Folia Microbiol. 30, 58- -64 (1985)

Poly-3-hydroxybutyratc Production and Changes of Bacterial Community in the Soil

A. HANZLiKOVX, A. JANDEI~A and F. Kv~c

Department of Microbial Ecology, Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4

Received March 1, 1984

ABSTRACT. Changes in the number of bacteria and relative distr ibution of strains producing poly-3-hydroxybutyra te (PHB) in soil were invest igated. Samples of ehernozem soil were cul- t iva ted with glucose in the presence of a mineral ni t rogen source (d iammonium hydrogen phos- phate) or in its absence, either in a batch or a heterocontinuous cult ivation system. In both cul t ivat ion sys tems the addit ion of glucose resulted in a roughly ten-fold increase of bacteria concentrat ion and an increase in the proportion of s t rains able to produce P t t B granules. W h e n the nitrogen source was added simultaneously with glucose, the concentrat ion of bacteria increased by two orders of magni tude in both cul t ivat ion systems. In the ba tch sys tem changes in the concentration of strains capable of P H B production were very small under these con- dit ions whereas in the heterocont inuous sys tem their number decreased by a lmost 50 %. The survival of bacteria in soil suspension after 57-d s tarvat ion was associated with P H B production which differed, depending on the previous t r ea tmen t of the soil samples. The concentrat ion of bacteria decreased least pronouncedly in the control with water and most significantly during cul t ivat ion with glucose and a ni trogen source, where the initial PH B content was~very low in spite of high numbers of bacteria.

The ability to survive is different in different microorganisms and is associated with the composition of the cultivation medium, growth rate, chemical composition of cells, etc. (Dawes 1976). I t is generally accepted that microorganisms isolated from a natural environment poor in nutrient sources (from soil or spring water) exhibit a higher survival ability (Boylen and Mulks 1978; Martin et al. 1979) than those living in the alimentary tract of higher organisms (Dawes and Ribbons 1965). The survival ability of micro- organisms depends primarily on the intensity of endogenous metabolism, rate of utilization of endogenous carbon and energy sources in the absence of an exogenous source (Boylen ar, d Mulks 1978).

The production and degradation of poly-3-hydroxybutyrate (PHB) and other reserve polymers in pure cultures of microorganisms were studied in connection with their survival under conditions of limited nutri t ion (Doudo- roff and Stanier 1959; Breznak et al. 1978). When studying the PHB pro- duction in soil it was demonstrated tha t the PHB content increases in soil samples supplemented with glucose during cultivation, both in batch and heterocontinuous systems (Hanzlikovs et al. 1984). During these studies the question arose, as to the microbial soil community changes with respect to

1985 POLY-3-HYDROXYBUTYRATE IN THE SOIL $$

the production of this polymer. Despite the fact that PHB was also detected in actinomycetes (Kannan and Rehs 1970) and yeasts (Nuti and Lepidi 1974), it is most often accumulated by bacteria of various morphological and physiological groups. Only the monomer was detected in the mycelium of micromycetes (I~uti et al. 1975). Therefore, in the present work changes in the bacterial community were followed in connection with PHB production, together with the occurrence of strains producing PHB granules in soil supplemented with glucose or glucose together with nitrogen and phosphorus sources, both in batch and heterocontinuous cultivation systems. The effect of the amount of PHB produced during the continuous cultivation on the survival of the bacterial community was also examined.

M A T E R I A L AND M E T H O D S

The chernozem soil and both the batch and heterocontinuous cultivation systems were described before (HanzlikovA et al. 1984). In the batch cul- tivation, 25-g samples of soil I (arable soil cellected after barley harvest) were air-dried, sieved and placed in 250-mL wide-mouth flasks. In the hetero- continuous cultivation, columns of 30 g of soil II (arable soil collected after harvest of clover-grass mixture) were separated into 3 layers with the aid of PVC nets. In the batch system, glucose was added at concentrations of 0.6, 1 or 2 %, together with 0.12 % diammonium hydrogen phosphate, in the heterocontinuous system 0.1 or 0.25 % glucose was added without or with 0.02 % diammonium hydrogen phosphate at a rate of 30--35 mL/d. The cultivation was carried out at 28 ~ in the dark in both cases.

