the mechanism of aggregate formation by selenomonas ruminantium

Appl Microbiol Biotechnol (1989) 32:350-355 Applied :,, Microbiology Biotechnology © Springer-Verlag 1989

The mechanism of aggregate formation by Selenomonas ruminantium

Ronald Mulder, M. Joost Teixeira de Mattos, and Oense M. Neijssel

Laboratorium voor Microbiologie, Universiteit van Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands

Summary. The mechanism of granule formation by Selenomonas ruminantium was investigated. A basic protein has been isolated from the lysate of S. ruminantium which triggers cluster formation (small aggregates of 20-100 cells) of suspended cells. Evidence is presented that these basic proteins were of ribosomal origin. It is suggested that ribosomes are released into the culture broth by lysis and that the associated basic proteins are subsequently dissociated by high monovalent ca- tion concentrations. It was found that these positively charged basic proteins interact with the negatively charged lipopolysaccharide of the organism to form the clusters. Adding lysate to suspended cells, followed by lowering of the pH from 5.8 to 4.5 also induced clustering. At dilution rates exceeding the maximum growth rate clusters were retained in anaerobic gas-lift reactors and grew into granules (1-3 mm). It is postulated that granules evolve from clusters. Within the dusters, lysis and a low pH are induced due to diffusion limitations. As a consequence dividing cells are entrapped within the clusters, resulting in growth.

Introduction

In a former paper it has been substantiated that Selenomonas ruminantium was the predominant organism within glucose-acidifying granular sludge (Mulder et al. 1989). When grown in pure culture in an anaerobic gas lift reactor (AGLR), S. ruminantiurn was able to form granules macrosco- pically identical to the mixed culture granules that evolve when activated sludge is inoculated in

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an AGLR. Furthermore, the fermentation pattern of these pure cultures was similar to that of mixed cultures. Hence, it has been concluded that in mixed cultures S. ruminantium was responsible for granule formation (Mulder et al. 1989).

In nature attachment of microorganisms to surfaces (immobilization) or to one another (granulation and flocculation) seems to be the rule rather than the exception. In general, adhesive properties are rationalized as either providing the organisms with some ecological advantage or hav- ing a sexual significance (Harris and Mitchell 1973; Calleja 1984). Clearly, the former explanation is applicable in the above-mentioned cultures as these AGLR's are run at dilution rates (D) that exceed the maximum growth rates for the bacteria present (Beeftink and van den Heuvel 1987). Thus, only those bacteria (or bacterial consortia) that are capable of aggregate formation, will be retained in the AGLR.

In general, cell-cell adherence is brought about by extracellular polymers or by fimbriae. Aggregation by fimbriae is often related to sexual processes and pellicle formation (Calleja 1984). Investigations on the role of extracellular polymers in bacterial aggregation have shown that polysaccharides are the most common polymers (Har- ris and Mitchell 1973). However, nucleic acids and proteins excreted in the medium by cellular lysis have been shown to play a significant role in bacterial aggregation as well (Nishikawa and Ku- riyama 1969; Weddle and Jenkins 1971). Here we report on the mechanism of aggregate formation by S. ruminantium.

Materials and methods

Growth conditions. Pure cultures of S. ruminantium were

R. Mulder et al.: Aggregation by S. ruminantium 351

grown in a mineral salts solution with 25% of the concentrations used by Evans et al. (1970). After sterilization (1 h, 120 ° C), a glucose solution (sterilized for 30 min at 110 ° C) was added to a final concentration of 10 g/1. The medium was supplemented with growth factors according to Linehan et al. (1978): NazCO3.10H~O, 4 g / l ; biotin, 25 ~tg/l; para-amino- benzoic acid, 100 ~tg/1; valeric acid, 1 ml/1 (S. ruminantium medium). The growth factors were added separately (sterilized for 30 rain at 110 ° C). The pH of the medium was set at 6.0 by titration with HC1. For 30 ml batch cultures the medium described above was used supplemented with 10 mM 2-[N-mor- pholino]ethanesulphonic acid (MES) buffer, 0.5% (w/v) yeast extract and 0.5% (w/v) casamino acids and the pH was set at 6.5. After inoculation the headspace was flushed with CO~ and 0.6 ml reducing agent was added. As reducing agent a mixture of cysteine hydrochloride (1.25% w/v) and Na2S (1.25% w/v) in 0.1 N NaOH was used and prepared according to Toerien and Siebert (1967).

