Journal of Applied Bacteriology 1985,59, 7-16 155914184

The effect of bacteria on the solubilization of silica in diatom frustules

SHEILA PATRICK 8z A.J. HOLDING Department of Agricultural and Food Bacteriology, The Queen’s University of Belfast, Newforge Lane, Belfast BT9 5 P X , U K

Received 18 April 1984, revised 28 January 1985 and accepted 12 February 1985

PATKICK, SHEILA & HOLDING, A.J. 1985. The effect of bacteria on the solu- bilization of silica in diatom frustules. Journal of Applied Bacteriology 59, 7-16.

Natural bacterial populations in samples of water from Loch Leven and Lough Neagh increased the rate of solubilization of frustule silica from an axenic Cyclotella meneghiniana culture, compared with sterile autolysis, at 25°C. In the inoculated cultures 50-60% of the silica was solubilized over a period of 30 d. Bacterial populations in Loch Leven water also enhanced the solubilization of silica from non-axenic cultures of Asterionella formosa, Tabellaria ffocculosa, Navicula pellicu- losa and C . meneghiniana, compared with control cultures sterilized with mercuric chloride. Similar results were obtained with Lough Neagh populations incubated with A . forrnosa. In comparison with untreated cells, the treatment of diatom cells with ultra-sonication did not increase the release of silica. Pure cultures of bacteria from Loch Leven water enhanced the release of silica from non-axenic A . forrnosa and axenic C. meneghiniana compared with sterile control treatments. The variation in the ability of cultures to solubilize the frustule silica appeared to be related to their potential to produce hydrolytic enzymes. Natural populations of Loch Leven and Lough Neagh water bacteria and certain bacterial cultures caused the diatoms to aggregate, which did not enhance the release of silica.

Little or no information is available about the effects of pure or mixed cultures of aquatic bac- teria on the rate of solubilization of frustule silica under laboratory conditions. In this paper we present the results of investigations on the effect of natural populations and pure cultures of aquatic bacteria from Loch Leven, Kinross, Scotland and Lough Neagh, Northern Ireland on the rate of solubilization of silica from diatom cultures.

The resolubilization and internal recycling of the silica in diatom frustules can provide the soluble silica that is essential for further growth of diatoms in freshwater lakes. This supply may be extremely important in localities such as Loch Leven (Bailey-Watts 1976) and Lough Neagh (Battarbee 1978), which receive an insig- nificant input of dissolved silica from external sources either on a general or seasonal basis. These two lakes are similar in their morphom- etry and gross chemistry despite their difference in area (Lough Neagh measures 367 km2 and

Loch Leven 13.3 km2) (Gibson et al. 1971). They are both shallow, continually mixed by local winds and have been subject to cultural eutrophication in recent years (Royal Society of Edinburgh 1974; Battarbee 1978).

Several factors, e.g. species of diatom, environmental temperature and pH have been shown to influence the solubility of the silica in diatom frustules under laboratory conditions. With non-axenic algal cultures, J4rgensen (1955), Kamatani (1969, 1971) and Kamatani & Riley (1979) showed that silica is solubilized at different rates and in different amounts from various diatom species. For example, Kamatani & Riley (1979) showed that Nitzschia Zinearis frustule silica was completely dissolved after 40 d, whereas only 20% of Thalassiosira nana silica was solubilized over the same period. Lewin (1961), Bailey-Watts (1976b) and Kama- tani & Riley (1979) reported increased silica dis- solution with a higher incubation temperature. Diatom silica is amorphous, and therefore