The concentration of bacteria in soil samples was determined using a com- mon dilution plate method on plates with agar medium containing yeast and soil extract and tryptone (Taylor 1951). The plates were cultivated for 5 d at 28 ~ The numbers of bacteria were determined always on 4--5 parallel dishes and expressed per 1 g dry soil.

From the grown colonies, sixty colonies were randomly selected, trans- ferred to meat-peptone agar and cultivated for 4 d at 28 ~ Well-developed colonies were transferred to a nutrient agar medium with minimal nitrogen content (42 ppm) and high content of glucose (2 %) (Schlegel et al. 1970). The plates were cultivated for 5 d at 28 ~ and stained for 20 min with 0.02 % Sudan black B and then bleached for 2 • 1 rain with ethanol. Colonies with cells containing PHB granules remained dark-blue, other colonies bleached to light-grey. The results are mean values obtained from two independently tested samplings in each variant.

Survival of the bacterial soil community was evaluated in soil samples from the heterocontinuous cultivation by comparing the concentrations of bacteria determined by the dilution method at the beginning and at the end of a 57-d incubation, in an environment without introduced nutrient sources. Soil samples (1 g) were transferred into flasks (500 mL) containing 100 mL of sterile water and incubated on a reciprocal shaker at 28 ~ the pH of the soil suspension was 6.7--6.8 and 6.6 at the beginning and at the end of the cultivation, respectively.

Isolation of PHB from the soil, as well as its determination after hydrolysis to crotonic~acid were described before (Hanzlikovs et al. 1984).

60 A. HANZL:[KOV~_ et al . Vol. 30

TABLE I. Concentrat ion of bacteria (N) and P H B content expressed as ~g of crotonic acid (C) in 1 g soil f rom batch cul t ivat ion sys tem

Subst ra te

Cult ivation time, d

3 8 57

C N C N C N ~g/g • 10-s ~g/g / 10 -8 ~g/g • 10-6

Control 2.36 3.8 2.16 2.1 2.04 8.3 Glucose

0.6 ~o 13.5 23 6.72 21 2.68 13 1 ~o 21.5 41 10.6 31 4.68 53 2 ~o 51.0 57 22.0 58 9.47 230

Glucose + (NH4)2HPO4 0.12 %

0.6 ~o 2.60 340 0.83 13 0.00 7.0 1 ~o 6.12 510 2.10 14 0.60 7.3 2 ~o 9.36 470 5.92 14 1.76 7.5

R E S U L T S A N D D I S C U S S I O N

Changes during batch cultivation

The content of P H B in soil I, air-ch'ied and previously untreated, was 2.48 ~g of crotonic acid per 1 g soil. This soil contained 8.1 • 106 bacteria in 1 g of soil. After wett ing to 60 % WHC, the concentration of bacteria in soil sample increased to 3.8• l08 during a 3-d cultivation, corresponding to a 4.5--5-fold division of the cells. In contrast, the P H B content decreased, even though very slowly (Hanzllkovs et al. 1984).

The content of P H B in soil samples increased only after an enrichment with glucose and, to a lesser extent, after addition of diammonium hydrogen phosphate to soil, simultaneously with glucose (Table I). The enrichment also led to quant i ta t ive changes in the bacterial soil community. The con- centration of bacteria was always highest after a 3-d cultivation; after enrichment with glucose its magnitude increased by an order of magnitude and in the presence of the nitrogen source, by two orders of magnitude. In

T)mLE I I . Relative occurrence of bacteria (~o) able to produce PI-IB granules during the ba tch cul t ivat ion

Subs t ra te Cult ivation time, d

3 8

Control 20 20

Glucose 1 ~o 33 26

Glucose 1 % + (NH4)2ttPO4 0.12 % 25 22

1985 P O L Y - 3 - H Y D R O X Y B U T Y R A T E I N T H E SOIL fi|

TABLE I I I . Relat ive occurrence of bacteria producing P H B granules (B) in per cent, concentra t ion of bacteria (N) and content of P H B in ~tg of crotonic acid (C) in 1 g soil dur ing the heterocon- t inuous cul t ivat ion

