6 PgP g- ’ and 394-455 FgCa g- i. The soil from five replicate plots of each of the cropping treatments was bulked, mixed, sieved ( < 2 mm) and stored moist (2628% w/w) at 5°C for a maximum of 4 months. :\n amendment rate of 5 mg glucose g-i soil was found to be adequate for substrate saturation and maximum initial respiration response (Anderson and Domsch. 1978). This rate was therefore used for all substrate additions. The phenolic acid or glucose was added to the soils as a finely-ground powder and thoroughly dispersed by vigorously shaking by hand for 10min. Soil from the barley, pea, potato or fallow plots was amended with glucose or one of the four phenolic acidsferulic. p-hydroxybenzoic, vanillic or caffeic acids. The latter acid, although not normally detected in soil, is chemically similar and was included for comparison. The soils were incubated at 22°C in IOO-
1980 455
456
G. P.
sely-stoppered 2 I conical flasks and subsamples taken for analyses.
SPARLING
were
Phenolic acids in the amended soils were extracted by shaking the soil with alkali (Wang et al., 1967) except that the NaOH was replaced by CaO. The presence of CaO produces an alkali solution of pH I1 5 and reduces variation in extraction efficiency caused by pH differences between the soils. The high pH also reduces sorption of phenolic acids by clays and the use of CaO rather than NaOH reduced the amount of brown humic material in the extracts. Yields of phenolic acids are increased by 334 times compared to extraction with water alone. After extraction of 5 g soil with I g CaO and 15 ml water. the samples were filtered and brought to pH 3.5 with HCI. then extracted twice with IOml ethyl acetate. The ethyl acetate solutions containing the phenolic acids were pooled and evaporated to dryness. The solids were taken up in IOml of a solution of methanol and water (I : I v/v). the pH of which was adjusted to 1.5 with HCI and the absorbance at 280nm recorded on a spectrophotometer (Unicam SP1800). The U.V. spectra of the soils amended with phenolic acids were compared with solutions of the pure phenolic acids. As a further check the phenolic acids were absorbed onto polyvinyl pyrrolidone (PVP) and the absorbance of the resulting solution was measured again at 280nm after removing the PVP by centrifugation. The measurement of total phenolic acid concentration by U.V. spectroscopy in conjunction with PVP absorption was found to agree well with total phenolic acid concentrations determined by GLC: the U.V. method was therefore adopted as a routine convenient technique for measuring total phenolic concentrations. Respiration rates at 22% were determined immediately after amendment (&6 h) and again after incubation for 1. 3. 7, 14 and 28days. Estimations of the biomass and amylase activity were also made at the same sampling times. The results presented for the respiration measurements are the means of two separate experiments made within 6 weeks of one another. The methods of measuring respiration, biomass and amylase activities were described by Sparling (1981) and Sparling et (11. (1981). In brief. respiration was measured by the rate at which CO* accumulated in the head-space of a sealed flask containing the soil sample. The head-space gas was sampled by syringe riLI a serum stopper and the COZ concentration determined by GC. The soil biomass was measured by the CHCI, fumigation method of Jenkinson and Powlson (1976) and the amount of CO2 released after fumigation measured by GC. The biomass was also estimated in soil immediately after amendment with glucose (5 mgg- ‘), using the method of Anderson and Domsch (1978). In order to differentiate between the CO2 normally respired by the soil and that evolved during the Anderson and Domsch biomass assay. the former has been designated “basal respiration”. Amylase activities were calculated from the amount of reducing sugars formed when soil was shaken with an aqueous solution of soluble starch (2”; w/v) in the presence of NaN, (O.l’?:, w/v) at 35-C for I8 h. After centrifugation. reducing sugar concentrations in the
rf ul.
supernatant were analysed with a Technicon AutoAnalyser using the alkaline ferricyanide method of Hoffman (1937).
