Foundations of Biochemical Engineering - ACS Publications

10

The

Role

of S p e c i a l i s t s a n d G e n e r a l i s t s

in

Microbial

Population

Interactions

Downloaded by CALIFORNIA INST OF TECHNOLOGY on December 5, 2016 | http://pubs.acs.org Publication Date: January 18, 1983 | doi: 10.1021/bk-1983-0207.ch010

J. G. KUENEN Delft University of Technology, Laboratory of Microbiology, Julianalaan 67a, 2628 BC Delft, The Netherlands In many environments highly specialized bacteria coexist with generalists, i.e. bacteria able to metabolize a large diversity of substrates. In order to understand the mechanisms of interaction between these types of bacteria, model experiments in continuous culture have been carried out with two typical specialists and one generalist. It could be shown that the generalist can compete successfully with specialists for growth limiting substrates when mixtures of substrates are available. Under these conditions the generalist can utilize these mixtures simultaneously. Another advantage of generalists might lie in their capability to continue to grow when the supply of different substrates a l ternate. In that case specialists would alternatively grow and starve. Model-competition experiments indicate that, in general, the success of specialists was favoured by increased length of growth and starvation periods. The breakdown of organic and i n o r g a n i c compounds i n nature i s c a r r i e d out by an enormous d i v e r s i t y of b a c t e r i a which are adapted to a v a r i e t y of p h y s i c a l and chemical environmental parameters. In a given environment, many microorganisms may c o e x i s t which o f t e n have completely d i f f e r e n t metabolic c a p a b i l i t i e s , but sometimes a l s o have overlapping p r o p e r t i e s . T y p i c a l l y , one can f i n d h i g h l y s p e c i a l i z e d organisms, able to metabolize only one or a few compounds, c o e x i s t i n g with very v e r s a t i l e organisms, termed general i s t s . The l a t t e r are able to metabolize a great d i v e r s i t y of o r ganic compounds. From the e c o l o g i c a l p o i n t of view one may ask how to e x p l a i n t h i s coexistence of microorganisms and how t h e i r r e s p e c t i v e phys i o l o g i c a l p r o p e r t i e s may give them an advantage or disadvantage under a given growth c o n d i t i o n . S i m i l a r l y , from the p o i n t of view of management of sewage treatment p l a n t s , one may ask how the 0097-6156/83/0207-0229$06.75/0 © 1983 American Chemical Society

Blanch et al.; Foundations of Biochemical Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