For growth on lactate as carbon source, glucose was re- placed by 1% (w/v) sodium DE-lactate and the medium was supplemented with 0.5% (w/v) yeast extract and 0.5% (w/v) casamino acids.

Chemostat cultures. Selenomonas ruminantium was grown anaerobically in chemostats (Bioflo C-30, New Brunswick Scientific, Edison, NJ, USA) with a working volume of 350 ml, operated at 30 ° C. The pH was controlled by automatic titration and set at 5.8 with 2 N NaECO3, which together with CO2 produced by the organism generated the gas atmosphere.

Hexose analysis. The hexose content of the biomass was determined with the anthrone method according to Herbert et al. (1971) with glucose as standard.

Protein determination. Protein was determined with the Biuret method according to Herbert et al. (1971) with bovine serum albumin as standard.

Product analyses. Fermentation products were analysed by HPLC (Kontron LC pump, London, UK) with an Aminex HPX 87H organic acid analyses column (Biorad, Richmond, Calif, USA) using a refractive index detector (Bruker, Berlin, FRG) an CI-10 integrator (LDC/Mil ton Roy, Shannon, Ire- land) and 4 mM HzSO4 (Merck, Darmstadt, FRG) as eluent (flow rate 0.5 ml/min), at a temperature of 60 ° C.

Enzyme experiments. Both lyophilized and fresh granules (ap- proximately 4 mg dry weight) were washed twice with deionized water by decantation and resuspended in the appropriate buffer (20 raM) containing the enzyme assayed. Deaggregation was monitored for 24 h. A-3514 amyloglucosidase (sodium acetate buffer, pH 4.5, 55 ° C), C-7502 cellulase (sodium acetate buffer, pH 5.0, 37°C), L-6876 lysozyme (+ 10 mM ethy- lenediaminetetraacetate (EDTA)) (MES buffer, pH 6.2, 25 ° C) and P-5147 protease (TRIS buffer, pH 7.5, 37°C) were obtained from Sigma (St. Louis, Mo, USA), I 104159 deoxyribo- nuclease (DNA-se) (TRIS buffer, pH 7.5, 25 ° C) and 109134 ribonuclease (RNA-se) (TRIS buffer, pH 7.5, 25°C) from Boehringer, Mannheim, FRG, pectinase (sodium acetate buffer, pH 4.0, 25 ° C) from Fluka (Buchs, Switzerland) and 24579 trypsin from Merck.

Determination of lysis in chemostats. Chemostat samples were taken and centrifuged. The supernatant was decanted and the cells were washed with deionized water and freeze dried. From the pellet RNA was extracted with 0.5 N perchloric acid using the modified Schneider method according to Herbert et al.

INFLUENT

GAS OUT ÷

EFFLUENT

GAS IN

Fig. 1. Schematic diagram of the mini anaerobic gas-lifl reactor

(1971). In both cell extract and supernatant, which was mixed 1 : 1 with 1 N perchloric acid, RNA was determined with the orcinol reagent according to Herbert et al. (1971) with RNA from yeast (Boehringer) as standard. Lysis was expressed as RNA extracellular/RNA total (intra- and extracellular) multiplied by 100%.