RESULTS
Phrnolic
AND DISCUSSION
acids
Only a small fraction (2.625”,,) of the phenolic acids was recovered in the extracting medium at the onset. i.e. day 0 (Fig. I). Extraction efficiency varied slightly according to the crop grown on the soil. but in general was in the order caffeic < ferulic < vanillit < p-hydroxybenzoic. Huang et rrl. (1977) reported sorption of phenolic acids by the clay minerals and hydroxy-aluminium or iron compounds from Taiwan soils. but in that case the acids were sorbed in the ferulic > syrorder p-hydroxybenzoic > coumaric ingic > vanillic acid. The concentrations of the phenolic acids, by the extraction method used by us, related well to their apparent availability as substrates for microbial growth and respiration. When the concentration of the acids declined after 3days (Fig. I) this corresponded to a decline in the rate of respiration (Fig. 2). In general. the more readily extractable acids such as p-hydroxybenzoic acid. caused greater increases in respiration. Similarly. both respiration and extractable acid concentrations had declined to a level similar to those of the unamended controls after 28 days incubation, The addition of glucose made no detectable difference to the phenolic acid concentration in the soils. Basal
respiration
All the amendments increased the rate of respiration. and in some cases this was still apparent after 28 days (Fig. 2). The immediate response of the biomass to substrate addition can be taken as a measure of the prior adaptation of the biomass for that particular substrate before selective growth has occurred. In all cases glucose gave the largest immediate respiration response, as expected for this generally nonspecific substrate. Of the phenolic acids tested, caffeic acid always caused the largest initial increase in respiration with 55-729; of the response given by glucose, whereas p-hydroxybenzoic acid gave the lowest immediate respiration response except in the potato plot soil. Expressed as a percentage of the respiration response given by glucose. the potato plot soil consistently gave higher immediate responses to phenolic acid amendments than any of the other soils (Table I). The highest rates of respiration were recorded after l-3 days in the glucose-amended soils. and generally after 3 or 7 days in the phenolic-acid-amended soils (Fig. 2). There were differences between the plots, the potato plot soil showing a much smaller respiration response to hydroxybenzoic acid on day 3 than the other three soils. and the response to ferulic acid was much less on the fallow soil than on the other cropping treatments. In all cases the rate of respiration had declined markedly by 14 days and by 28 days was only slightly greater than in unamended soil. Increased soil respiration following amendment with p-hydroxybenzoic acid was also noted by Anderson and Domsch (1974) who attributed much of the increase to the selective growth of fungi.
Changes
in microbial
biomass
and activity
in soils amended
with phenolic
acids
457
Table 1. The effect of phenolic acid amendment on the rate of respiration of soils of different cropping history, expressed as a proportion of the respiration increase following glucose amendment. The rates of respiration were measured t&6 h after amendment (5 mg g- ’ soil) and for each crop treatment the rate of respiration of the glucose amended soil was taken as maximum i.e. lOtIS; Crop Amendment Control (nil) Caffeic acid Ferulic acid p-Hydroxybenzoic Vanillic acid
acid
Fallow
Barley
Peas
14 67 40 25 30
13 55 35 25 27
18 55 40 39 43
Amduse The soil amendments altered the amylase activity with the fallow plot behaving differently from the three cropping treatments (Fig. 3). In all soils amended with phenolic acids there was an initial increase in activity followed by a decrease. The decreases were greatest at 3, 7 and 14days and were particularly marked in the pea and barley soils. The continuous reduction in amylase activity in soils
Potatoes 22 72 43 53 45
amended with glucose may have been caused by catabolite repression, but the reason for the decreased activity after 3 days in the phenolic acid-amended soils is not clear. This repression by phenolic acids did not occur on the fallow soil. The rapid fluctuations in amylase activities suggest a high rate of turnover and indicate that the bulk of the activity was from endogenous cellular amylase rather than accumulated extracellular soil enzyme. The amylase activity at
I .4
im
1.2
F -
1.0
ii 0 u
0.6
n G Q
0.6
u 2
0.4
5 I a
0.2
1.4
iD
1.2
i-l
r -
I.0
Y s
POTATO
0.6
n c Q
0.6
u
i
is
0.4
w aI
0.2
L
01
3
7
DAYS
DAYS
soil) of extractable phenolic acids in Countesswells soil from four Fig. 1. The concentration (pgg-’ at 22’C, and sampled at cropping treatments. amended with phenolic acids (5 mg g- ’ soil). incubated various times up to 28 days. Control and glucose-amended soil, -0; p-hydroxybenzoic acid * --_* : vanillic acid q----0; ferulic acid A-A; calTeic acid -0. S”.”
136
R
45x
DAYS
DAYS
Fig. 2. The rate of respiration (~1 COa g-t soil h-r) of Countesswells soil from four crop treatments. amended with phenolic acid or glucose (5 mg g- ’ soil) and incubated at 22-C for 28 days. (Mean of two expertments). ‘@ Fallow: il Peas; n Potato: 0 Barley.