8 S . Patrick and

should be more soluble at pH > 9 (Stumm & Morgan 1970), and J4rgensen (1955) reported optimum dissolution of frustules in carbonate buffer at pH 10. Lewin (1961) showed an increase in the rate of silica dissolution from acid-cleaned frustules with an increase in pH. From pH 6.2 to 7.2, the concentration of silica released during 10 d increased from approx- imately 7 mg Si/l to 23 mg Si/l, whereas at a pH of 9.2 44 mg Si/l was released. This author also reported that when a Nauicula pelliculosa culture was heat-killed or treated with protein de-naturing agents, the release of silica increased from 1-2 mg Si/l to 6 9 mg Si/l in 50 d at 19°C. Up to 46 mg Si/l (a saturated solution) was rel- eased after all the organic matter had been removed with acid treatment and the treatment of heat-killed cells with oxalate or EDTA had a similar effect. This increased release could be prevented by the addition of solutions contain- ing aluminium, iron, or titanium (0.0037 molil). Lewin (1961) also reported that heat-killed marine phytoplankton released more silica than a heat-killed axenic Navicula pelliculosa culture. All the N. pelliculosa experiments were carried out in 0.01 mol/l Tris buffer at pH 9-9.3 (Golterman 1960) showed that only 2&30% of the silica in an axenic Stephanodiscus hantzschii culture went into solution during incubation for 21 d whereas 60% of the silica was released after the death of a diatom bloom in an experi- mental tank. These differences may be attribut- able to the activity of bacteria associated with the natural phytoplankton population, although this was not investigated.

The possible importance of bacteria on the solubilization process has been noted by Krum- bein (1978). Electron dense X-ray spot analyses and scanning electron microscopy were used to correlate the points of bacterial attachment with a reduction in the silica content in that area of the frustule. These studies were undertaken within algal mats in Solar Lake, Sinai.

Materials and Methods

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

Non-axenic cultures of Tabellaria Jocculosa (Roth) Kutzing var. asterionelloides Knudsen CCAP1081/1, Asterionella forrnosa Hassall CCAP 1005/1 b and Nauicula pelliculosa Breb CCAP1050/3a were obtained from the Culture

A . J . Holding

Centre of Algae and Protozoa (CCAP) Storey’s Way, Cambridge. Cyclotella rneneghiniana was isolated from the River Cam, Cambridge by Dr H. Belcher, CCAP and an axenic culture was produced by the method of Pringsheim (1946). All the strains were subcultured monthly into 100 ml flasks containing the Woods Hole MBL (WH) medium (Nichols 1973) and placed on a north facing window sill, protected from direct sunlight. Duplicate cultures were kept in incu- bators artificially illuminated and maintained at 12-15°C. The C. rneneghiniana culture did not survive at low temperatures and was maintained at 20°C. Diatoms were grown in bulk for the silica dissolution experiments, either in 2 1 flasks containing the WH medium or in the case of N . pelliculosa, in Roux bottles on Chu 10 agar medium (George 1976).

B A C T E R I A L P R O C E D U R E S

The isolation, routine maintenance and counting of bacteria were carried out on casein peptone starch (CPS) agar (Collins & Wil- loughby 1962). The identification scheme of Gibson et a/. (1977) was used for the preliminary identification of bacteria. Fat and protein hydrolysis were detected on media containing (g or ml/l) peptone, 5 ; yeast extract, 3; agar (Oxoid No. l), 10; and either glycerol tributyrate (BDH), 10; or antibiotic-free skim milk powder (Oxoid L54), 10. Starch hydrolysis was deter- mined by flooding starch nutrient agar plates with an iodine solution. Proteolytic activity was also determined by stab-inoculating tubes of gelatin and by the addition of charcoal gelatin discs to broth cultures. A range of hydrolase activities was also determined with the API ZYM system (API Laboratory Products Limited, Basingstoke).

