Microb Ecol (1983) 9:317-328
MICROBIALs 9 1983 Springer-Verlag
Bacterial Activity at the A i r / W a t e r Interface Malte H e r m a n s s o n and B j r r n Dahlb/ick Department of Marine Microbiology, Botanical Institute, University of Grteborg, Carl Skottsbergs Gata 22, S-41319 Grteborg, Sweden
Abstract. By using substrate molecules o f varying degrees o f surface activity, we were able to measure some features o f bacterial activity in the surface microlayers (SM) and in the subsurface (bulk) water. The fraction o f active cells was d e t e r m i n e d by a c o m b i n e d m i c r o a u t o r a d i o g r a p h y - e p i fluorescence (ME) method. Measurements were m a d e o f 14CO 2 evolution to determine the rate o f respiration. Results from in situ m e a s u r e m e n t s showed no significant difference between fraction o f active cells in the SM and in the bulk. This m a y be due to an exchange o f bacteria between SM and bulk. This exchange was assessed by spreading a film o f 3H-palmitic acid on the surface and, after incubation, measuring the a m o u n t o f labeled cells at the surface and in the bulk. Test bacteria showing a high accumulation at the surface also showed a low exchange between the 2 strata. W h e n low concentrations o f added ~4C-protein were used, the respiration m e a s u r e m e n t s showed a lower value for bulk than for interface localized protein. At higher concentrations, the evolved 14CO2 was the same whether the protein was m i x e d in the bulk or spread at the surface. W h e n 2 . 4 - 1 2 n g . c m -2 o f ~4C-palmitic acid was spread on the surface, there was a linear relation between t u r n o v e r time and a m o u n t o f added substrate. At higher substrate concentrations there was a deviation f r o m the straight line. Resuits are discussed in terms o f the unique habitat found at an interface.
Introduction The p h e n o m e n o n o f bacterial accumulation at the air/water interface is well d o c u m e n t e d [6, 39, 45]. There is also an increasing a m o u n t o f work being done on the mechanisms o f adhesion o f bacteria to this interface [29, 39]. T h e question o f microbial activity at the air/water interface has been addressed by several investigators. Measurements have been done on bacterial uptake and respiration o f various types ofsubstrates [4, 8, 1 l, 38, 4 l, 43], algal photosynthetic activity [2, 14, 20], biochemical activities o f bacterial isolates [9, 25, 30, 44], nitrification [26], the fraction o f respiring bacteria [22], as well as n u m e r o u s estimates o f viable counts [2 l, 36]. It is clear that there are differences a m o n g bacteria in a natural habitat with respect to the surface characteristics that are related to adhesion [ 10]. T h e r e appears to be a correlation between these characteristics and the scavenging
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a b i l i t y o f b a c t e r i a a t a s o l i d / l i q u i d i n t e r f a c e [28]. It f o l l o w s t h a t a c t i v i t y m e a surements of bacteria at an interface should be put into an adhesion context. In most investigations of the activity of microorganisms in the surface mic r o l a y e r s , t h e s u r f a c e s a m p l e s a r e i n c u b a t e d i n t h e s a m e w a y as t h e b u l k s a m p l e s [2, 4, 11, 20, 26, 38, 41, 43]. F o r e x a m p l e , a v o l u m e o f t h e s u r f a c e m i c r o l a y e r s is c o l l e c t e d w i t h a s c r e e n - s a m p l e r a n d s u b s e q u e n t l y i n c u b a t e d i n a flask. T h i s means that microorganisms from the surface are transferred to a totally new e n v i r o n m e n t . I n c o n t r a s t , t h e m e t h o d s w e h a v e u s e d a r e in s i t u i n c u b a t i o n s . T h i s r e v e a l s n e w a s p e c t s o f b a c t e r i a l a c t i v i t i e s a t i n t e r f a c e s . A l s o , t h e use o f different methods are discussed. In these experiments, exchange of molecules a n d o r g a n i s m s b e t w e e n t h e i n t e r f a c e a n d t h e b u l k is still p o s s i b l e t h r o u g h o u t t h e i n c u b a t i o n , a n d t h e s t r a t i f i c a t i o n o f t h e s y s t e m is n o t d i s r u p t e d . T h e a p proach of measuring on a relatively undisturbed interracial stratum has also b e e n u s e d b y G a l l a g h e r [14]. W e b e l i e v e t h a t t h i s is a v e r y i m p o r t a n t p o i n t b e c a u s e t h e q u e s t i o n o f h o w t h e i n t e r f a c e affects t h e b a c t e r i a l a c t i v i t y c a n b e s t be answered by investigations of bacteria actually at the interface. We have carried out measurements on bacterial activity using amino acids, a f a t t y a c i d , a n d a p r o t e i n m i x t u r e as s u b s t r a t e s . T h e 2 l a t t e r s u b s t r a t e s a r e c o n s i d e r e d to b e i m p o r t a n t c o n s t i t u e n t s o f t h e s u r f a c e m i c r o l a y e r [3, 19, 27, 35]. T h e u s e o f f a t t y a c i d s a n d p r o t e i n s is a n a t t e m p t to s p e c i a l l y c o n s i d e r t h e u n i q u e e n v i r o n m e n t o f t h e s u r f a c e m i c r o l a y e r . I n t h i s s t u d y w e u s e d (1) a c o m b i n e d m i c r o a u t o r a d i o g r a p h y - - e p i f l u o r e s c e n c e m e t h o d [47] to d e t e r m i n e t h e f r a c t i o n o f a c t i v e cells a n d (2) m e a s u r e m e n t s o f e v o l v e d 14CO z to d e t e r m i n e t h e r a t e o f r e s p i r a t i o n in t h e s u r f a c e m i c r o l a y e r s a n d i n t h e s u b s u r f a c e w a t e r .
M a t e r i a l and M e t h o d s Radiochemicals, Test Bacteria, M e d i u m a n d N i n e Salt Solution ( N S S ) The radiochemicals used were L- 14,5-3H I-leucine, 1.0 mCi- ml- ~, 136 Ci. mmol-~, Amersham; L- IG-all [-glutamic acid, 1.0 mCi- ml- ~, 38 Ci" mmol- t, Amersham; 19,10-3H(N) I-palmitic acid, 10.0 mCi-ml -~ 15.2 Ci-mmol -~, New England Nuclear; [ 1-t4C I-palmitic acid, 100 ~Ci.ml -t, 55 mCi.mmol -j, Amersham; and I~'C(U)I-protein from E. coil B/r, 52.6 ~zCi.mg-~, New England Nuclear. Serratia marcescens EF 190 wildtype (w) and a mutant (m) and Pseudomonas halocrenae ATCC 19712 were used as test bacteria. The mutant of S. rnarcescensis devoid of a red pigment (prodigiosin) which makes it less hydrophobic [Kjelleberg et al. 33]. The bacteria were cultivated in V medium [49]: peptone 1.0 g; yeast extract 0.5 g; glucose 0.5 g; starch (soluble) 0.5 g; FeSO4. 7H20 0.01 g; Na2HPO4 0.01 g; nine salt solution (NSS) 1,000 ml. NSS consists of NaC1 23.48 g; Na2SO4 1.96 g; NaHCO3 0.10 g; KC1 0.33 g; KBr 0.05 g; MgCI2-2H20 2.49 g; CaCI2-2H20 0.55 g; SrCI2.6H20 0.01 g; H3BO 3 0.01 g; double-distilled water 1,000 ml.