Mini anaerobic gas-lift reactor experiments. Small anaerobic gas-lift reactors (8 ml) were developed to assay the aggregating potential of a chemostat culture (see Fig. 1). As lift gas a mixture of N2 (85%), COz (10%) and H2 (5%) was pumped through the reactors by means of a peristaltic pump 100 ml/h/reactor). To remove all traces of oxygen the gas was led over a catalyst at 150°C (R 3-11, BASF, Ludwigshafen, FRG). The reactors were kept at 30°C in a water bath. All media were reduced and buffered. For this purpose 10 mM MES buffer was added to the S. ruminantium medium and the pH was set at the de- sired value before sterilization. After sterilization and addition of the separately sterilized glucose solution the medium was

N2

- - , ~ 4

5

1

w

Fig.2. Schematic diagram of the experimental set-up: 1, gas cylinder; 2, catalyst; 3, medium; 4, medium pump; 5, mini anaerobic gas-lift reactors; 6, lift gas pump

352 R. Mulder et al.: Aggregation by S. ruminantium

flushed with the lift-gas overnight after which 200 ml of the reducing agent described above was added. Use was made of butyl rubber tubing; the peristaltic medium pump was placed in an anaerobic bag which was flushed with N2. The reactors were run at D = 0.77 + 0.07 h-1. A schematic diagram of the experimental set-up is given in Fig. 2. Each experiment was started by adding 5 ml inoculum to an empty reactor.

Lysate production. Cells were lysed with a French press (4 ° C) at 16,000 psi, cell debris was spun down at 39,000 9, and the supernatant was used as lysate.

Extraction of basic protein. A 20-1 batch culture was grown overnight at pH 7 with 100 m M phosphate buffer. No yeast extract or casamino acids were added. Cells were concentrated 20-fold by means of centrifugation. The concentrated cells were lysed in the French press and lysate was obtained as described above. The acid extraction procedure for protamines according to Felix (1960) was applied to the lysate. The lysate was set at pH 4.5 with concentrated acetic acid and left at 4 ° C for 1 h. Flocs were spun down for 10min at 4,0009. The supernatant was decanted and to the flocs five volumes of 1% (V/V) H2SO4 was added and stirred for 15 rain. After this, un- dissolved material was spun down (10,000 g) and three volumes of ethanol were added to the supernatant to precipitate the protein sulphate. The precipitate was recovered by centrifugation (4,000 9), dried under a stream of N2 to evaporate the alcohol and dissolved in water, after which it was freeze dried. For all experiments, 15 mg of the powdery material was dissolved in 10 ml water.

Extraction of lipopolysaccharides. Cells from a 10-1 batch were centrifuged (5,000 g) and resuspended in 175 ml water. The phenol extraction according to Sutherland and Wilkinson (1971) was performed. One repetition of the ultracentrifuga- tion procedure was necessary to obtain a pure fraction. The recovered lipopolysaccharide (LPS) was dissolved in deminer- alized water (3 mg/ml) and experiments were carried out with this solution.

Results and discussion

In a previous publication (Mulder et al. 1989) granule formation by S. ruminantium in AGLR's that were especially developed to retain biomass, was described. On a simple salts medium with glucose as growth-rate-limiting carbon source (1% w/v) a 100-ml AGLR was inoculated with a batch culture of S. ruminantium. When glucose was completely converted, the reactor was set at D = 0.2 h-1 and run for 2 weeks. At this dilution rate the bacteria grew in apparent suspension, but after an increase to D = 0.6 h - 1, wash-out of bacteria took place. However, after 1 day small granules were formed which increased in size and number. After 3 days more glucose conversion was complete again and the granules had a typical diameter of 1-3mm. Induction of granular growth was found to occur only when D exceeded the maximal growth rate.

These granules were chemically analysed and found to contain 15%-20% (w/w) hexose. This result possibly implied that the adhesive polymer was a polysaccharide. However it is known that the organism is capable of accumulating glycogen intracellularly up to 25% of its dry weight even under glucose-limiting conditions (Wallace 1980).

The organism is able to grow on lactate as carbon and energy source (Paynter and Elsden 1970). As polysaccharide formation from this carbon source requires energy, it was not expected that S. ruminantium would form polysaccharides when grown carbon-limited on lactate. Therefore, it was investigated whether granular growth would occur on this substrate. Under this condition granules were still formed, yet the hexose content was found to be only 3%. This is an average value for carbon-limited grown cells (Herbert 1961). We concluded that polysaccharide had no function as an adhesive material.