230

BIOCHEMICAL ENGINEERING

regime of a p l a n t may s e l e c t c e r t a i n metabolic types and how t h i s may have a bearing on p r o p e r t i e s of the p l a n t such as s u s c e p t i b i l i t y to pulse charges and loading c a p a c i t y . In order to understand the complicated r e a c t i o n s occuring between s p e c i a l i s t s and g e n e r a l i s t s w i t h i n a complex mixture of other organisms, a thorough i n s i g h t i n t o t h e i r b a s i c metabolic p r o p e r t i e s , that i s t h e i r p h y s i o l o g i c a l behaviour, i s needed. Unf o r t u n a t e l y the physiology of most microorganisms i s impossible to study i n the very complicated mixtures i n which they n a t u r a l l y occur. Therefore, as a i n i t i a l approach, i t has been necessary to study s p e c i a l i s t s and g e n e r a l i s t s i n pure c u l t u r e s , and i n a r t i f i c i a l composite-mixtures of organisms i n order to obtain i n s i g h t i n t o t h e i r e c o l o g i c a l niches. When grown on t h e i r s p e c i f i c substrate i n the laboratory, the s p e c i a l i s t s are c h a r a c t e r i z e d by very high s p e c i f i c growth rates (ymax), whereas the g e n e r a l i s t s turn out to possess r e l a t i v e l y low maximum s p e c i f i c growth r a t e s on the substrates they can u t i l i z e . Even at very low concentrations of a s u b s t r a t e , as they g e n e r a l l y occur i n the environment, the v e r s a t i l e organisms grow r e l a t i v e l y slowly. F i g u r e 1 shows a g e n e r a l i z e d p i c t u r e of the r e l a t i o n s h i p between the growth l i m i t i n g substrate and the s p e c i f i c growth r a t e of a s p e c i a l i s t and a g e n e r a l i s t . I t may be asked whether the g e n e r a l i s t s might be able to grow f a s t e r than s p e c i a l i s t s i n mixtures of s u b s t r a t e s . However, under such c o n d i t i o n s the g e n e r a l i s t s o f t e n show s e q u e n t i a l substrate u t i l i z a t i o n , known as d i a u x i e . A w e l l known example i s the growth of the bacterium Escherichia coli on mixtures of glucose and l a c tose. F i r s t the glucose i s u t i l i z e d , and only when t h i s compound i s completely metabolized, the enzymes needed f o r l a c t o s e u t i l i z a t i o n w i l l be induced, allowing growth on l a c t o s e . Thus at high concentrations of mixtures of s u b s t r a t e s , simultaneous u t i l i z a t i o n i n t h i s case i s not p o s s i b l e . One should r e a l i z e that i n many n a t u r a l and seminatural e n v i ronments concentrations of substrates are g e n e r a l l y low, u s u a l l y below the mM and even o f t e n below the pM range. Under such condit i o n s , the growth r a t e of microorganisms w i l l be l i m i t e d by the concentration of t h e i r s u b s t r a t e s , and mixed substrate u t i l i z a t i o n might be p o s s i b l e . In the l a b o r a t o r y , growth under dual subs t r a t e l i m i t a t i o n can be conveniently created i n a f l o w - c o n t r o l l e d chemostat, or continuous c u l t u r e . In the growth medium supplied to the c u l t u r e , a l l i n g r e d i e n t s necessary f o r growth are i n excess except f o r the two substrates i n question. I t has been shown for E. coli by S i l v e r and Mateles (1) that under such c o n d i t i o n s simultaneous u t i l i z a t i o n of glucose and l a c t o s e was p o s s i b l e . Analogous r e s u l t s have been obtained f o r other b a c t e r i a , such as Pseudomonas oxalaticus growing on mixtures of formate and acetate (2) and Thiobacillus s t r a i n A2 growing on t h i o s u l f a t e and acetate (J3). Our own work on the g e n e r a l i s t Thiobacillus A2 may serve as



Microbial

Specialists and

Generalists

Specialist

Generalist

-> s

Figure 1. The relationship between the concentration (s) of the growth-limiting substrate and the specific growth rate (μ) of a typical specialist and a typical gen eralist bacterium growing on the same substrate.