In Situ Incubations In the field study, the in situ uptake of 3H-leucine by bacteria in the surface microlayers and in the bulk was determined. Near-shore seawater (0.5 liter) was collected at 4 separate sampling times in July and August, with glass dishes (15.5 cm, d.). The dishes were placed in shallow water. After 2 hours, radioactive substrate was added with a syringe into the bulk phase. Five or 25 ul 3Hleucine was diluted in 5 ml of seawater and injected through the surface. This corresponds to an addition of the amino acid in the picomolar range. Samples were taken after 1, 2, or 3 hours. Bulk
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319
Table 1. Fraction of active cells, determined by the combined microautoradiography-epifluorescence method, in samples taken from in situ incubations with 3H-leucine at 4 stations on the Swedish west coast Active bacteria % _+ SD Stations a
1 2 3 4
Surface 40 39 31 20
___ 1 _+ 6 + 6 + 3
No. o f experiments
(4) (5) (6) (5)
a Stations 1, 2, 3 (near-shore): 57~ 4: 57"38'N, 11"36'E
Bulk 50 41 42 29
+_ 9 + 5 + 10 ___7
No. of experiments
(6) (4) (6) (6)
11"56'E; station
samples were taken with a syringe and fixed with formalin (37% formaldehyde solution filtered through a 0.2 #m Nuclepore filter; final concentration 2% formalin). The surface microlayers were sampled with the Teflon-sheet technique [34]. A Teflon sheet (18 cm, d.) was put on the surface, withdrawn, and transferred to a Petri dish containing 10 ml NSS (filtered through 0.2 #m Nuclepore filter) with a final concentration o f 2% formalin. Controls fixed with formalin simultaneously with the substrate addition were treated in the same way as the samples. Between 50 and 200 ~1 of fixed bulk samples were filtered through 0.2 ~tm Nuclepore filter, 13 ram, d. Ten milliliters of a 4% Tween-8C solution in NSS (2% formalin) was added to the Petri dishes with the Teflon sheets. The sheets wer, vigorously rotated to suspend the collected bacteria. Between 0.4 and 2 ml of this solution was filtered. Duplicates were taken from each dish. One dish corresponded to o n e combination of the amino acid concentration and incubation time used (number of experiments, Table 1). On 3 different occasions, we incubated parallel seawater samples with 3H-glutamic acid and 3H-leucine, respectively. Amounts between 40 and 160 ~1 of the 2 amino acids were added to seawater samples that were brought to the laboratory. For sampling technique, see above.
Combined Microautoradiography and Epifluorescence Method (ME Method) The technique proposed by Meyer-Reil [37] and modified by Tabor and Neihof [47] was used for combined counting (1) total numbers of microorganisms (by acridine orange staining) and (2) individual organisms with active transport function (by microautoradiography). The Nuclepore filters (see above) were placed upside-down on coverslips covered with autoradiography emulsion and exposed for 3 days at 5"C. After development, the preparations were stained with acridineorange and destained (to reduce the background) in a series of citrate buffers. Filters were removed and the emulsions were examined by epifluorescence (for total bacterial count) and by )ransmitted light (for counting o f silvergrain clusters associated with active bacteria). At least 10 fields were examined on each filter. From the 2 different counts, the proportion of active bacteria was obtained. Each Teflon sheet withdrew 55 #1 of water. This figure is used to express the bacterial enrichment in the surface microlayers in c o n c e n t r a t i o n terms.
Determination of the Exchange of Bacteria Between Surface Microlayer and Subsurface Water To assess the downward transport of surface bacteria that have been active in the uptake at the surface microlayer, ~H-palmitic acid was spread on the surface of I liter seawater and on suspensions o f 3 different marine bacteria (with different surface characteristics) in 1 liter NSS. The experiments
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with seawater were run indoors 30 min after the water was collected (near-shore, see Table l for position). Overnight cultures were harvested and washed in NSS. Between 2 and 12 ~g of 3Hpalmitic acid dissolved in hexane was spread on the surface of the water samples with a syringe and 7/~g was spread on the surfaces of the bacterial suspensions. The surface area was 254 cmL With the ME method we determined the fraction of active bacteria in the surface and in the bulk.
Respiration Measurements with Radioactive Substrates To compare the surface microlayers with the subsurface water as a potential site for breakdown of organic matter, respiration experiments were carried out with palmitic acid and a protein mixture. Seawater samples were taken at a near-shore station (see Table 1 for position). Experiments were started within 30 rain after sampling. Samples were equilibrated for 2 hours before the substrate was introduced. ~4C-palmitic acid was dissolved in hexane and spread on the water surface with a syringe. ~4C-protein mixture was dissolved in 52% isopropanol, 40% hexane, 8% water and spread in the same way on the surface. In parallel experiments, the protein mixture was also dissolved in the bulk phase. An airtight lid was fastened to the dishes both during the incubation and the subsequent stripping of the CO2. Twenty milliliters of 4 M H2SO4 was added before COz was stripped by an airflow through the closed dish. Incubation and stripping times were 2 hours, respectively. The CO2 was trapped in 15 ml scintillation cocktail (Oxifluor, New England Nuclear) and the t4CO2 was counted in a Packard Tri-Carb Liquid Scintillation Counter Mod. 3255. Turnover times were calculated according to Wright and Hobble [52].
Control Experiments To determine whether the protein mixture could be spread as a film on the surface, we conducted experiments in the following way. The protein (dissolved as above) was spread on the surface of 500 ml autoclaved seawater. After 4 hours, 400 ml of the bulk was carefully transferred to a new vessel. After the volume had been adjusted to 500 ml, both vessels were inoculated with a washed, overnight culture of S. marcescens. The vessels were shaken for 1 hour. The evolved t4CO2 (after acidification) originating from the 400 ml of bulk solution was less than 10% of the total t4CO2 evolved from the 2 vessels. Thus, less than 10% of the protein spread at the surface was dissolved in the bulk. By adding 20 ml 4 M H2SO 4 to 1 liter NSS augmented with ~4C-carbonate, the recovery of ~4COz was shown to he at least 98%. Seawater samples that were formalin-killed showed no ~4CO2 evolution. The Tween solutions and the NSS used in the ME method were determined to be bacteria- and particle-free by epifluorescence microscopy. Formalin-killed samples showed no detectable active bacteria in surface or bulk samples whether fatty acid or amino acids were used as radioactive substrates in the ME method.
Results
Fraction of Active Bacteria in Surface Microlayers and Subsurface Water The results for the in situ measurements of the fraction of active cells in the bulk and in the surface samples, obtained by the ME method, are shown in T a b l e 1. 3 H - l e u c i n e w a s u s e d a s a s u b s t r a t e . T h e d i f f e r e n t i n c u b a t i o n t i m e s u s e d g a v e n o s i g n i f i c a n t d i f f e r e n c e s i n f r a c t i o n o f a c t i v e cells, n o r c o u l d w e s e e a n y e f f e c t o f d i f f e r e n t s u b s t r a t e c o n c e n t r a t i o n s . A t s t a t i o n s 1 a n d 4, t h e r e w a s a small but significant difference between surface and bulk samples. Lower percentages were noted for both bulk and surface at station 4 compared with the other stations. This station is located further out in the archipelago. Enr i c h m e n t v a l u e s (E), d e f i n e d a s t h e r a t i o b e t w e e n t h e t o t a l n u m b e r o f b a c t e r i a
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Table 2. Results from incubations with 3H-paimitic acid, spread as a surface film, using the combined microautoradiography--epifluorescence method Active bacteria % No. of No. of Sur- experiexpefiNce ments Bulk ments Seawater samples ~
20
(8)
2.8
(11)
54 42 65
(2) (1) (2)
0.5 2.0 0.9
(2) (1) (2)
E
ACB/ ACS
12
189
87 6.0 1.0
1.8 145 412
Test bacteria b
Serratia marcescens (w)c S. marcescens(m)c Pseudomonas halocrenaea
Water samples from a near-shore station were incubated in the laboratory The test bacteria were suspended in NSS (see Material and Methods) c The wildtype (w) has a surface-located pigment that increases its tendency for accumulation at the interface compared with the mutant (m) that lacks this pigment. 3- 106 bacteria/ml a 1.5- 107 bacteria/ml For calculation of enrichment factors (E), see Results. Ratios between the total number of labeled bacteria in the bulk and the total number in the surface (ACB/ ACS) are calculated
Table 3. Comparison between fraction of active cells (% active bacteria of the total numbers), as measured by the microautoradiography-epifluorescence method, when samples were incubated with 3H-leucine or 3Hglutamic acida Experiment 1
2 3
Surface
Bulk
Leu
Glu
Leu
Glu
26 41 14"
13 27 16
21 48* 24*
20 22* 21
a Mean values from 2 parallels, except when marked by * where only 1 experiment was made. Leucine and glutamic acid was added in the range of 0.3-1 nmol and 0.3-2 nmol, respectively
p e r m l in t h e s u r f a c e m i c r o l a y e r s a n d t h e t o t a l n u m b e r o f b a c t e r i a p e r m l in t h e bu l k , r a n g e d f r o m 1 4 - 9 5 , w i t h t h e h i g h e s t v a l u e s at s t a t i o n 4. T h e t o t a l n u m b e r s o f b a c t e r i a in t h e b u l k r a n g e d f r o m 6 . 9 . 1 0 5 m1-1 t o 6 .1 - 1 0 6 m l - I , a n d in t h e s u r f ace f r o m 3.5- 107 m l -~ to 2 . 7 . 1 0 s m l ~.