As proteins and nucleic acids have also been thought to function in aggregate formation (Ni- shikawa and Kuriyama 1969; Weddle and Jenkins 1971), the following depolymerizing enzymes were assayed for deaggregating properties: amyloglucosidase, celtulase, pectinase, lysozyme ( + EDTA), protease, trypsin, RNA-se and DNA- se. None of the enzymes tested could deaggregate the granules. However, it should be realized that the polymers might be ionically or covalently bound within the granule, and therefore could be protected from enzymatic breakdown. A similar mechanism has been described by Nishikawa and Kuriyama (1969) for activated sludge flocs.

Aggregated growth by S. ruminantiurn has been observed before in chemostat cultures (Hob- son and Summers 1972). Indeed, when chemostats were inoculated and kept as pH-controlled batch cultures overnight, microscopically small aggregates of 20-100 cells (clusters) could be observed in the culture broth. However, we would like to stress the point that a clear distinction can be made between these clusters and the granules formed in an AGLR, the latter being visible to the eye (with a typical diameter of 1-3 mm). It is no- teworthy that when the medium pump was turned on (D=0.01 h - 1 and D=0 .1 h-a), clusters washed out of the chemostats and after approxi- mately five volume exchanges the culture broth contained suspended cells only.

A major difference between a steady-state chemostat culture and a pH-controlled batch culture resides in a gradual increase in the concentration of monovalent cations in the latter culture, due to

R. Mulder et al.: Aggregation by S. rurninantium 353

the addition of Na2CO3 needed to neutralize the production of acetic acid and propionic acid. From the final concentrations of these products it could be calculated that the concentration of Na + increased with 80 mM. It was found that addition of 80 mM NaC1 to samples from steady state chemostat cultures (both at D=0.01 h - I and D = 0 . 1 h -1) containing suspended cells could bring about clustering. Ionic strength effects could be ruled out as addition of FeC13 (80 mM) to a chemostat sample did not invoke clustering.

Interestingly, if the cells were washed three times and suspended in a physiological salt solution (155 mM NaC1, 25 mM MES buffer, pH 5.8) no clustering was observed. This suggested that some component from the culture broth was necessary for clustering as well. As pointed out in the introduction, lysis products are implied in certain cases of aggregation. Indeed, addition of lysate (see Materials and methods) to the above-mentioned washed cell suspensions (1 : 1, v/v) resulted in immediate clustering. This result implied that lysis products were present in significant amounts in steady-state chemostat cultures. High lysis per- centages (see Materials and methods) were found, 31.0%+7.5% at D=0.01h-1 ; 12.0%+1.8% at D=0.1 h -1 (in the steady state, clusters were washed out). Selenomonas ruminantium is known to lyse readily and Kamio and Takahashi (1980) ascribe this to the fact that the organism lacks the Brown lipoprotein that connects the petidoglycan layer to the outer membrane. According to Kings- ley and Hoeniger (1973) there is no prolonged sta- tionary phase in the growth curve of the organism, the decline phase sets in almost immediately after the peak of growth has been reached. It can be appreciated therefore that in the chemostat run as a batch culture overnight, a large part of the population was lysed.

Nucleic acids and proteins might be components responsible for clustering (Nishikawa and Kuriyama 1969; Weddle and Jenkins 1971). In order to separate the two, lysate was titrated with protamines (commercial basic proteins with a high affinity for nucleic acids). Surprisingly, when in a control experiment protamines were added to a washed cell suspension (0.15 mg/ml suspension), clustering was invoked. Hence, it seemed logical to assume that basic proteins in the lysate were responsible for aggregation. In order to obtain these proteins the extraction procedure for protamines (Felix 1960) was applied to lysate. A fraction (30 mg/g dry wt.) was obtained, which mainly consisted of protein (70% w/w). When added (0.15 mg protein extract/ml chemostat cul-

ture) to suspended chemostat cells of S. ruminantium, this fraction induced immediate cluster formation. Similarly, protein fractions capable of in- voking cluster formation with S. rurninantium could be obtained from other chemostat-grown Gram-negative organisms (Escherichia coli, Kleb- siella aerogenes and Pseudomonas putida) and extraction yields were comparable with S. ruminantium (data not shown). These results imply that the protein extracted from S. ruminantium was not specific.