S I L I C A A N A L Y S E S

Soluble silica was determined colorimetrically by the method of Strickland & Parsons (1968) using a Pye Unicam SP6-400 spectrophotom- eter. This method detects soluble silica at con- centrations between 0.1 and 140 pg-atoms Si/l. For total silica measurements, washed diatom cells in silica-free WH medium were dried over- night at 80°C in platinum crucibles. The cells

Bacterial solubilization of frustules were then ashed by placing in a cold furnace, heating to 1000°C and then fusing with excess sodium carbonate (cu 0.2 g) over a fully oxidized Bunsen burner flame. The melt produced was dissolved in distilled water with gentle heating. The resulting alkaline solution was neutralized with molar sulphuric acid and the solution made up to 25 ml in acid-soaked volumetric flasks which had been rinsed with distilled water immediately before use. This solution was immediately diluted to prevent poly- condensation of the concentrated silica solution and a soluble silica analysis performed. With this method, 99% of the silica in a standard of dried, spectrophotometrically pure silicon dioxide was recovered.

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

D I S S O L U T I O N

In general, four-week old cultures of diatoms were used for the decomposition experiments, although cultural conditions varied throughout the year. As the silica content of diatom cells and silica release during decomposition can vary with cultural conditions (Werner 1977) the percentage silica released in different experi- ments is not directly comparable. Before each experiment the diatom cultures were examined under the light microscope ( x 1200 magnification) to ensure that the cells were still viable and morphologically typical. The cultures were washed three times in silica-free WH medium or lake water. When required, diatoms were heat-killed by incubating in a 60°C water bath for 3 h (Golterman 1960). The diatoms were suspended in the appropriate medium (e.g. lake water) and soluble silica was determined in 5 ml samples removed after the diatoms had been pelleted by centrifugation. The 5 ml amount was then replaced with sterile medium.

The silica concentration in lake water and sterile lake water, without added diatom culture, was also monitored. Any difference in silica con- centration resulting from the decomposition of the natural diatom population was therefore accounted for. The results are expressed as the percentage soluble silica released from the added diatom culture. Four replicates of each treatment were set up and the results are expressed as the mean +standard error, unless otherwise stated.

9 Results

S I L I C A RELEASE FROM D I A T O M C U L T U R E S

To determine the effect of lake bacteria on frus- tule solubility, an (initially bacteria-free or) axenic culture of C. meneghiniunu was examined in the presence and absence of aquatic bacteria. Active and heat-killed diatoms were compared. The diatom culture was suspended in silica-free WH medium, untreated or heat-sterilized Loch Leven water (1.6 x lo9 cells/ml; 2.6 pg total diatom culture SiOJml) and in Lough Neagh water (5.0 x lo4 cells/ml; 5.8 pg total diatom culture SiOJml). Incubation was in the dark at 25°C. The percentages of the initial total diatom silica solubilized during the experiments are pre- sented in Figs la, b and c.

In the presence of the Loch Leven water and associated microflora, the release of soluble silica from an initially active culture increased rapidly from day 11 to day 25 and by day 42 60% of the frustule silica had been solubilized (Fig. la). The reduction in concentration of soluble silica detected in the first few days with the sterile Loch Leven water and untreated diatom culture treatment may have been caused by the uptake of released silica by viable diatom cells. The process is known to occur in the dark (Werner 1977). The subsequent slow release is clearly far less than in the presence of the bac- teria. In these experiments 40% or more of the silica remained undissolved. Using Lough Neagh water as the inoculant (Fig. lb) the sub- stantial increase in the rate of solubilization with both the active and heat-killed culture in the presence of bacterial populations is again apparent. The results show a similar increase in the release of silica from heat-killed and active cells in the presence of lake bacteria; however, more silica was released from the heat-killed culture in either sterile lake water (Fig. lb) or WH medium (Fig. lc). Lewin (1961) also report- ed an increase in silica release after heat-killing axenic N . pelliculosa. The pH of the WH medium remained the same throughout the experimental period but in all the Loch Leven water treatments the pH increased from pH 7.3-8.0 and the Lough Neagh treatments decreased from pH 8.3-8.0. The differences in silica release among the treatments were there- fore not related to gross pH change. A change of pH in the microenvironment, however, may be

S . Patrick and A . J . Holding

Time ( d )