E x c h a n g e o f Bacteria Between Surface Microlayers a n d Subsurface Water By spreading 3H-palmitic acid on the surface o f seawater and on the surface o f bacterial suspensions and then counting the labeled bacteria (ME m e t h o d ) in t h e s u r f a c e a n d i n t h e b u l k , w e t r i e d t o d e t e r m i n e t h e e x c h a n g e o f b a c t e r i a
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3O 9
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I~C-PALHITIs ACID (ng.cm -2) Fig. 1. Turnover time o f palmitic acid plotted against added amount of 14C-palmitic acid spread as a surface film on seawater samples (1 liter, 254 cm2).
between the 2 strata. Palmitic acid spread as a surface film by this technique has been shown not to dissolve in the subsurface water [31]. These experiments revealed a small fraction in the bulk o f labeled cells ( < 12%, mean: 2.8%, Table 2). Nevertheless, they represent a large total n u m b e r o f labeled cells c o m p a r e d with those at the surface. T h e m e a n percent o f active cells in the surface from 3 experiments with seawater with at least 2 parallels was 20%. E n r i c h m e n t values were between 8 and 15. T h e ratio between the absolute n u m b e r o f active cells in the bulk (1 liter) and the absolute n u m b e r o f active cells at the surface (254 cm 2) (ACB/ACS) was 189. This means that for each labeled cell in the surface there are 189 labeled cells in the bulk. T h e E values show large differences between the different test bacteria and are negatively correlated to the ratio ACB/ACS. The palmitic acid was taken up readily by all 3 bacteria as seen from the fraction active cells in the surface.
The Use o f Different Amino Acids in the M E Method W h e n comparing different a m i n o acids as substrates in the bulk, we found very similar percentages in the bulk for the 2 a m i n o acids (Table 3) except for 1 measurement. For the surface samples, incubation with leucine gave significantly higher fraction o f active cells in 2 o f 3 experiments. As with leucine, different concentrations o f glutamic acid gave no difference in the fraction o f active ceils.
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Table 4. Results from respiration measurements using ~'C-protein applied either in the bulk or at the surface o f seawater samples Amount t4C-protein added, ~tg
Surface
Bulk
Experiment I
1.5 3.0
1,200 1,500
650 1,600
Experiment II
1.5 4.5
500 470
14CO2-DPM
80 ~ 500
Less than 1.5 times higher than background values Bulk volume 1 liter, surface area 254 cm 2
Respiration Experiments The results o f the respiration m e a s u r e m e n t s with 14C-palmitic acid spread as a film on the surface o f natural water samples in the laboratory are presented in Fig. l, where t u r n o v e r time is plotted against a d d e d a m o u n t s [52] o f p a l m i t i c acid. It can be seen that the plot is linear between 2.4 and 12 n g . c m -2 o f added substrate. T h e data for 24 n g . c m -2 deviates from the line. 24 n g . c m -2 o f palmitic acid correspond to a tenth o f a condensed monolayer. T h e t u r n o v e r time for respiration at natural substrate concentration was 3.1 hours (the intercept o f the line with the y-axis). Table 4 shows that for the lower a m o u n t o f added protein, the values for evolved 14CO2 are lower when the protein is mixed in the bulk c o m p a r e d with when spread at the surface. F o r the higher a m o u n t s added, the values are the same for bulk and surface.
Discussion In situ incubations revealed only small differences between surface and bulk samples in the fraction o f active bacteria measured with the ME m e t h o d (Table 1). Incubations were carried out during both intense sunlight and cloudy conditions but we could not see any effects o f sunlight on the activity o f bacteria in the surface samples. Fujioka et al. [13] have shown that fecal streptococci and coliforms were affected by sunlight down to a depth o f 3.3 m in seawater. T h e y conclude that visible light rather than the U V spectrum was responsible for the bactericidal effects. The approach o f in situ incubation gives a m o r e realistic picture o f the air/ sea interface-bulk system, but it also involves an increased complexity which to some extent makes it m o r e difficult to interpret. T h e small differences m a y be due to an exchange o f bacteria between bulk and interface. The labeled bacteria in the surface samples could have taken up the a m i n o acids both when in the bulk or at the interface. By spreading 3H-palmitic acid on the surface o f natural water and counting
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labeled cells at the interface and in the bulk we could see, at least qualitatively, that there was an exchange o f bacteria between the 2 strata. In an a t t e m p t to reveal some o f the m e c h a n i s m s b e h i n d this p h e n o m e n o n we used 3 model bacteria, thoroughly characterized with respect to their surface properties and their tendencies to accumulate at the air/water interface, reflected in different E values [23, 24, 33, 46]. T h e accumulation o f cells at the interface is negatively correlated to the ratio ACB/ACS. This indicates for a bacterium that a high accumulation involves a firmer adhesion to the surface and gives a situation o f low exchange between interface and bulk. These findings seem to be in agreement with the proposal o f Fletcher and Marshall [ 12] that, for solid/liquid interfaces, a t t a c h m e n t is not an all-or-none p h e n o m e n o n . D o w n w a r d transport o f fatty acids mediated by extracellular surface active agents is unlikely in our system. T h e 2 strains o f S. marcescens used here were shown to retain their individual e n r i c h m e n t values when m i x e d together [5]. This fact indicates that neither o f the 2 strains produces extracellular surface active agents u n d e r these conditions. The difference in ACB/ACS values obtained here is therefore unlikely due to bacterial products bringing palmitic acid into solution. It has been shown by surface balance technique [31 ] that Serratia marinorubra, which reacts in a very similar m a n n e r to Serratia marcescens used in this study, could penetrate the surface film o f a phospholipid when the film was compressed to a condensed m o n o l a y e r , whereas P s e u d o m o n a s halocrenae was squeezed out o f the film when the surface pressure was increased. This would suggest that the rate o f exchange o f bacteria between the interface and the bulk is different for different types o f bacteria, m o s t probably due to the same surface characteristics that govern the adhesion o f bacteria to the surface microlayer. This also means that bacteria like Serratia will be m o r e affected by the conditions at the surface since they reside for a longer time at the surface. The similarities o f the activity percentages for bulk and surface (Table 1) might then be explained by an exchange between the 2 strata o f some part o f the bacterial population that is less firmly adhered. T h e m o r e firmly adhered bacteria might be responsible for the often reported differences in distribution o f bacterial species between the bulk and the surface microlayers [39]. Investigations o f bacterial uptake or respiration o f labeled substrates show higher activities in bulk than in surface samples [4, 11, 41 ], which is in contrast to this work. R o m a n e n k o et al. [43], studying a model system, revealed a higher uptake o f h y d r o l y z e d protein per v o l u m e in surface samples than in subsurface samples. However, when correlated to CFU, uptake was lower for surface than bulk. In their investigations o f a salt marsh e n v i r o n m e n t , H a r v e y and Young [22], using reduced intracellular f o r m a z a n deposits as a measure o f an active respiratory chain, showed that 16% o f the total cells in the surface were active c o m p a r e d with 5% in the bulk. T h e authors suggested that this was due to a higher content o f particles and a higher fraction o f particle-bound bacteria at the surface. T h e model for substrate uptake based on the Michaelis-Menten kinetics put forward by Parsons and Strickland [40] has been used widely [for review see 48]. However, for m a n y systems, measured data do not seem to fit the model [15]. It can be seen from Fig. 1 that when 0.6-3.0/~g o f 14C-palmitic acid was spread on the surface, the calculated t u r n o v e r times seem to fit a straight line.