28 days on the fallow soil amended with vanillic or hydr~~ybenzoic acid was much greater than for other substrates. The other soils showed little response to these amendments. although after 28 days the amylase activity of all glucose-amended soils was greater than the controls and the other phenohc-amended soils were also showing signs of increase. Kiss et &. (1978) reported increases in amylase activity in gfucoseamended soils 21-63 days after amendment. and amylase activities have been increased by amendments of other non-starch substances such as oxidized coal. sewage siudge. mineral fertilizers and Lime (Kiss et trl.. !978).
The biomass, estimated after 28 days incubation :i~kn most of the added substrate had been metabolized. is Lawn in Table 2. At earlier sampling times anomatcusiy low or negative estimates of the biomass
of amended soils were obtained using the fumigation technique. This was not attributable to any toxic effect of the phenoiic acids, but to the high rate of respiration of the non-fumigated and amended soils. Further, in the fumigated soils there was a lag period while the inoculated organisms recolonised the soil. and these reinoculated organisms frequently seemed slower at metabolizing the remaining substrate than the original microorganisms. An attempt was made to allow for the lag period by prolonging the incubation beyond lOdays. but because of the rapidly changing rate of respiration of the non-fumigated amended soils. it was difficult to establish a “base line” to determine the duration of the extra incubation period. These factors resulted in the CO, respired from the fumigated samples frequently being lower or only slightly higher than from the non-fumigated samples. This inability to detect the “flush” of CO2 following fumigation resulted in anomalously low estimates of biomass and these results are not presented.
Table 2. Effect of soil phenolic acids or glucose amendments (5 mg g-’ soil) on the biomass (JI~C II_-’ soil) estimated by fumigation or respiration. in Countesswells soil from four cropping treatments. Estimations were made 28days after initial amendment and incubation at 22
Amendment Control (nil) Caffeic acid Ferulic acid p-Hydroxy benzoic actd Vanillic acid Glucose
Fallow Fumigation Respiration
Fumigation
Barley Respiration
Fumigation
Peas Respiration
Potatoes Fumigation Respiration
196 0 0
212 370 344
235 94 246
336 552 468
199 71 122
290 489 525
191 69 I30
352 474 402
0 0 118
390 286 368
394 225 254
622 630 493
304 224 272
615 517 508
251 187 351
493 419 385
Changes
in microbial
biomass
and activity
FALLOW
in soils amended
with phenolic
acids
459
l
PEA
h
DAYS
DAYS
f
r POTATO
kc3 DAYS
DAYS Fig. 3. Amylase activity (pg reducing sugar gg ’ cropping treatments. amended with phenolic acids sampled at various times up to 28 days. Control acid O---Cl; ferulic acid A.---A;
Paul and Voroney
(1980)
reported
similar
difficul-
soil and suggested that an estimate of the biomass could be made without subtraction of the unfumigated soil respiration. Such a step does not appear valid in our experiments because substrate was present throughout the earlier stages of the incubation and would have been contributing to the CO, level. Unless the CO2 flush from the fumigated soil can be distinguished from CO2 resulting from substrate utilization, the k factors derived by Jenkinson and Powlson (1976) or Anderson and Domsch (1978) may not be valid and estimation of biomass could be in error. Biomass estimated by the respiration method was consistently much higher than that estimated by fumigation, and there was poor agreement between the biomass estimated by the two methods. The fallow soil. which had higher levels of substrate remaining after 28 days than the other soils, gave negative results for all the phenolic acid amendments when tested by the fumigation method, and all fumigated soils previously amended with caffeic acid gave anomalously low estimates of the biomass. The poor correlation between the respiration and fumigation methods of biomass estimation was noted by Sparling et al. (1981) in amended soils, and possible reasons for the discrepancies were suggested. ties with
glucose-amended
soil hh’ at 35C) in Countesswells soil from four or glucose (5 mgg-’ soil), incubated at 22-C. and -0; p-hydroxybenzoic acid * -*; vanillic caffeic acid *-0; glucose n--n.
In conclusion, our results show that the soil biomass is well able to utilize soil phenolic acids as substrates, and that the acids are quickly metabolized. There was no evidence of any overall toxic effect at the concentrations used, and interaction of the acids with the soil by sorption provided a buffering effect on extractable phenolic acid concentration. The differences in the initial rates of soil respiration following amendment strongly suggest that some crops, particularly potatoes, stimulated phenolic acid-degrading organisms. Such changes in the soil microflora may be of significance in crop rotations where plants differ in their sensitivity to phenolic acids. Ackno~ledyement~We cal assistance.
thank
Susan Henderson
for techni-
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