232


an example, T. A2 i s a very v e r s a t i l e organism able to grow on at l e a s t 25-30 d i f f e r e n t organic s u b s t r a t e s , and, i n a d d i t i o n , a l s o capable of a u t o t r o p h i c growth on i n o r g a n i c reduced s u l f u r com pounds. (See Table I)· Under the l a t t e r c o n d i t i o n s i t o x i d i z e s , f o r example, t h i o s u l f a t e or s u l f i d e as a source of energy while using carbon d i o x i d e as the only carbon source f o r growth. When grown i n batch c u l t u r e i n the presence of high concentrations of acetate and t h i o s u l f a t e Τ. A2 c l e a r l y shows a b i p h a s i c u t i l i z a t i o n of the two compounds (Figure 2). However when grown i n the chemos tat under growth l i m i t a t i o n by t h i s mixture, simultaneous u t i l i z a t i o n of acetate and t h i o s u l f a t e i s p o s s i b l e i r r e s p e c t i v e of the a v a i l a b l e r a t i o of t h i o s u l f a t e and acetate (Figure 3). Under such c o n d i t i o n s , acetate and t h i o s u l f a t e concentrations i n the c u l t u r e are below the d e t e c t i o n l e v e l . The metabolic machinery of T. A2 adapts to the r e q u i r e d turnover rates of the r e s p e c t i v e sub s t r a t e s . For example, the a b i l i t y to o x i d i z e t h i o s u l f a t e i s pre sent at maximum c a p a c i t y when only t h i o s u l f a t e i s s u p p l i e d to the c u l t u r e , whereas no t h i o s u l f a t e r e s p i r a t i o n c a p a c i t y i s present when only acetate i s a v a i l a b l e . I n t e r e s t i n g l y , the a b i l i t y of the c e l l s to a s s i m i l a t e C O 2 f o r c e l l carbon i s e f f i c i e n t l y adapted to the a v a i l a b l e organic carbon i n the c u l t u r e . This implies that when the r a t i o of t h i o s u l f a t e to acetate i s r e l a t i v e l y h i g h , ace tate i s used p r i m a r i l y as a carbon source to "save" energy f o r C O 2 f i x a t i o n . F u r t h e r work i n our laboratory by others (Ji and j ) has shown that simultaneous u t i l i z a t i o n of mixtures of other substrates i s e q u a l l y p o s s i b l e . The aim of our research was then focussed on the question of whether t h i s g e n e r a l i s t would be able to compete s u c c e s s f u l l y f o r g r o w t h - l i m i t i n g substrates with s p e c i a l i s t s during mixotrophic growth under n u t r i e n t l i m i t a t i o n . For our purpose we used the three model organisms shown i n Table I I . These included a t y p i c a l g e n e r a l i s t , namely the v e r s a t i l e Thiobacillus A2, and two s p e c i a l i s t s . The f i r s t s p e c i a l i s t was the chemolithoautotroph, Thioba cillus neap olitonus which can grow very f a s t i n m i n e r a l s - t h i o s u l f a t e medium using C O 2 as i t s carbon source, and the second was a chemoorganoheterotroph Spirillum G7, which can grow very r a p i d l y on m i n e r a l s - a c e t a t e medium using acetate as carbon and energy source. (See a l s o Table I ) . A s e r i e s of experiments was c a r r i e d out i n continuous c u l t u r e to study the competition between sets of two and three organisms. 9

In

the

first

experiment, Thiobacillus

A2

and

Thiobacillus

neapoli-

tanus were each grown s e p a r a t e l y i n a t h i o s u l f a t e - l i m i t e d chemos t a t . Once steady s t a t e s had been e s t a b l i s h e d at a f i x e d d i l u t i o n r a t e , the c u l t u r e s were mixed one to one (v/v) and the change i n the percentages of the two organisms was followed u n t i l no f u r t h e r change could be observed f o r one or two volume changes. F i g u r e 4 shows that i n m i n e r a l s - t h i o s u l f a t e medium, Thiobacillus A2 was out-competed by the s p e c i a l i s t . T. A2 was not completely e l i m i n a ted s i n c e i t has been shown that the s p e c i a l i s t excretes g l y c o l l a t e (6) which can be consumed by T. A2.


10.

KUENEN

Microbial

Specialists

and

233

Generalists

Table I


11

Types of metabolism of "model b a c t e r i a used f o r the study of competition between " s p e c i a l i s t s " and " g e n e r a l i s t s " .

GENERALIST: Facultative

chemolitho(auto)troph ("mixotroph") (Thiobacillus

S 0 2

Energy source:

3

= (or S )

°2

> S0

4

and/or

Λ U

(many) organic compound(s) CO2 — * Carbon source:

A2)

2

> CO^

cellmaterial

and/or (many) organic compound(s) — ^ c e l l m a t e r i a l

SPECIALIST I O b l i g a t e chemolitho(auto)troph (Thiobacillus 0

2 =-> SO,

S20 3 «

Carbon source:

CO^—^ c e l l m a t e r i a l

u

?