Ribosomal proteins are known to be basic (B6ck 1985). Furthermore, high monovalent ca- tion concentrations are known to bring about re- moval of protein from ribosomes (Furano 1966). To investigate whether the adhering protein was of ribosomal origin, pure ribosomal 30S subunits from S. ruminantium cells were isolated. Subse- quently, the protamine extraction procedure was applied to these subunits and again a protein fraction was obtained with which clustering of suspended cells could be invoked (0.15 mg protein extract/ml chemostat culture). Although a role of basic cytosolic proteins cannot be excluded at this point, our results strongly indicate that basic ribosomal proteins function as the adhesive material in clusters of S. ruminantiurn.

The question then arises as to the site of attachment for the positively charged protein. Since in the Gram-negative S. ruminantium lipopolysaccharide (LPS) is the main outer cell wall polymer (Kingsley and Hoeniger 1973), it was investigated

IOO

8 0 " 0

~ ~o

C:~ ._ N ~a 0

4 ~ I~ I~

N ae r

~ eo ~1.

~O

O~ 0 . ~

r i I I

o~o o.~o o.~o oao ~.oo

mg LPS/mg protein

Fig. 3. Affinity experiment between extracted protein and extracted lipopolysaccharide (LPS). Protein and LPS were mixed in severa! ratios, flocs were spun down and protein was determined in the supernatant

354 R. Mulder et al. : Aggregation by S. ruminantium

whether LPS showed any affinity for the isolated protein. Pure LPS was isolated from S. ruminantium and incubated in various ratios with isolated protein. This resulted in flocculation. As can be seen in Fig. 3, with LPS/protein ratios (w/w) of 0.1 to 0.5 more than 60% of the protein was extracted from the solution by the LPS. Moreover, at these ratios the supernatant lost the ability to aggregate suspended cells. A decrease in the amount of protein spun down can be observed at higher ratios (Fig. 3). This can be ascribed to the increasing viscosity of the solution at higher LPS concentrations.

The negatively charged carboxyl and phosphate groups of the LPS form a potential site of attachment for the positively charged protein. A further indication of the involvement of LPS in aggregation was found; a chemostat culture that had been run for 3 months at D = 0.1 h-1 lost the ability for protein-induced aggregation. An LPS extraction performed on these cells yielded 16 mg LPS/10 g dry weight of cells as compared to 60 mg LPS/10 g dry weight of cells from the origi- nal cells. Probably a selection had taken place for cells not wasting energy on cell wall components that are superfluous in a chemostat.

Escheriehia coli, K. aerofenes and P. putida were tested for protein-induced aggregation. Ad- dition of the protein extracted from S. ruminantium to chemostat samples containing suspended cells (0.15 mg protein/ml chemostat culture) did not lead to cluster formation. However, when the cells were washed with a physiological salt solution prior to protein addition, E. coli and P. putida showed immediate clustering. An explanation could be that divalent cations, which stabilize the

Table 1. Granular growth in four mini anaerobic gas-lift reactors (D = 0.77 h-1) in relation to protein extract addition, medium pH and growth rate of inoculum

Growth Protein Medium Granular Reactor pH rate of added pH biomass in after 12 h inoculum 12 h ( h - 1) (rag/reactor)

0.01 + 5.9 4.0±0.4 5.9±0.0 0.01 - 5.9 0.0±0.0 5.9±0.0 0.01 + 6.2 3.9±0.4 6.0±0.1 0.01 - 6.2 1.4±1.4 6.2±0.0 0.01 + 6.4 1.6±0.5 6.2±0.1 0.01 - 6.4 0.7±0.6 6.4±0.0