70

60

p 50

n N .- - - 3

40 " .- - - m

30

20 -

10 ... B' P

-

I 1 I 1 I0 20 30 40 50

Time [ d )

Fig. 1. The percentage of silica solubilized from active and heat-killed (H-K) Cyclotella rneneghiniona cells (axenic culture); the influence of untreated (solid symbols) and heat-sterilized water (open symbols) from (a) Loch Leven (b) Lough Neagh and (c) silica- free Woods Hole MBL (WH) culture medium. 0, ., Active cells; 0, m, H-K cells. I = standard error in all figures (n = 4).

important. It is interesting that the rate of silica release from Lough Neagh sediment core samples increased 21% over the pH range 7.8& 9.05 (Rippey 1977) although Dickson (1975) had found no correlation between water pH changes and soluble silica flux in the lough itself.

During the course of these experiments the diatoms and bacteria formed aggregates or clumps, which were visible to the naked eye after 3-5 d incubation. These aggregates were formed with active or heat-killed cultures in Loch Leven or Lough Neagh water, but not in sterile lake water or WH medium. It was there- fore impossible to determine the morphological state of the diatoms with the light microscope ( x 1200 magnification), although frustular material could be identified within the clumps. The cells in the other treatments gradually lost their cytoplasmic contents; the frustules, however, remained largely intact. Axenic cul- tures of diatoms other than C . meneghiniana were not available. Therefore, cultures of Tabel- laria Jlocculosa, Asterionella formosa and Navic- ula pelliculosa were heat-killed to reduce

Bacterial solubilizution of frustules 1 1 baterial contamination to a low level and sus- pended in both untreated lake water and water sterilized with mercuric chloride ( 1 g/l). The effect of untreated lake water, containing bac- teria, is again evident (Figs 2a and b). The importance of biological as opposed to purely chemical and physical processes in silica solu- bilization is emphasized by the low rates ob- tained in all cultures in the presence of HgCI, . Strict comparison of the amounts of silica rel- eased from these diatoms may not be valid as different cultural conditions can result in solu- bility differences with only one diatom type (Werner 1977). The percentage of solubilization from N . pelliculosa, a small diatom is, however, significantly lower than with the larger diatoms. It is possible that the nutrients available for bac- terial growth vary with the size and content of the diatom cell. Therefore, the interrelationship of diatom cell volume, surface area and frustule thickness could relate to the amount of silica solubilized if the diatom cell contents provide growth limiting nutrients for the bacteria and

1 D,

silica solubilization is mediated by bacteria attached to the frustule surface. The surface in this case could be defined as the area available for bacterial attachment and not the ‘real’ surface area of the complex pore structure of the frustule.

E F F E C T OF U L T R A S O N I C T R E A T M E N T O N

S I L I C A S O L U B I L I Z A T I O N

Invertebrate grazing may influence silica release by physical disruption and breakage of frustules. Tessenow (1966) obtained enhanced silica release from Asterionellu sp. incubated in the presence of Daphnia magna. Invertebrate grazing in the water column in Loch Leven affects the diatom population for only short periods, soluble silica availability being the major con- trolling factor (Bailey-Watts 1976a). Grazing, however, could affect the decomposition of diatoms in the benthos. To assess the possible importance of physical disruption, cells of C. meneghiniana were ultra-sonicated in an MSE

Time ( d ) Fig. 2. The percentage of silica solubilized from heat-killed (H-K) cells of Tabellaria ffocculosa, Asterionellu fbrmosa, Nauicula pellicrtlosa and Cyclotella meneghiniana cultures; the influence of untreated (solid symbols) and HgCI, sterilized (open symbols) (a) Loch Leven water (b) Lough Neagh water. 0 , 0, Asterionellaformom; 8 , 0 . Tabellaria,flocc.ulosa; A, A , Cyclotella meneghiniana and V, V, Navicula pelliculosa.