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For the higher amounts of added palmitic acid, we can only hypothesize that since palmitic acid is very surface active it does not only mix with the molecules at the surface, as more commonly used substrates (like glucose) would do in the bulk, but also to some extent changes the composition of the molecules at the interface by competing successfully with less surface active substances. This again points out the unique properties found at an interface. Both the ME method (Table 2) and the respiration (Fig. 1) show that bacteria at the air/sea interface are active in that they take up palmitic acid located at the interface. Attempts to measure ~4CO2 evolution from fatty acids in the bulk [ 1] were not successful. This indicates that surface-active molecules are more easily degraded at an interface than in the bulk. As we had no corresponding bulk values to compare with, the parameter fatty acid respiration per active cell was not very useful to us. However, in other cases we believe the normalization of activity parameters to "per active cell," measured by the ME method, might prove even more useful than the approach o f "activity per cell," proposed by Wright [51]. Protein was chosen as a substrate because it can be both surface localized and dissolved in the water. These experiments were focused more on the fate o f substrate, depending on which strata it was localized in, than on the bacterial activity. Graham and Phillips [16-18] calculated for/~-casein, BSA, and lysozyme that 2-3 mg protein m -2 would give a condensed monolayer. In our experiments, 0.1 mg. m -2 or less o f the protein mixture was spread. The first step in protein degradation is mediated by exoenzymes that cut the protein into smaller molecules [42]. By the action ofexoenzymes, shorter fragments might be distributed in the bulk from protein originally spread at the interface. This might be one part of the explanation o f the similarities in the amounts o f CO2 evolved for surface and bulk at the higher protein concentration. On the other hand, exoenzymes have a greater chance of finding a substrate molecule in the surface microlayer than in the bulk [53]. Also, the uptake of the products by the bacteria might be more efficient at the surface. The results indicate that, at low substrate concentrations, degradation occurs more rapidly at the surface; it might even be possible, at least in some situations, that the only environment where large surface active molecules are degraded in the sea is at interfaces (see also discussion above on palmitic acid). It was shown by Kjelleberg et al. [32], in a model experiment, that bacteria at the interface could use lower concentrations of nutrients for growth than bacteria in the bulk. This is in agreement with our results for the lower concentration o f protein. In the bulk, we could see no differences in uptake with the ME method for the 2 amino acids tested. Other workers [7, 50] have found higher uptakes for glutamic acid than leucine. The higher percentage of active cells for leucine than for glutamic acid incubations shows that the uptake is affected at the interface, which may be explained by the accumulation o f the more hydrophobic leucine at the interface. It also shows that one should be aware o f possible differences between substrates when generalizations are made on the basis o f incubation studies. Although local environmental factors certainly affect the results, discrepancies between results obtained here and those reported by others could partly be explained by differences in methods. The importance of the delicate strat-
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ification at an interface should not be overlooked when performing experiments. T a k i n g t h i s i n t o c o n s i d e r a t i o n l e d us t o a c o m p l e x b u t h o p e f u l l y m o r e r e a l i s t i c p i c t u r e o f t h e m i c r o b i a l i n t e r a c t i o n s at t h e a i r / w a t e r i n t e r f a c e .
Acknowledgments. We wish to thank L. Adler, S. Kjelleberg, and B. Norkrans for constructive criticism, and the Interface group for helpful discussions. A special thanks to the crew of R/V Falsterbo. Grants from the Swedish Natural Science Research Council are gratefully acknowledged.
References 1. Andrews P, Williams PJ leB (1971) Heterotrophic utilization of dissolved organic compounds in the sea. III. Measurement of the oxidation rates and concentrations of glucose and amino acids in sea water. J Mar Biol Ass U K 51:111-125 2. Albright LJ (1980) Photosynthetic activities of phytoneuston and phytoplankton. Can J Microbiol 26:389-392 3. Baler RE, Goupil DW, Perlmutter S, King R (1974) Dominant chemical composition of seasurface films, natural slicks and foams. J Rech Atmos 8:571-600 4. Bell CR, Albright LJ (1982) Bacteriological investigation of the neuston and plankton in the Fraser River estuary, British Columbia. Estuarine Coastal and Shelf Sci 15:385-394 5. Blanchard DC, Syzdek LD (1978) Seven problems in bubble and jet drop researches. Limnol Oceanogr 23:389-400 6. Carlucei AF, Williams PM (1965) Concentration of bacteria from sea water by bubble scavenging. J Cons Perm Int Explor Mer 30:28-33 7. Crawford CC, Hobble JE, Webb KL (1974) The utilization of dissolved free amino acids by estuarine microorganisms. Ecology 55:551-563 8. Crawford RL, Johnson LeeAnn, Martinson M (1982) Numbers and metabolic activities of bacteria in surface films of freshwater lakes. Abstr Ann Meet Am Soc Microbiol, N2, p 178 9. Dahlbiick B, Gunnarsson L/~H, Hermansson M, Kjelleberg S (1982) Microbial investigations of surface microlayers, water column, ice and sediment in the Arctic Ocean. Mar Ecol Progr Series 9, 101-109 10. Dahlblick B, Hermansson M, Kjelleberg S, Norkrans B (1981 ) The hydrophobicity of bacteria-an important factor in their initial adhesion at the air-water interface. Arch Microbiol 128: 267-270 11. Dietz AS, Albright LJ, Tuominen T (1976) Heterotrophic activities of bacterioneuston and bacterioplankton. Can J Microbiol 22:1699-1709 12. Fletcher M, Marshall KC (1982) Are solid surfaces ofecological significance to aquatic bacteria? In: Marshall KC (ed) Advances in microbial ecology, Vol. 6., Plenum Press, New York and London, pp 199-236 13. Fujioka RS, Hashimoto HH, Siwak EB, Young RHF (1981) Effect of sunlight on survival of indicator bacteria in seawater. Appl Environ Microbiol 41:690-696 14. Gallagher JL (1975) The significance of the surface film in salt marsh plankton metabolism. Limnol Oceanogr 20:120-123 15. Gocke K (1977) Comparison of methods for determining the turnover times of dissolved organic compounds. Mar Biol 42:131-141 16. Graham DE, Phillips MC (1979) Proteins at liquid interfaces. I. Kinetics of adsorption and surface denaturation. J Colloid Interface Sci 70:403-414 17. Graham DE, Phillips MC (1979) Proteins at liquid interfaces. II. Adsorption isotherms. J Colloid Interface Sci 70:415-426 18. Graham DE, Phillips MC (1979) Proteins at liquid interfaces. III. Molecular structures of adsorbed films. J Colloid Interface Sci 70:427-439 19. Gucinski H, Goupil DW, Baier RE (1981) Sampling and composition ofthe surface microlayer. In Eisenreich SJ (ed) Atmospheric pollutants in natural waters. Ann Arbor Science Pub, Michigan, pp 165-180