Cult ivation time, d

Subs t ra te Layer a 3 10

B C N B C N % [zg/g X 10 -s % ~zg/g x 10 -s

Control

Glucose 0.1%

Glucose 0.1% + (NH4)2HPO4 0.02 %

1 28 3 26

1 26 3 26

1 28 3 28

1.56 3.1 28 1.20 2.1 1.58 3.2 30 1.32 2.6

5.10 10.8 42 23.0 91.0 3.25 4.1 45 9.62 12.0

1.67 450 15 0 840 2.90 11.7 42 4.97 380

a Layers of the soil column numbered f rom above.

samples cult ivated with glucose in the presence of nitrogen the concentration of bacteria after an 8-d cultivation decreased to tha t observed in variants with glucose (Table I) and further slowly decreased in the course of the whole time period investigated. After 57 d, the concentration decreased in all variants with glucose and nitrogen to the initial value in the control with water and it remained elevated only in soil samples with glucose alone, in particular when glucose was added at the highest concentration. In this case the content of P H B remained increased even further (Table I), indicating its function as a reserve polymer.

The addition of nutritional sources represents a certain selective pressure with respect to the biological soil population. I t results in differences in the density of colonization and in the occurrence of microbial types and species, including strains capable of the production of P H B granules.

In soil which was only wetted, roughly 20 % colonies of a randomly selected collection could produce P H B (after 3 and 8 d). A similar state was observed also at the beginning of the cultivation (18 ~/o). After enrichment with glucose (1 ~o), the proportion of strains producing P H B granules in- creased to 33 ~o and after supplementation with glucose simultaneously with a nitrogen source, 25 ~o colonies accumulated PHB. After 8 d, these numbers decreased to the initial values (Table II).

However, the method employed enabled us to determine the numbers of P H B producing colonies in the cultivation medium with a high content of carbon and energy sources and a minimal content of nitrogen (the C : N ratio in the cultivation medium was 190 : 1), rather than the number of colonies actually synthesizing P H B in the soil sample. During cultivation in soil supplemented with glucose and nitrogen, the relative representation of strains capable of the production of P H B granules slightly increased; their absolute concentration was higher by an order of magnitude, similarly to the total concentration of bacteria. However, the amount of P H B was lower here

62 A. HANZL~[KOV_~ et al. Voh 30

TAI3LE IV. Survival of the soil bacterial populat ion. Soil samples were cont inuously enriched for 10 d wi th subs t ra tes and the P]:IB content in [zg of crotonic acid (C) per 1 g soil was then determined. Concentrat ion of bacteria (N) was determined after the end of the cul t ivat ion and after a 57-d s ta rva t ion

Substra te

After a 10-d cont inuous After a 57-d cul t ivat ion s t a rva t ion

C N N N [zg/g x 10 -s • 10 -6 ~/o a

Control 1.28 1.8 8.7 4.83 Glucose

0.1 ~o 15.3 68 18 0.26 0.25 ~o 57.0 76 34 0.44

Glucose 0 . 1 % + (NH4)2HPO4 0.02 % 1.90 460 2.6 0.0056

a Viable bacter ia in ~o of the concentrat ion at the beginning of s tarvat ion.

(Table I). Thus, the PHB production by bacteria in the presence of nitrogen sources decreases more than is indicated by the changes in the amount of PHB alone.

From these da ta it is possible to evaluate the theoretical amount of PHB corresponding to a microbial cell tha t is able to produce P H B granules. This amount is higher in the variant with 1 ~/o glucose than in the variant with glucose and 0.12 ~ nitrogen. However, the highest amount of PHB per cell was found in the variant wetted only with water.