12 S . Patrick and sonicator until 90% of the frustules were shat- tered into small fragments (10 x 30 s exposures). Despite the increase in frustule surface available for bacterial attachment, solubilization from broken cells was approximately the same as from heat-killed cells in the presence of the water bacteria (Fig. 3). A similar result was obtained when whole and broken C. meneghin- iana and N . pelliculosa were incubated in Loch Leven water (Patrick 1981). Lewin (1961), found no difference in the silica released from heat- killed and axenic N . pelliculosa cells which had been broken in a French pressure cell.

70

60

p 50- N - - - n

2 40- = - 0

v, - - -

30-

20

10

-

-

-

-

L I 1 I 1 1 H) 20 30 40 50

Fig. 3. The percentage of silica solubilized from heat- killed (H-K) and ultra-sonicated (U-s) Cyclotella meneghiniana cells (axenic culture); the influence of untreated (solid symbols) and heat-sterilized (open symbols) Lough Neagh water. 0, 0, U-S cells; .,

, H-K cells.

EFFECT O F P U R E B A C T E R I A L C U L T U R E S O N

T H E S O L U B I L I Z A T I O N O F T H E S I L I C A

The solubilization of silica from diatom cultures inoculated with pure cultures of bacteria from Loch Leven water was investigated to determine if individual bacterial types were active in the process. Cultures of A. formosa and C. meneghin-

A. J. Holding iana suspended in heat sterilized lake water, were inoculated with five pure cultures of Loch Leven bacteria (approximately lo5 cfu/ml). All the isolates were Gram-negative rods. They were identified on the basis of routine biochemi- cal tests as enterobacteria (Yl, W1 and W2), Flavobacterium (Y2) and a Pseudornonas spp.

Figures 4a and b illustrate that the pure cul- tures did not enhance silica release to the same extent as untreated lake water. This could reflect either the more efficient action of the complete populations or denatured nutrients in the heat- sterilized lake water. In all instances the silica release was faster than in the sterile controls. There was an initial drop in silica concentration in the presence of the pseudomonad (C). The reason for this apparent uptake is not clear, although bacterial assimilation of silica has been reported (Heinen 1962). Isolate Y1 was the most efficient solubilizer of A . formosa, although W2 was as efficient in the C. meneghiniana suspen- sion (Fig. 4b). Isolates W2 and Y2 formed aggre- gates with the diatom cells, although Y1 and W1 did not, they were equally efficient at silica solubilization. All the isolates were recoverable from the suspensions at the end of the experi- ment. Differences in the silica released, however, were not related directly to the total viable counts of the bacteria at the end of the experi- ment.

(C).

H Y D R O L Y T I C P O T E N T I A L OF T H E B A C T E R I A L C U L T U R E S FROM L O C H LEVEN

The range of extracellular enzymes produced by these organisms was compared to determine if the potential to produce these enzymes was related to the release of silica from the diatoms. Breakdown of starch, casein, tributyrin and gelatin were examined and the API ZYM system was also used. Noticeable differences occurred in the range of amidases, sugar hydro- lases and proteolytic enzymes produced by the isolates (Table 1). The pseudomonad (C) which was least efficient in silica release did not produce any of these enzymes, whereas Y1 which was most efficient produced the widest range. Although the enzymes tested may not necessarily be active against components of the diatom cells, there is some indication that hydrolytic enzyme activity and silica solu- bilization are related. The possible implications

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14 S . Patrick and A . J . Holding

Fig. 4. The percentage of silica solubilized from cells of (a) heat-killed Asterionella,formosa and (b) active Cyclo- rellu mrnryhiniana (axenic culture): the influence of untreated, HgCI, sterilized and heat-sterilized Lough Neagh inoculated with pure cultures of Loch Leven bacteria. (Each point in 4a represents the mean of duplicate treatments). 0, Untreated water; V, HgCI, sterilized water; 0, C ; A, Y2; 0, Y1; A, W1; m, W2; V, heat sterilized water. The final total viable count cfu/ml (millions) for (a) untreated water, 14; Y1, 3.4; Y2, 1.8; WI, 4.7; W2,0.45; C, 1.7 and (b) untreated water, 0.4; Y1, 5.0; W2, 2.0.

of these findings are discussed briefly below in relation to general biochemical features of the diatom cell wall.