(or S )

o

Energy source:

5

neapolitanus)

SPECIALIST I I Chemo(organo)heterotroph (Spirillum

G7)

°

2

Energy source:

(few) organic compounds

> CO^

Carbon source:

(few) organic compounds—^ c e l l m a t e r i a l


234



0

2

4

6

8 time (h)

10

12

%

16

Figure 2. Maximum substrate oxidation capacities ( Q o ^ ' j and substrate concen tration during growth of T. A2 on a mixture of acetate and thiosulfate in batch culture. The inoculum consisted of cells from an acetate-grown culture. Key: · , q ™*-thiosulfate; A , Qog^'-acetate; O , thiosulfate concentration; Δ , acetate concentration. Reproduced, with permission, from Ref. 3. Copyright 1980, SpringerVerlag. 0



10.

KUENEN

Microbial

Specialists and

0

2

4

40

36

32

6

235

Generalists

8 10 12 14 acetate (mM) 28 24 20 16 12 thiosulfate (mM)

16

18 20

8

4

0

Figure 3. Maximum substrate oxidation potentials and carbon dioxide fixation potential of whole cells of T . A2 as a function of different acetate and thiosulfate concentrations in the reservoir medium of the chemostat cultures. Key: · , Qo™*thiosulfate; A , QoJ™ *-acetate; M, C0 -fixation potential. Reproduced, with permission, from Ref. 3. Copyright 1980, Springer-Verlag. 1

2

Data were obtained with cells from thiosulfate- and/or acetate-limited chemostat cultures in steady state at a dilution rate of 0.05 h~ . Acetate and thiosulfate concentrations in the cultures were below the detection level. Cellular carbon in the cultures ranged from 110 mg C/L (40 mM thiosulfate) to 200 mg C/L (20 mM acetate). 1



236


When acetate was s u p p l i e d to the i n f l o w i n g medium, the number of T. A 2 c e l l s increased s i n c e T. neapolitanus cannot u t i l i z e acetate. F i g u r e 5 shows that with i n c r e a s i n g acetate c o n c e n t r a t i o n the percentage of T. A 2 g r a d u a l l y increased and coexistence of the two organisms became p o s s i b l e . Above a c o n c e n t r a t i o n of about 10 mM acetate, the s p e c i a l i s t was e l i m i n a t e d from the c u l t u r e . The explanation f o r t h i s l i e s i n the a b i l i t y of T. A 2 to u t i l i z e acetate and t h i o s u l f a t e simultaneously under these growth c o n d i t i o n s . With i n c r e a s i n g acetate c o n c e n t r a t i o n , the absolute concent r a t i o n of the T. A 2 c e l l s increased and allowed these organisms to c l a i m an i n c r e a s i n g l y l a r g e r share of t h i o s u l f a t e . E v e n t u a l l y , the r a t i o of acetate to t h i o s u l f a t e became such that T. A 2 was able to reduce the t h i o s u l f a t e c o n c e n t r a t i o n to below the l e v e l at which T. neapolitanus could maintain the r e q u i r e d growth r a t e , r e s u l t i n g i n the wash out of the s p e c i a l i s t . Very s i m i l a r r e s u l t s were obtained with mixtures of t h i o s u l f a t e and g l y c o l l a t e ( 4 ) or t h i o s u l f a t e and glucose ( 5 ) . Competition experiments between the s p e c i a l i s t Spirillum G7 and T. A 2 gave analogous r e s u l t s . In acetate medium Spirillum G7 out-competed T. A 2 completely. Thiobacillus A 2 out-competed the s p e c i a l i s t when more than 10 mM t h i o s u l f a t e was present i n the acetate-minerals medium. Obviously, under " n a t u r a l " conditions i n the environment, or i n sewage treatment p l a n t s , the g e n e r a l i s t would have to compete with both s p e c i a l i s t s at the same time. Therefore, three-membered c u l t u r e s were a l s o s t u d i e d . The r e s u l t s , as summarized i n F i g u r e 6, show that coexistence of the three organisms was p o s s i b l e over a large range of c o n c e n t r a t i o n r a t i o ' s with T. A 2 dominating the c u l t u r e . The coexistence of three organisms does not agree with t h e o r e t i c a l p r e d i c t i o n s which w i l l be discussed below. In any case the outcome of these experiments c l e a r l y i n d i c a t e d that when mixed substrates are s u p p l i e d , general i s t s have metabolic advantages which allow them to c l a i m a "niche", that i s , a r i g h t of existence i n the n a t u r a l environment. The v a l i d i t y of t h i s g e n e r a l i s a t i o n was confirmed by the r e s u l t s of enrichment c u l t u r e s c a r r i e d out i n the chemostat i n o c u l a t e d with n a t u r a l samples. Table I I I shows the outcome of such e n r i c h ment c u l t u r e s performed with d i f f e r e n t mixtures of t h i o s u l f a t e and a c e t a t e . In a l l cases when f r e s h water i n o c u l a were used, a dominant c u l t u r e of a g e n e r a l i s t Thiobacillus was obtained. I n t e r e s t i n g l y , i n 4 out of 5 cases the g e n e r a l i s t was a f a c u l t a t i v e chemolithotroph able to grow a u t o t r o p h i c a l l y . However, i n one case when r e l a t i v e l y high amounts of acetate were presented to the c u l t u r e , a bacterium able to o b t a i n energy from t h i o s u l f a t e but not able to f i x C O 2 came to the f o r e . This i s not s u r p r i s i n g , s i n c e at t h i s r a t i o C O 2 f i x a t i o n i s not r e q u i r e d (compare F i g u r e 3 f o r T. A2). As mentioned b e f o r e , a somewhat p u z z l i n g r e s u l t of the compet i t i o n experiments with the three-membered c u l t u r e was that coexistence of three organisms seemed p o s s i b l e . In s e v e r a l theore-