Changes during the heterocontinuous cultivation

During the first 3 d of cultivation (when water flowed through all the columns to remove the mineral components and set an equilibrium in the soil biological component), the PHB content in the soil slightly decreased (from 1.68 to 1.57 ~g crotonic acid in 1 g of soil) and the concentration of bacteria increased proportionally to 4.5--5 divisions, similarly as in the batch cultivation. During further 10 d of cultivation with water, the amount of bacteria did not further increase but the PHIB content permanent ly slowly decreased. When the columns were supplemented with glucose their P H B content increased gradually during the following 10 d. However, its amount reached only about 1/3 of the maximal amount determined during the batch cultivation, despite the fact tha t the amount of added glucose was compar- able (Hanzlikov~ et al. 1984). I t is necessary to stress here tha t only a part of the soil bacterial population able to grow and produce P H B in the used cultivation media was studied and tha t processes of P H B production and degradation proceed simultaneously in the soil system. The increased PHB content was most significant in the upper layer; the concentration of bacteria increased also first in the upper layer of the soil column (Table III). After 10 d the concentration of bacteria increased by an order of magnitude in both studied layers. When supplementing the soil column with glucose together with diammonium hydrogen phosphate, the content of P H B in the upper layer was negligible and in the bot tom layer it reached roughly a three-fold

1985 P O L Y - 3 - H Y D R O X Y B U T Y R A T E IN TH E SOIL 63

value of the initial determined in soil II. In this case the concentration of bacteria increased more rapidly than without a nitrogen source in both layers.

The relative occurrence of bacterial strains producing P H B granules was determined after a 3- and 10-d cultivation. After 3 d, the changes were insignificant. In the non-enriched variant 28 % of colonies, on the average, produced PI-IB at the beginning, after 3 and even after l0 d of cultivation. When glucose was added in the course of 10 d, the number of these colonies increased to 42--45 %; however, after cultivation with glucose in the presence of nitrogen only 15 % of the colonies in the upper soil layer could produce P H B , whereas 42 % colonies produced P H B in the bot tom layer of the soil column. As ammonium ions, nitrate and nitrites were not detected in the heterocontinuous system in the solution passing through the column during the whole cultivation t ime (whereas glucose occurred already after a 11-d cultivation) it may be assumed that nitrogen was exhausted in the upper layers of the soil column. Nitrogen deficiency might be associated with the increased occurrence of strains producing P H B in the bot tom layer of the soil column (also the P H B content in this layer is determinable; see Table III). A possibility of changes of the bacterial population in time and a simultaneous distribution of its components in space, i.e. in layers of the soil column, were described before (Kune and Macura 1965; Macura and Kunc 1965).

When supplementing glucose and nitrogen source, the total number of cells producing P H B was higher (wi'th respect to the overal concentration of bacteria) as compared with glucose alone, in contrast to its amount. Similarly as in the batch system, in this experimental arrangement it can be shown that the amount of P H B per cell tha t is able to produce P H B granules is higher in the variant with 0 . 1 % glucose than in the variant with glucose and 0.02 % nitrogen sources. The decreased P t t B production after the addition of a nitrogen source together with glucose is more pronounced in the continuous system than in the batch system. Nevertheless, the highest P H B amount per one cell that is able to produce P H B was detected in the variant with water even in the heterocontinuous system.

Survival of the soil bacterial population

As a result of the selection pressure of added nutrients on the soil biological population only a certain part of the microbial population differing from the original one multiplies. When using the heterocontinuous cultivation system, this selection pressure decreases (Macura 1969). Therefore, when investigating the ability to survive a long-term starvation, the population from the hetero- continuous system which resembles in its composition and density the natural one, was used.

After a 57-d incubation of soil samples, supplied for the previous 10 d with water, glucose or glucose in the presence of diammonium hydrogen phosphate, in the environment without added nutrit ional sources relatively high amounts of bacteria remained viable (Table IV). The concentration of bacteria decreased most pronouncedly in the variant precult ivated with glucose in the presence of a nitrogen source. In spite of the fact tha t the concentration of bacteria determined at the end of the incubation was roughly 10s per 1 g dry soil, this was only 56 ppm of the original concen-

64 A. HANZL][KOV-A. et al. Vol. 30

tration, representing thus an almost 50 000-fold decrease. When the soil was precultivated with glucose, the concentration of bacteria decreased roughly to 1 : 500. The highest number of viable cells (with respect to the initial state) remained in the non-enriched soil precultivated with water; the concentration decreased only to 1 : 23. The results are in agreement with the conclusions of the previous discussion (amount of P H B per 1 cell producing this polymer).