Discussion

The results provide laboratory data which indi- cate that bacteria associated with diatom cells might play an important role in the solu- bilization and re-cycling of silica and that bac- terial hydrolytic activity is associated with silica release. The precise mechanism(s), however, used by the bacteria to dissolve the silica is not known. Several different mechanisms may be responsible.

The primary site of the bacterial colonization of diatoms is likely to be the organic layer which covers the frustule. Hecky et al. (1973) described the content of this layer and postu- lated the following chemical structure; an outer,

mannose-rich polysaccharide layer, covering an unspecified region containing lipid and under- neath a protein layer consisting of an amino acid ‘template’ chemically bound to the amor- phous silica frustule. Assuming that bacteria are capable of decomposing this organic material, its removal may be sufficient to enhance solu- bility; Lewin (1961) reported that removal of the organic component by acid treatment enhanced solubility. Bacteria which produce hydrolytic enzymes specific for the polysaccharides of higher algae have been reported (Percival & McDowell 1967). Active solubilization of the silica by metabolic by-products of the bacteria may also occur. For example, pH changes in the micro-environment could result in localized sites of solubilization. The release of cytoplasmic contents, which may be associated with dis- ruption of the outer organic layer, may further enhance bacterial growth. The production of

Bacterial solubilization of frustules 15 chelating agents by bacteria, resulting in the solubilization of inorganic silicates and phos- phates has been reported (Webley & Duff 1965). A similar mechanism could be important in frustule solubilization as metal ions retard silica release (Lewin 1961).

In the presence of lake bacteria, the breaking of frustules by sonication to simulate any inver- tebrate action did not increase the silica relrase compared with unbroken cells. It would be interesting to determine the role of bacteria within the digestive tracts of invertebrates. Diatom cells can be seen in faecal pellets of invertebrates. These cells are surrounded by a layer of organic material which is subject to bacterial decomposition after excretion (Ferrante & Parker 1977).

The formation of detrital clumps containing diatoms and other plankton has been reported (e.g. by Paer! 1973). The results of the present study indicate that aquatic bacteria are associ- ated with this aggregation. Under laboratory conditions these aggregates did not influence the rate of silica release. In the natural environment, however, where diatom and bacterial concentra- tions may be lower, it could be important. For example, sedimentation may be faster, resulting in enhanced sediment microbial activity.

Eventually physical and chemical changes in the frustule convert the amorphous silica of diatoms to the relatively insoluble ‘opaline’ silica of fossil diatoms (Lewin 1961). Whether diatom silica is solubilized or not depends on the balance between this aging process and the factors involved in frustule solubilization.

The results and discussion emphasize the complexity of the factors determining the rate of recycling of the silica. Bacterial growth could influence frustule solubility in the following eco- logical niches: (a) when the diatom cells are colonized either after death within the water column or during sedimentation to the benthos; (b) within the digestive tracts of invertebrates and in association with faecal pellets, after they are voided. Finally, nutrients, including glyco- proteins, proteins and other organic molecules accumulate at solid/liquid interfaces in aquatic environments (Marshall 1980). If this secondary accumulation of nutrients occurs at the frustule surface, bacterial decomposition of these com- pounds could increase the associated rate of silica release. The relative importance of these factors is yet to be determined.

The authors wish to acknowledge the help and advice received from the following: Mr M. Saunders, Department of Geology, University of Edinburgh, the staff of the Wetlands Research Group, Institute of Terrestrial Ecology, Edin- burgh and the staff of the Culture Centre for Algae and Protozoa, Cambridge. Part of this research was carried out in the Department of Microbiology, University of Edinburgh and S.P. was the holder of a Natural Environmental Research Council Research Studentship.

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