0.1 + 5.9 0.0±0.0 5.9±0.0

A 5 ml sample from a chemostat was inoculated in a mini anaerobic gas-lift reactor with or without addition of protein (0.15 mg protein extract/ml chemostat culture)

negatively charged carboxyl and phosphate groups in the Gram-negative cell wall (Schindler and Osborn 1979), were washed away. Thereby the negatively charged groups became available for the positively charged protein. This suggests that divalent cations are relatively weakly bound to the LPS of S. rurninantium.

To test whether S. ruminantium clusters from a chemostat culture could function as nuclei for the formation of granules, small anaerobic gas-lift reactors (mini-AGLR's) were constructed. Ino- cula from chemostats from which clusters had not been washed out completely gave rise to granular growth in these reactors (D=0.77+0.07 h - l ) within 12 h. Inocula from chemostats containing suspended cells only were completely washed out in this period of time unless protein extract from S. ruminantium was added. Thus, it seems justi- fied to suppose that granules evolve from clusters. As can be seen in Table 1 granulation by protein addition was pH-dependent. At pH 6.4 granular growth could not be induced. In support, the start-up procedure to invoke granular growth in a 100-ml AGLR (see above), failed at pH 6.4. The bacteria washed out of the reactor completely when the dilution rate was raised to 0.6 h-1.

Surprisingly, cells grown at D = 0.1 h - 1 showed immediate clustering when protein was added but inoculation in the mini-AGLR's (pH 5.9) did not lead to granular growth within 12 h (Table 1) and the cells washed out completely. Electron microscopical observations showed that cells grown at D = 0 . 0 1 h -1 lacked flagella whereas at D=0.1 h -1 flagella were present. These constantly moving organelles possibly served as a counter force opposing aggregation.

The effect of pH decrease on cluster formation was assayed. Whilst no effect was observed with suspended cells from a chemostat culture, a lowering of the pH from 5.8 to 4.5 of a 1 : 1 mixture of a chemostat sample and lysate resulted in immediate clustering. When the lysate was preincu- bated with protease or trypsin (0.3 mg/ml, incu- bation time 1 h at 37 ° C), this was not observed.

The results presented in this paper suggest the following mechanism for granule formation by S. rurninantium. Firstly, clusters are formed due to the release of ribosomal proteins. Once clusters have formed, physiological gradients will emerge due to diffusion limitations. Going from the out- side to the inside of the cluster, substrate concentrations as well as pH will decrease. While the former will promote lysis the latter could induce aggregation. By such a mechanism dividing cells could be entrapped and thus contribute to the

R. Mulder et al.: Aggregation by S. ruminantium 355

growth of a cluster. This mechanism is in agree- ment with the observation that substantial parts of the granules consist of lysed cells (Mulder et al. 1989).

In this paper the mechanism underlying aggregation by S. ruminant ium is clarified. It can be concluded that the specific feature of S. ruminantiurn to form aggregates firstly lies with its ten- dency to lyse easily and secondly with its characteristic lipopolysaccharide.

Since the organism is responsible for granule formation in a glucose acidifying AGLR, this knowledge could contribute to better control and maintenance of acidifying reactors containing im- mobilized biomass (Beeftink and van den Heuvel 1987; Heijnen 1984). Although granulation by S. ruminant ium is a specific characteristic of the organism, the underlying mechanism is rather non- specific and might be applicable to other Gram- negative microorganisms.

Acknowledgements. These investigations were supported by the Netherlands Technology Foundation (STW), Technical Science Branch of the Netherlands Organization for the Ad- vancement of Pure Research and by Gist-brocades. The expert technical assistance in electron microscopy of E. Pas (Univer- sity of Amsterdam) is gratefully acknowledged.

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Received 13 April 1989/Accepted 14 July 1989

the mechanism of aggregate formation by selenomonas ruminantium

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