It may be assumed that PHB produced by the microflora under suitable nutritional conditions might be one of the possible factors helping the cells to survive unfavorable conditions of starvation, although it is not the only and decisive factor. The endogenous metabolic activity of the soil bacterial population appears to be the main factor determining the survival under conditions of limited nutrition in the environment (Hattori 1973). In fact, some organisms of the oligotrophic bacterial soil population actually ex- hibited a minimal endogenous metabolic activity (Boylen and Mulks 1978). It is problematic, to what extent these ecologico-trophical groups of micro- organisms can be detected on the used cultivation media (Nikitin and Niki- tina 1978). In connection with the above facts it is possible to explain the slow decrease of the PHB content as a possible endogenous carbon and energy source during the cultivation of soil samples with water.

R E F E R E N C E S

BOYLEN C.W., MULXS M.H. : Coryneform bacteria during periods of prolonged nut r ien t s tarvation. J.Gen.Microbiol. 105, 323 (1978).

BREZNAK J.A., POTRIKUS C.J., PFENNm N., ENSIGX J.C.: Viability and endogenous substrates used during s tarvat ion survival of Rhodospirillum rubrum. J.Bacteriol. 134, 381 (1978).

DAWES E.A.: Endogenous metabolism and the survival of starved prokaryotes. Symp.Soc.Gen. Microbiol. 26, 19 (1976).

DAWES E.A., RIBBONS D.W.: Studies on the endogenous metabolism of Escherichia coli. Biochem. J. 95, 332 (1965).

DOUDOROFle M., STANIER R.Y.: Role of poly-~-hydroxybutyrie acid in the assimilation of organic carbon by bacteria. Nature 183, 1440 (1959).

HANZLIKOV.4 A., JANDERA A., Ku~-c F.: Format ion of poly-3-hydroxybutyrate by a soil micro- bial communi ty (luring batch and heteroeontinuous cultivation. Folia Microbiol. 2.% 233 (1984).

HATTORI T.: Microbial Life in the Soil. An Introduction (A.D. McLaren, Ed.). M. Dekker, New York 1973.

KANt-AN L.V., ~EI~I/kCEK Z.: Format ion of poly-~-hydroxybutyrate by actinomycetes. Ind, J. Biochem. 7, 126 (1970).

KUNC F., MACURA J.: Decomposition of root exudates in soil. Folia Microbiol. l l , 239 (1966). MACURA J.: Heterocont inuous cultivation of microorganisms in soil. (In Czech) DSc Thesis,

Ins t i tu te of Microbiology, Czech.Acad.Sci., Prague 1969. MACURA J. , KUNC F.: Continuoas flow method in soil microbiology. V. Nitrification. Folia

Microbiol. 10, 125 (1965). MATIN A., VELDHUIS C., STEGEMAN V., VEENHUIS M. : Selective advantage of a Spirillum species

in a carbon limited environment . Accumulat ion of poly-~-hydroxybutyrie acid and its role in starvation. J.Gen.Microbiol. 112, 349 (1979).

NIKITIN D.I., NIKITINA E.S.: Self-cleaning Processes of Environment and Bacterial Parasites (Genus Bdellovibrio). (In Russian) Nauka, Moscow 1978.

N~rTI M.P., B~OOKS J.B. , LEPIm A.A. : Occurrence of ~-, ~-, and "~,-hydroxybutyrates in some soil microfungi. Trans.Brit.Mycol.Soc. 64, 79 (1975).

N~TTZ M.P., LEPIDI A.A.: Poly-~-hydroxybutyr~tes occurrence in Saccharomyces cerevisiae and its significance in the fermentat ion process, p. 123 in Proc.Fourth Int.Symp. of Yeasts, Par t I, Vienna 1974.

SCHLEGEL H.G., LAFElCTY I~., KR~USS I.: The isolation of mu ta n t s not accumulat ing poly-~-hy- droxybutyrie acid. Arcl,~.Microbiol. 71, 283 (1970).

TAYLOJ~ C.B.: The nutr i t ional requirements of predominant flora of the soil. Proc.Soc.Appl.Baet. 14, 101 (1951).