10.

Microbial

KUENEN

Specialists

and

237

Generalists

Table I I Maximum s p e c i f i c growth rates ν^οχ (h ^) of the o b l i g a t e l y c h e m o l i t h o ( a u t o ) t r o p h i c Thvobacillus

specialized, neapolitanus,

the v e r s a t i l e , f a c u l t a t i v e l y chemolithοtrophic Thiobacillus A2 (a g e n e r a l i s t ) , and a s p e c i a l i z e d h e t e r o t r o p h i c Spirillum G7, during growth i n minerals medium supplemented with t h i o s u l f a t e ( t ) , acetate (a) or mixtures of both substrates ( t + a ) . Downloaded by CALIFORNIA INST OF TECHNOLOGY on December 5, 2016 | http://pubs.acs.org Publication Date: January 18, 1983 | doi: 10.1021/bk-1983-0207.ch010

T. neapolitanus

and Spirillum

sulfate respectively

G7 cannot grow on acetate or t h i o

(10). Maximum s p e c i f i c growth r a t e P t

T.

neapolitanus

Thiobacillus Spirillum

a

0.35 A2

G7

-

3

A

5

0.35

0.22

0.22

0.43

0.43

6

(h ^)

t +a

-

0.10

m a x

7

8

Volume changes Figure 4. Competition in continuous culture between T. neapolitanus (the special ist) and T. A2 (the generalist) for thiosulfate as the only growth-limiting substrate. Key: Φ, relative cell number of Τ. A2; Ο , T. neapolitanus. Reproduced, with per mission, from Ref. 4. Copyright 1979, Springer-Verlag. 1

The chemostat was run at a dilution rate of 0.05 h' with a 40 mM thiosulfate concentration in the reservoir medium. Organisms had been pregrown separately in continuous culture at Ό = 0.05 h' and at zero time mixed in a 1:1 rate. 1



238


Figure 5A. Effect of different concentrations of organic substrate on the outcome of the competition between T . A 2 and T. neapolitanus for thiosulfate. Protein concentration and organic content in the cultures are shown, limited by thiosulfate plus acetate or glycollate. Reproduced, with permission, from Ref. 4. Copyright 1979, Springer-Verlag. 1

The chemostat was run at a dilution rate of 0.07 h' . The inflowing medium contained thiosulfate (40 mM) together with either acetate or glycollate at concentrations ranging from 0-7 mM. Relative cell numbers, protein content, and organic cell carbon in the culture were determined after steady states had been established.