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20. Hardy JT (1973) Phytoplankton ecology of a temperate marine lagoon. Limnol Oceanogr 18: 525-533 21. Harvey RW, Young LY (1980) Enrichment and association of bacteria and particulates in salt marsh surface water. Appl Environ Microbiol 39:894-899 22. Harvey RW, Young LY (1980) Enumeration of particle-bound and unattached respiring bacteria in the salt marsh environment. Appl Environ Microbiol 40:156-160 23. Hermansson M, Kjelleberg S, Korhonen TK, Stenstrrm T-A (1982) Hydrophobic and electrostatic characterization of surface structures of bacteria and its relationship to adhesion to an air-water interface. Arch Microbiol 131:308-312 24. Hermansson M, Kjelleberg S, Norkrans B (1979) Interaction of pigmented wildtype and pigmentless mutant of S e r r a t i a m a r c e s c e n s with lipid surface film. FEMS Microbiol Lett 6:129132 25. Hoppe H-G (I 977) Analysis of actively metabolizing bacterial populations with the autoradiographic method. In: Rheinheimer G (ed) Microbial ecology of a brackish water environment. Ecological studies 25. Springer-Verlag Berlin, Heidelberg, New York 26. Horrigan SG, Carlucci AF, Williams PM (1981) Light inhibition of nitrification in sea-surface films. J Mar Res 39:557-565 27. Kattner GG, Brockmann U H (1978) Fatty-acid composition of dissolved and particulate matter in surface films. Mar Chem 6:233-241 28. Kefford B, Kjelleberg S, Marshall KC (1982) Bacterial scavenging: utilization of fatty acids localized at a solid-liquid interface. Arch Microbiol 133:257-260 29. KjellebergS (1983) Mechanisms ofbacterialadhesion at air/waterinterfaces. In: Fletcher MM, Savage DC (eds) Mechanisms and physiological significance of bacterial adhesion. Plenum Press, New York 30. Kjelleberg S, H~kansson N (1977) Distribution oflipolytie, proteolytic, and amylolytic marine bacteria between the lipid film and the subsurface water. Mar Biol 39:103-109 31. Kjelleberg S, StenstrSm TA (1980) Lipid surface films: interaction of bacteria with free fatty acids and phospholipids at the air/water interface. J Gen Microbiol 116:417-423 32. Kjelleberg S, Humphrey BA, Marshall KC (1982) Effect of interfaces on small, starved marine bacteria. Appl Environ Microbiol 43:1166-1172 33. Kjelleberg S, Lagercrantz C, Larsson TH (1980) Quantitative analysis of bacterial hydrophobicity studied by the binding of dodecanoic acid. FEMS Microbiol Lett 7:41-44 34. Kjelleberg S, Stenstr~m, TA, Odham G (1979) Comparative study of different hydrophobic devices for sampling lipid surface films and adherent microorganisms. Mar Biol 53:21-25 35. Larsson K, Odham G, SSdergren A (1974) On lipid surface films on the sea. I. A simple method for sampling and studies of composition. Mar Chem 2:49-57 36. Marumo R, Taga N, Nakai T (1971) Neustonic bacteria and phytoplankton in surface microlayers of the equatorial waters. Bull Plankton Soc Japan 18:36-41 37. Meyer-Reil L-A (1978) Autoradiography and epifluorescence microscopy combined for the determination of number and spectrum of actively metabolizing bacteria in natural waters. Appl Environ Microbiol 36:506-512 38. Mitamura O, Matsumoto K (I 981) Uptake rate of urea nitrogen and decomposition rate of urea carbon at the surface microlayer in Lake Biwa. Verh Internat Verein Limnol 21:556-564 39. Norkrans B (1980) Surface microlayers in aquatic environments. In: Alexander M (ed) Advances in microbial ecology. Vol. 4. Plenum Publishing Corporation, New York, pp 51-85 40. Parsons TR, Strickland JDH (1962) On the production of particulate organic carbon by heterotrophic processes in seawater. Deep Sea Res 8:211-222 41. Passman FJ, Novitsky TJ, Watson SW (1979) Surface microlayers of the North Atlantic: microbial populations, heterotrophic and hydrocarbonoclastic activities. In: Bourquin AW, Pritchard PH (eds) Microbial degradation of pollution in marine environments, U.S. Environmental Protection Agency, EPA-600/9-79-012 Gulf-Breeze, Florida, pp 214-226 42. Rogers HJ (1961) The dissimilation of higher molecular weight substances. In: Gunsalus IC, Stanier RY (eds) The bacteria, Vol. 2. Academic Press, New York and London, pp 257-318 43. Romanenko VI, Pubienes MA, Daukshta AS (1978) Growth and activity of bacteria on the surface film of water under experimental conditions. Mikrobiologiya 47:149-157
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44. Sieburth JMcN (1971) An instance of bacterial inhibition in oceanic surface water. Mar Biol 11:98-100 45. Sieburth JMcN, Willis P-J, Johnson KM, Burney CM, Lavoie DM, Hinga KR, Caron DA, French III FW, Johnson PW, Davis P G (1976) Dissolved organic matter and heterotrophic microneuston in the surface microlayers of the North Atlantic. Science 194:1415-1418 46. Syzdek LD (1982) Concentration of Serratia in the surface microlayer. Limnol Oceanogr 27: 172-177 47. Tabor PS, NeihofRA (1982) Improved microautoradiographic method to determine individual microorganisms active in substrate uptake in natural waters. Appl Environ Microbio144:945953 48. Van Es FB, Meyer-Reil L-A (1982) Biomass and metabolic activity of heterotrophic marine bacteria. In: Marshall KC (ed) Advances in microbial ecology, Vol. 6. Plenum Press, New York and London, pp 111-170 49. Vii/tt~lnen P (1976) Microbiological studies in coastal waters of the Northern Baltic Sea. I. Distribution and abundance of bacteria and yeasts in the Tvlirminne area. Walter and Andrre de Nottbeck Found Scient Rep 1:1-58 50. Williams PJ leB, Berman T, Holm-Hansen O (1976) Amino acid uptake and respiration by marine heterotrophs. Mar Biol 35:41-47 51. Wright RT (1978) Measurement and significance of specific activity in the heterotrophic bacteria of natural waters. Appl Environ Microbiol 36:297-305 52. Wright TT, Hobbie JE (1966) Use of glucose and acetate by bacteria and algae ecosystems. Ecology 47:447-464 53. ZoBell CE (1943) The effect of solid surfaces upon bacterial activity. J Bacteriol 46:39-54
MICROBIALs 9 1983 Springer-Verlag
Bacterial Activity at the A i r / W a t e r Interface Malte H e r m a n s s o n and B j r r n Dahlb/ick Department of Marine Microbiology, Botanical Institute, University of Grteborg, Carl Skottsbergs Gata 22, S-41319 Grteborg, Sweden
Abstract. By using substrate molecules o f varying degrees o f surface activity, we were able to measure some features o f bacterial activity in the surface microlayers (SM) and in the subsurface (bulk) water. The fraction o f active cells was d e t e r m i n e d by a c o m b i n e d m i c r o a u t o r a d i o g r a p h y - e p i fluorescence (ME) method. Measurements were m a d e o f 14CO 2 evolution to determine the rate o f respiration. Results from in situ m e a s u r e m e n t s showed no significant difference between fraction o f active cells in the SM and in the bulk. This m a y be due to an exchange o f bacteria between SM and bulk. This exchange was assessed by spreading a film o f 3H-palmitic acid on the surface and, after incubation, measuring the a m o u n t o f labeled cells at the surface and in the bulk. Test bacteria showing a high accumulation at the surface also showed a low exchange between the 2 strata. W h e n low concentrations o f added ~4C-protein were used, the respiration m e a s u r e m e n t s showed a lower value for bulk than for interface localized protein. At higher concentrations, the evolved 14CO2 was the same whether the protein was m i x e d in the bulk or spread at the surface. W h e n 2 . 4 - 1 2 n g . c m -2 o f ~4C-palmitic acid was spread on the surface, there was a linear relation between t u r n o v e r time and a m o u n t o f added substrate. At higher substrate concentrations there was a deviation f r o m the straight line. Resuits are discussed in terms o f the unique habitat found at an interface.
Introduction The p h e n o m e n o n o f bacterial accumulation at the air/water interface is well d o c u m e n t e d [6, 39, 45]. There is also an increasing a m o u n t o f work being done on the mechanisms o f adhesion o f bacteria to this interface [29, 39]. T h e question o f microbial activity at the air/water interface has been addressed by several investigators. Measurements have been done on bacterial uptake and respiration o f various types ofsubstrates [4, 8, 1 l, 38, 4 l, 43], algal photosynthetic activity [2, 14, 20], biochemical activities o f bacterial isolates [9, 25, 30, 44], nitrification [26], the fraction o f respiring bacteria [22], as well as n u m e r o u s estimates o f viable counts [2 l, 36]. It is clear that there are differences a m o n g bacteria in a natural habitat with respect to the surface characteristics that are related to adhesion [ 10]. T h e r e appears to be a correlation between these characteristics and the scavenging
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a b i l i t y o f b a c t e r i a a t a s o l i d / l i q u i d i n t e r f a c e [28]. It f o l l o w s t h a t a c t i v i t y m e a surements of bacteria at an interface should be put into an adhesion context. In most investigations of the activity of microorganisms in the surface mic r o l a y e r s , t h e s u r f a c e s a m p l e s a r e i n c u b a t e d i n t h e s a m e w a y as t h e b u l k s a m p l e s [2, 4, 11, 20, 26, 38, 41, 43]. F o r e x a m p l e , a v o l u m e o f t h e s u r f a c e m i c r o l a y e r s is c o l l e c t e d w i t h a s c r e e n - s a m p l e r a n d s u b s e q u e n t l y i n c u b a t e d i n a flask. T h i s means that microorganisms from the surface are transferred to a totally new e n v i r o n m e n t . I n c o n t r a s t , t h e m e t h o d s w e h a v e u s e d a r e in s i t u i n c u b a t i o n s . T h i s r e v e a l s n e w a s p e c t s o f b a c t e r i a l a c t i v i t i e s a t i n t e r f a c e s . A l s o , t h e use o f different methods are discussed. In these experiments, exchange of molecules a n d o r g a n i s m s b e t w e e n t h e i n t e r f a c e a n d t h e b u l k is still p o s s i b l e t h r o u g h o u t t h e i n c u b a t i o n , a n d t h e s t r a t i f i c a t i o n o f t h e s y s t e m is n o t d i s r u p t e d . T h e a p proach of measuring on a relatively undisturbed interracial stratum has also b e e n u s e d b y G a l l a g h e r [14]. W e b e l i e v e t h a t t h i s is a v e r y i m p o r t a n t p o i n t b e c a u s e t h e q u e s t i o n o f h o w t h e i n t e r f a c e affects t h e b a c t e r i a l a c t i v i t y c a n b e s t be answered by investigations of bacteria actually at the interface. We have carried out measurements on bacterial activity using amino acids, a f a t t y a c i d , a n d a p r o t e i n m i x t u r e as s u b s t r a t e s . T h e 2 l a t t e r s u b s t r a t e s a r e c o n s i d e r e d to b e i m p o r t a n t c o n s t i t u e n t s o f t h e s u r f a c e m i c r o l a y e r [3, 19, 27, 35]. T h e u s e o f f a t t y a c i d s a n d p r o t e i n s is a n a t t e m p t to s p e c i a l l y c o n s i d e r t h e u n i q u e e n v i r o n m e n t o f t h e s u r f a c e m i c r o l a y e r . I n t h i s s t u d y w e u s e d (1) a c o m b i n e d m i c r o a u t o r a d i o g r a p h y - - e p i f l u o r e s c e n c e m e t h o d [47] to d e t e r m i n e t h e f r a c t i o n o f a c t i v e cells a n d (2) m e a s u r e m e n t s o f e v o l v e d 14CO z to d e t e r m i n e t h e r a t e o f r e s p i r a t i o n in t h e s u r f a c e m i c r o l a y e r s a n d i n t h e s u b s u r f a c e w a t e r .
M a t e r i a l and M e t h o d s Radiochemicals, Test Bacteria, M e d i u m a n d N i n e Salt Solution ( N S S ) The radiochemicals used were L- 14,5-3H I-leucine, 1.0 mCi- ml- ~, 136 Ci. mmol-~, Amersham; L- IG-all [-glutamic acid, 1.0 mCi- ml- ~, 38 Ci" mmol- t, Amersham; 19,10-3H(N) I-palmitic acid, 10.0 mCi-ml -~ 15.2 Ci-mmol -~, New England Nuclear; [ 1-t4C I-palmitic acid, 100 ~Ci.ml -t, 55 mCi.mmol -j, Amersham; and I~'C(U)I-protein from E. coil B/r, 52.6 ~zCi.mg-~, New England Nuclear. Serratia marcescens EF 190 wildtype (w) and a mutant (m) and Pseudomonas halocrenae ATCC 19712 were used as test bacteria. The mutant of S. rnarcescensis devoid of a red pigment (prodigiosin) which makes it less hydrophobic [Kjelleberg et al. 33]. The bacteria were cultivated in V medium [49]: peptone 1.0 g; yeast extract 0.5 g; glucose 0.5 g; starch (soluble) 0.5 g; FeSO4. 7H20 0.01 g; Na2HPO4 0.01 g; nine salt solution (NSS) 1,000 ml. NSS consists of NaC1 23.48 g; Na2SO4 1.96 g; NaHCO3 0.10 g; KC1 0.33 g; KBr 0.05 g; MgCI2-2H20 2.49 g; CaCI2-2H20 0.55 g; SrCI2.6H20 0.01 g; H3BO 3 0.01 g; double-distilled water 1,000 ml.
In Situ Incubations In the field study, the in situ uptake of 3H-leucine by bacteria in the surface microlayers and in the bulk was determined. Near-shore seawater (0.5 liter) was collected at 4 separate sampling times in July and August, with glass dishes (15.5 cm, d.). The dishes were placed in shallow water. After 2 hours, radioactive substrate was added with a syringe into the bulk phase. Five or 25 ul 3Hleucine was diluted in 5 ml of seawater and injected through the surface. This corresponds to an addition of the amino acid in the picomolar range. Samples were taken after 1, 2, or 3 hours. Bulk
Bacterial Activity at the Air/Water Interface
319
Table 1. Fraction of active cells, determined by the combined microautoradiography-epifluorescence method, in samples taken from in situ incubations with 3H-leucine at 4 stations on the Swedish west coast Active bacteria % _+ SD Stations a
1 2 3 4
Surface 40 39 31 20
___ 1 _+ 6 + 6 + 3
No. o f experiments
(4) (5) (6) (5)
a Stations 1, 2, 3 (near-shore): 57~ 4: 57"38'N, 11"36'E
Bulk 50 41 42 29
+_ 9 + 5 + 10 ___7
No. of experiments
(6) (4) (6) (6)
11"56'E; station
samples were taken with a syringe and fixed with formalin (37% formaldehyde solution filtered through a 0.2 #m Nuclepore filter; final concentration 2% formalin). The surface microlayers were sampled with the Teflon-sheet technique [34]. A Teflon sheet (18 cm, d.) was put on the surface, withdrawn, and transferred to a Petri dish containing 10 ml NSS (filtered through 0.2 #m Nuclepore filter) with a final concentration o f 2% formalin. Controls fixed with formalin simultaneously with the substrate addition were treated in the same way as the samples. Between 50 and 200 ~1 of fixed bulk samples were filtered through 0.2 ~tm Nuclepore filter, 13 ram, d. Ten milliliters of a 4% Tween-8C solution in NSS (2% formalin) was added to the Petri dishes with the Teflon sheets. The sheets wer, vigorously rotated to suspend the collected bacteria. Between 0.4 and 2 ml of this solution was filtered. Duplicates were taken from each dish. One dish corresponded to o n e combination of the amino acid concentration and incubation time used (number of experiments, Table 1). On 3 different occasions, we incubated parallel seawater samples with 3H-glutamic acid and 3H-leucine, respectively. Amounts between 40 and 160 ~1 of the 2 amino acids were added to seawater samples that were brought to the laboratory. For sampling technique, see above.
Combined Microautoradiography and Epifluorescence Method (ME Method) The technique proposed by Meyer-Reil [37] and modified by Tabor and Neihof [47] was used for combined counting (1) total numbers of microorganisms (by acridine orange staining) and (2) individual organisms with active transport function (by microautoradiography). The Nuclepore filters (see above) were placed upside-down on coverslips covered with autoradiography emulsion and exposed for 3 days at 5"C. After development, the preparations were stained with acridineorange and destained (to reduce the background) in a series of citrate buffers. Filters were removed and the emulsions were examined by epifluorescence (for total bacterial count) and by )ransmitted light (for counting o f silvergrain clusters associated with active bacteria). At least 10 fields were examined on each filter. From the 2 different counts, the proportion of active bacteria was obtained. Each Teflon sheet withdrew 55 #1 of water. This figure is used to express the bacterial enrichment in the surface microlayers in c o n c e n t r a t i o n terms.
Determination of the Exchange of Bacteria Between Surface Microlayer and Subsurface Water To assess the downward transport of surface bacteria that have been active in the uptake at the surface microlayer, ~H-palmitic acid was spread on the surface of I liter seawater and on suspensions o f 3 different marine bacteria (with different surface characteristics) in 1 liter NSS. The experiments
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with seawater were run indoors 30 min after the water was collected (near-shore, see Table l for position). Overnight cultures were harvested and washed in NSS. Between 2 and 12 ~g of 3Hpalmitic acid dissolved in hexane was spread on the surface of the water samples with a syringe and 7/~g was spread on the surfaces of the bacterial suspensions. The surface area was 254 cmL With the ME method we determined the fraction of active bacteria in the surface and in the bulk.