10.

KUENEN

Microbial

Specialists

and

Generalists

239

T.A (• glycollate) 2

J. neapolitanus (•glycollate)

Oh

-L

-L

JL

3 4 5 6 7 8 9 GLYCOLLATE OR ACETATE (mM)

10

Figure 5B. Effect of different concentrations of organic substrate on the outcome of the competition between T . A2 and T. neapolitanus for thiosulfate. Conditions as in Figure 2A. The percentage of T . A2 cells and of T . neapolitanus cells in cultures are shown, with thiosulfate plus acetate or glycollate in the reservoir medium. Reproduced, with permission, from Ref. 4. Copyright 1979, SpringerVerlag.



240


mM

THIOSULFATE

Figure 6. Results of competition between the generalist T . A2, and two specialists, T. neapolitanus and Spirillum G7 for thiosulfate and acetate as growth-limiting substrates in the chemostat at a dilution rate of 0.07 h' . Concentrations in the inflowing medium ranged from 0-20 mM for acetate and from 0-40 mM for thiosulfate. After a steady state had been established relative cell numbers were determined. Solid line, experimental data; dashed line, outcome of the competition as predicted from mathematical modeling. 1


Blanch et al.; Foundations of Biochemical Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 1983. 1

sample

canal canal canal ditch ditch

Number

I II III IV V

30 10 30 20 10

t t t t t

+ 5a + 15 a + 5a + 10 a + 15 a

substrate concentration i n medium (mM) 82 75 85 50 86

% of t o t a l

chemolithotroph It ft II

type

chemolithoheterotroph

facultative tt tt It

metabolic

Dominant population

Results of enrichment c u l t u r e s (I - V) from f r e s h water samples a f t e r 15-20 volume changes i n the chemostat under dual substrate l i m i t a t i o n of t h i o s u l f a t e ( t ) and a c t e t a t e (a) at a d i l u t i o n r a t e of 0.05 h " , using d i f f e r e n t mixtures of t + a. Adapted from G o t t s c h a l and Kuenen, ( Π ) .

Table I I I



242


t i c a l treatments of the growth of m i c r o b i a l populations on mix tures of substrates i t has been p r e d i c t e d that the maximum number of organisms which can c o e x i s t can never be more than the number of g r o w t h - l i m i t i n g substrates supplied to the c u l t u r e . This holds i f no i n t e r a c t i o n other than mere competition takes place i n the c u l t u r e . Based on previous p u b l i c a t i o n s of other workers G o t t s c h a l and Things tad (_7) have developed a mathematical model f o r growth of the three organisms i n the p a r t i c u l a r case. The model i s based on simple Monod k i n e t i c s f o r growth. The s p e c i f i c growth r a t e of the s p e c i a l i s t autotroph (A) on t h i o s u l fate (t) i s

U

max (8 ) = t A · t y

tA

S

i n which s i s the c o n c e n t r a t i o n of the t h i o s u l f a t e and K ^ the substrate s a t u r a t i o n constant of the autotroph f o r t h i o s u l f a t e . S i m i l a r l y , f o r growth of the heterotroph (H) on acetate (a) one obtains t

t

max M

aH

I •I ' I ' I

0

ε C02-f'xotion

Ô ^

CM

ο

4-+ * -1000k

Foundations of Biochemical Engineering - ACS Publications

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