Respiration Measurements with Radioactive Substrates To compare the surface microlayers with the subsurface water as a potential site for breakdown of organic matter, respiration experiments were carried out with palmitic acid and a protein mixture. Seawater samples were taken at a near-shore station (see Table 1 for position). Experiments were started within 30 rain after sampling. Samples were equilibrated for 2 hours before the substrate was introduced. ~4C-palmitic acid was dissolved in hexane and spread on the water surface with a syringe. ~4C-protein mixture was dissolved in 52% isopropanol, 40% hexane, 8% water and spread in the same way on the surface. In parallel experiments, the protein mixture was also dissolved in the bulk phase. An airtight lid was fastened to the dishes both during the incubation and the subsequent stripping of the CO2. Twenty milliliters of 4 M H2SO4 was added before COz was stripped by an airflow through the closed dish. Incubation and stripping times were 2 hours, respectively. The CO2 was trapped in 15 ml scintillation cocktail (Oxifluor, New England Nuclear) and the t4CO2 was counted in a Packard Tri-Carb Liquid Scintillation Counter Mod. 3255. Turnover times were calculated according to Wright and Hobble [52].
Control Experiments To determine whether the protein mixture could be spread as a film on the surface, we conducted experiments in the following way. The protein (dissolved as above) was spread on the surface of 500 ml autoclaved seawater. After 4 hours, 400 ml of the bulk was carefully transferred to a new vessel. After the volume had been adjusted to 500 ml, both vessels were inoculated with a washed, overnight culture of S. marcescens. The vessels were shaken for 1 hour. The evolved t4CO2 (after acidification) originating from the 400 ml of bulk solution was less than 10% of the total t4CO2 evolved from the 2 vessels. Thus, less than 10% of the protein spread at the surface was dissolved in the bulk. By adding 20 ml 4 M H2SO 4 to 1 liter NSS augmented with ~4C-carbonate, the recovery of ~4COz was shown to he at least 98%. Seawater samples that were formalin-killed showed no ~4CO2 evolution. The Tween solutions and the NSS used in the ME method were determined to be bacteria- and particle-free by epifluorescence microscopy. Formalin-killed samples showed no detectable active bacteria in surface or bulk samples whether fatty acid or amino acids were used as radioactive substrates in the ME method.
Results
Fraction of Active Bacteria in Surface Microlayers and Subsurface Water The results for the in situ measurements of the fraction of active cells in the bulk and in the surface samples, obtained by the ME method, are shown in T a b l e 1. 3 H - l e u c i n e w a s u s e d a s a s u b s t r a t e . T h e d i f f e r e n t i n c u b a t i o n t i m e s u s e d g a v e n o s i g n i f i c a n t d i f f e r e n c e s i n f r a c t i o n o f a c t i v e cells, n o r c o u l d w e s e e a n y e f f e c t o f d i f f e r e n t s u b s t r a t e c o n c e n t r a t i o n s . A t s t a t i o n s 1 a n d 4, t h e r e w a s a small but significant difference between surface and bulk samples. Lower percentages were noted for both bulk and surface at station 4 compared with the other stations. This station is located further out in the archipelago. Enr i c h m e n t v a l u e s (E), d e f i n e d a s t h e r a t i o b e t w e e n t h e t o t a l n u m b e r o f b a c t e r i a
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Table 2. Results from incubations with 3H-paimitic acid, spread as a surface film, using the combined microautoradiography--epifluorescence method Active bacteria % No. of No. of Sur- experiexpefiNce ments Bulk ments Seawater samples ~
20
(8)
2.8
(11)
54 42 65
(2) (1) (2)
0.5 2.0 0.9
(2) (1) (2)
E
ACB/ ACS
12
189
87 6.0 1.0
1.8 145 412
Test bacteria b
Serratia marcescens (w)c S. marcescens(m)c Pseudomonas halocrenaea
Water samples from a near-shore station were incubated in the laboratory The test bacteria were suspended in NSS (see Material and Methods) c The wildtype (w) has a surface-located pigment that increases its tendency for accumulation at the interface compared with the mutant (m) that lacks this pigment. 3- 106 bacteria/ml a 1.5- 107 bacteria/ml For calculation of enrichment factors (E), see Results. Ratios between the total number of labeled bacteria in the bulk and the total number in the surface (ACB/ ACS) are calculated
Table 3. Comparison between fraction of active cells (% active bacteria of the total numbers), as measured by the microautoradiography-epifluorescence method, when samples were incubated with 3H-leucine or 3Hglutamic acida Experiment 1
2 3
Surface
Bulk
Leu
Glu
Leu
Glu
26 41 14"
13 27 16
21 48* 24*
20 22* 21
a Mean values from 2 parallels, except when marked by * where only 1 experiment was made. Leucine and glutamic acid was added in the range of 0.3-1 nmol and 0.3-2 nmol, respectively
p e r m l in t h e s u r f a c e m i c r o l a y e r s a n d t h e t o t a l n u m b e r o f b a c t e r i a p e r m l in t h e bu l k , r a n g e d f r o m 1 4 - 9 5 , w i t h t h e h i g h e s t v a l u e s at s t a t i o n 4. T h e t o t a l n u m b e r s o f b a c t e r i a in t h e b u l k r a n g e d f r o m 6 . 9 . 1 0 5 m1-1 t o 6 .1 - 1 0 6 m l - I , a n d in t h e s u r f ace f r o m 3.5- 107 m l -~ to 2 . 7 . 1 0 s m l ~.
E x c h a n g e o f Bacteria Between Surface Microlayers a n d Subsurface Water By spreading 3H-palmitic acid on the surface o f seawater and on the surface o f bacterial suspensions and then counting the labeled bacteria (ME m e t h o d ) in t h e s u r f a c e a n d i n t h e b u l k , w e t r i e d t o d e t e r m i n e t h e e x c h a n g e o f b a c t e r i a
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3O 9
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24
I~C-PALHITIs ACID (ng.cm -2) Fig. 1. Turnover time o f palmitic acid plotted against added amount of 14C-palmitic acid spread as a surface film on seawater samples (1 liter, 254 cm2).
between the 2 strata. Palmitic acid spread as a surface film by this technique has been shown not to dissolve in the subsurface water [31]. These experiments revealed a small fraction in the bulk o f labeled cells ( < 12%, mean: 2.8%, Table 2). Nevertheless, they represent a large total n u m b e r o f labeled cells c o m p a r e d with those at the surface. T h e m e a n percent o f active cells in the surface from 3 experiments with seawater with at least 2 parallels was 20%. E n r i c h m e n t values were between 8 and 15. T h e ratio between the absolute n u m b e r o f active cells in the bulk (1 liter) and the absolute n u m b e r o f active cells at the surface (254 cm 2) (ACB/ACS) was 189. This means that for each labeled cell in the surface there are 189 labeled cells in the bulk. T h e E values show large differences between the different test bacteria and are negatively correlated to the ratio ACB/ACS. The palmitic acid was taken up readily by all 3 bacteria as seen from the fraction active cells in the surface.
The Use o f Different Amino Acids in the M E Method W h e n comparing different a m i n o acids as substrates in the bulk, we found very similar percentages in the bulk for the 2 a m i n o acids (Table 3) except for 1 measurement. For the surface samples, incubation with leucine gave significantly higher fraction o f active cells in 2 o f 3 experiments. As with leucine, different concentrations o f glutamic acid gave no difference in the fraction o f active ceils.
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Table 4. Results from respiration measurements using ~'C-protein applied either in the bulk or at the surface o f seawater samples Amount t4C-protein added, ~tg
Surface
Bulk
Experiment I
1.5 3.0
1,200 1,500
650 1,600
Experiment II
1.5 4.5
500 470
14CO2-DPM
80 ~ 500
Less than 1.5 times higher than background values Bulk volume 1 liter, surface area 254 cm 2
Respiration Experiments The results o f the respiration m e a s u r e m e n t s with 14C-palmitic acid spread as a film on the surface o f natural water samples in the laboratory are presented in Fig. l, where t u r n o v e r time is plotted against a d d e d a m o u n t s [52] o f p a l m i t i c acid. It can be seen that the plot is linear between 2.4 and 12 n g . c m -2 o f added substrate. T h e data for 24 n g . c m -2 deviates from the line. 24 n g . c m -2 o f palmitic acid correspond to a tenth o f a condensed monolayer. T h e t u r n o v e r time for respiration at natural substrate concentration was 3.1 hours (the intercept o f the line with the y-axis). Table 4 shows that for the lower a m o u n t o f added protein, the values for evolved 14CO2 are lower when the protein is mixed in the bulk c o m p a r e d with when spread at the surface. F o r the higher a m o u n t s added, the values are the same for bulk and surface.
Discussion In situ incubations revealed only small differences between surface and bulk samples in the fraction o f active bacteria measured with the ME m e t h o d (Table 1). Incubations were carried out during both intense sunlight and cloudy conditions but we could not see any effects o f sunlight on the activity o f bacteria in the surface samples. Fujioka et al. [13] have shown that fecal streptococci and coliforms were affected by sunlight down to a depth o f 3.3 m in seawater. T h e y conclude that visible light rather than the U V spectrum was responsible for the bactericidal effects. The approach o f in situ incubation gives a m o r e realistic picture o f the air/ sea interface-bulk system, but it also involves an increased complexity which to some extent makes it m o r e difficult to interpret. T h e small differences m a y be due to an exchange o f bacteria between bulk and interface. The labeled bacteria in the surface samples could have taken up the a m i n o acids both when in the bulk or at the interface. By spreading 3H-palmitic acid on the surface o f natural water and counting
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labeled cells at the interface and in the bulk we could see, at least qualitatively, that there was an exchange o f bacteria between the 2 strata. In an a t t e m p t to reveal some o f the m e c h a n i s m s b e h i n d this p h e n o m e n o n we used 3 model bacteria, thoroughly characterized with respect to their surface properties and their tendencies to accumulate at the air/water interface, reflected in different E values [23, 24, 33, 46]. T h e accumulation o f cells at the interface is negatively correlated to the ratio ACB/ACS. This indicates for a bacterium that a high accumulation involves a firmer adhesion to the surface and gives a situation o f low exchange between interface and bulk. These findings seem to be in agreement with the proposal o f Fletcher and Marshall [ 12] that, for solid/liquid interfaces, a t t a c h m e n t is not an all-or-none p h e n o m e n o n . D o w n w a r d transport o f fatty acids mediated by extracellular surface active agents is unlikely in our system. T h e 2 strains o f S. marcescens used here were shown to retain their individual e n r i c h m e n t values when m i x e d together [5]. This fact indicates that neither o f the 2 strains produces extracellular surface active agents u n d e r these conditions. The difference in ACB/ACS values obtained here is therefore unlikely due to bacterial products bringing palmitic acid into solution. It has been shown by surface balance technique [31 ] that Serratia marinorubra, which reacts in a very similar m a n n e r to Serratia marcescens used in this study, could penetrate the surface film o f a phospholipid when the film was compressed to a condensed m o n o l a y e r , whereas P s e u d o m o n a s halocrenae was squeezed out o f the film when the surface pressure was increased. This would suggest that the rate o f exchange o f bacteria between the interface and the bulk is different for different types o f bacteria, m o s t probably due to the same surface characteristics that govern the adhesion o f bacteria to the surface microlayer. This also means that bacteria like Serratia will be m o r e affected by the conditions at the surface since they reside for a longer time at the surface. The similarities o f the activity percentages for bulk and surface (Table 1) might then be explained by an exchange between the 2 strata o f some part o f the bacterial population that is less firmly adhered. T h e m o r e firmly adhered bacteria might be responsible for the often reported differences in distribution o f bacterial species between the bulk and the surface microlayers [39]. Investigations o f bacterial uptake or respiration o f labeled substrates show higher activities in bulk than in surface samples [4, 11, 41 ], which is in contrast to this work. R o m a n e n k o et al. [43], studying a model system, revealed a higher uptake o f h y d r o l y z e d protein per v o l u m e in surface samples than in subsurface samples. However, when correlated to CFU, uptake was lower for surface than bulk. In their investigations o f a salt marsh e n v i r o n m e n t , H a r v e y and Young [22], using reduced intracellular f o r m a z a n deposits as a measure o f an active respiratory chain, showed that 16% o f the total cells in the surface were active c o m p a r e d with 5% in the bulk. T h e authors suggested that this was due to a higher content o f particles and a higher fraction o f particle-bound bacteria at the surface. T h e model for substrate uptake based on the Michaelis-Menten kinetics put forward by Parsons and Strickland [40] has been used widely [for review see 48]. However, for m a n y systems, measured data do not seem to fit the model [15]. It can be seen from Fig. 1 that when 0.6-3.0/~g o f 14C-palmitic acid was spread on the surface, the calculated t u r n o v e r times seem to fit a straight line.
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325
For the higher amounts of added palmitic acid, we can only hypothesize that since palmitic acid is very surface active it does not only mix with the molecules at the surface, as more commonly used substrates (like glucose) would do in the bulk, but also to some extent changes the composition of the molecules at the interface by competing successfully with less surface active substances. This again points out the unique properties found at an interface. Both the ME method (Table 2) and the respiration (Fig. 1) show that bacteria at the air/sea interface are active in that they take up palmitic acid located at the interface. Attempts to measure ~4CO2 evolution from fatty acids in the bulk [ 1] were not successful. This indicates that surface-active molecules are more easily degraded at an interface than in the bulk. As we had no corresponding bulk values to compare with, the parameter fatty acid respiration per active cell was not very useful to us. However, in other cases we believe the normalization of activity parameters to "per active cell," measured by the ME method, might prove even more useful than the approach o f "activity per cell," proposed by Wright [51]. Protein was chosen as a substrate because it can be both surface localized and dissolved in the water. These experiments were focused more on the fate o f substrate, depending on which strata it was localized in, than on the bacterial activity. Graham and Phillips [16-18] calculated for/~-casein, BSA, and lysozyme that 2-3 mg protein m -2 would give a condensed monolayer. In our experiments, 0.1 mg. m -2 or less o f the protein mixture was spread. The first step in protein degradation is mediated by exoenzymes that cut the protein into smaller molecules [42]. By the action ofexoenzymes, shorter fragments might be distributed in the bulk from protein originally spread at the interface. This might be one part of the explanation o f the similarities in the amounts o f CO2 evolved for surface and bulk at the higher protein concentration. On the other hand, exoenzymes have a greater chance of finding a substrate molecule in the surface microlayer than in the bulk [53]. Also, the uptake of the products by the bacteria might be more efficient at the surface. The results indicate that, at low substrate concentrations, degradation occurs more rapidly at the surface; it might even be possible, at least in some situations, that the only environment where large surface active molecules are degraded in the sea is at interfaces (see also discussion above on palmitic acid). It was shown by Kjelleberg et al. [32], in a model experiment, that bacteria at the interface could use lower concentrations of nutrients for growth than bacteria in the bulk. This is in agreement with our results for the lower concentration o f protein. In the bulk, we could see no differences in uptake with the ME method for the 2 amino acids tested. Other workers [7, 50] have found higher uptakes for glutamic acid than leucine. The higher percentage of active cells for leucine than for glutamic acid incubations shows that the uptake is affected at the interface, which may be explained by the accumulation o f the more hydrophobic leucine at the interface. It also shows that one should be aware o f possible differences between substrates when generalizations are made on the basis o f incubation studies. Although local environmental factors certainly affect the results, discrepancies between results obtained here and those reported by others could partly be explained by differences in methods. The importance of the delicate strat-
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ification at an interface should not be overlooked when performing experiments. T a k i n g t h i s i n t o c o n s i d e r a t i o n l e d us t o a c o m p l e x b u t h o p e f u l l y m o r e r e a l i s t i c p i c t u r e o f t h e m i c r o b i a l i n t e r a c t i o n s at t h e a i r / w a t e r i n t e r f a c e .
Acknowledgments. We wish to thank L. Adler, S. Kjelleberg, and B. Norkrans for constructive criticism, and the Interface group for helpful discussions. A special thanks to the crew of R/V Falsterbo. Grants from the Swedish Natural Science Research Council are gratefully acknowledged.
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