Microbial hydrolytic enzyme activities in deep-sea sediments

Helgoland Marine Research, May 2019

The potential hydrolysis rates of five different hydrolytic enzymes were determined in deep-sea sediments from the northeast Atlantic (BIOTRANS area) in March 1992. Fluorogenic substrates were used to assay extracellular α- and β-glucosidase, chitobiase, lipase and aminopeptidase. The potential activity of most of the enzymes investigated decreased to a minimum within the upper two centimetre range, whereas aminopeptidase was high over the upper five centimetre range. Exceptions were found when macrofaunal burrows occurred in the cores, always increasing the activities of some hydrolases, and therefore indicating the impact of bioturbation on degradation rates. The most striking feature of the investigated enzyme spectrum was the 50–2000 times higher specific activity of the aminopeptidase, compared with the other hydrolases. The activity of hydrolytic enzymes most likely reflects the availability of their respective substrates and is not a function of bacterial biomass.

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Microbial hydrolytic enzyme activities in deep-sea sediments

HELGOLANDER MEERESUNTERSUCHUNGEN Helgol~nder Meeresunters. M i c r o b i a l h y d r o l y t i c e n z y m e a c t i v i t i e s in d e e p - s e a s e d i m e n t s A. B o e t i u s 0 9 Biologische Anstalt Helgoland, Hamburg 0 Institut ffir Hydrobiologie und Fischereiwissenschaft, Abteilung Biologische Ozeanographie; Zeiseweg 9 , 22765 Hamburg , Germany The potential hydrolysis rates of five different hydrolytic enzymes were determined in deep-sea sediments from the northeast Atlantic (BIOTRANS area) in March 1992. Fluorogenic substrates were used to assay extracellular c~- and [~-glucosidase, chitobiase, lipase and aminopeptidase. The potential activity of most of the enzymes investigated decreased to a minimum within the upper two centimetre range, whereas aminopeptidase was high over the upper five centimetre range. Exceptions were found when macrofaunal burrows occurred in the cores, always increasing the activities of some hydrolases, and therefore indicating the impact of bioturbation on degradation rates. The most striking feature of the investigated enzyme spectrum was the 50-2000 times higher specific activity of the aminopeptidase, compared with the other hydrolases. The activity of hydrolytic enzymes most likely reflects the availability of their respective substrates and is not a function of bacterial biomass. - short i n c u b a t i o n times of the samples, "snapshots" of the potential e n z y m e activities within the sediments could be obtained. Hydrolytic e n z y m e s are n a m e d after the substrates they respectively cleave (IUB E n z y m e Nomenclature, 1973) . Some e n z y m e s m a y h a v e a broader specificity, e n a b l i n g t h e m to hydrolyse other substrates with a lower activity. METHODS The BIOTRANS area is situated in the northeast Atlantic at 47~ a n d 19~ at 4500 m dept h (Pfannkuche et al., 1990 ). The investigations were carried out on s e d i m e n t samples recovered with a multiple corer (Barnett et al., 1984) during the "Meteor" cruise 21/1 i n March/April 1992 . Methylumbelliferyl (MUF) a n d m e t h y l c o u m a r i n y l a m i d (MCA) derivatives were used to m e a s u r e the potential enzymatic activity of different hydrolases: M U F - ~ - a n d MUF-~glucoside for the respective glucosidases, M U F - N - a c e t y l g l u c o s a m i n e for chitobiase, MUF-stearate for lipase a n d L - l e u c i n e - M C A for aminopeptidase. The e n z y m e s liberate the fluorochrome from the n o n f l u o r e s c e n t substrate in a 1 : 1 ratio. The m e t h o d u s e d is b a s e d on previous work of Hoppe (1983) and Meyer-Reil (1986). Three to five s e d i m e n t cores from one multiple corer were sliced directly after sampling in o n e - c e n t i m e t r e thick layers, a n d slices from the same s e d i m e n t depth w e r e mixed to avoid small-scale variations. From the mixtures, s e d i m e n t slurries were p r e p a r e d with sterile filtered d e e p - s e a water in a 1 : 1 dilution. Samples from each slurry were a m e n d e d with concentrations of substrate at saturation level which had b e e n tested in advance. The samples were i n c u b a t e d at in-situ t e m p e r a t u r e (2 ~ with different M U F / M C A substrates. At five time-intervals d u r i n g four hours of incubation, s u b s a m p l e s were r e m o v e d a n d diluted 1 : 2 with sterile filtered deepsea water. After the addition of 0.25 ml of borate buffer (pH 10), the s u b s a m p l e s were centrifuged (10 min, 4000 U/rain). The fluorescence was i m m e d i a t e l y m e a s u r e d at 365 n m excitation a n d 455 n m emission. The fluorescence units were calibrated by m e a s u r i n g k n o w n concentrations of methylumbelliferone. The e n z y m e activity is expressed as velocity of hydrolysis (nmol m1-1 s e d i m e n t h - l ) . Data from 6 different multiple corer samples t a k e n over a time-interval of e l e v e n days (March 25th to April 4th, 1992) are p r e s e n t e d as m e a n values of potential e n z y m e activities in the BIOTRANS area. Samples with significant deviations from the usual distribution of e n z y m e activities due to the occurrence of biogenic structures in the cores were e x c l u d e d a n d are separately shown. RESULTS A c t i v i t i e s of s p e c i f i c h y d r o l a s e s a n d t h e i r p r o f i l e s w i t h s e d i m e n t d e p t h ol-Glucosidase and fi-Glucosidase These e n z y m e s hydrolyse two groups of saccharides: a-glucosidic b o n d s occur in storage c o m p o u n d s of plants a n d animals like starch a n d glycogen w h i c h are easily degradable; ~-glucosidic b o n d s m a k e up structural c o m p o u n d s like cellulose, as in plantwall material, a n d mucopolysaccharides, as in i n v e r t e b r a t e slimes. Not m a n y organisms b e s i d e bacteria can digest ~-glucosidic substrates. The potential activity of c~-glucosidase was very low a n d did not c h a n g e m u c h with s e d i m e n t depth (Fig. 1). The activity of ~-glucosidase was ten times higher, sharply decreasing after a m a x i m u m at the s e d i m e n t surface (Fig. 2). Chitobiase Lipase Chitin is an a b u n d a n t structural polysaccharide. Crustacean shells, as well as their fecal m e m b r a n e s , consist of chitin. Chitobiase hydrolyses the bonds b e t w e e n the Nacetyl-glucosamine monomers; it also cleaves the m u r a m i n disaccharides in bacterial cell walls. The potential activity of chitobiase was five times higher t h a n ~-glucosidase; their profiles were alike in the e x p o n e n t i a l decrease with s e d i m e n t depth (Fig. 3). The substrate used (MUF-Stearate) is the monoacylester of a long-chain fatty acid, as in waxes, the typical storage lipids of animals. The enzymatic activity of lipase was about as high as [~-glucosidase at the sediment surface, rapidly decreasing to detection l i m i t b e l o w 2 cm sediment depth (Fig. 4). Aminopeptidase This e n z y m e has a broad specificity a n d cleaves nearly all L-peptides. Peptide bonds occur in a multitude of compounds, in the easily d e g r a d a b l e proteins as well as in very refractory aromatic substances like humic complexes. A comparison of the investigated hydrolases revealed that a m i n o p e p t i d a s e had a 50-2000 times h i g h e r potential t h a n the other enzymes. Furthermore, its profile was Fig. 1. Hydrolysis of MUF-a-glucoside. Sediment profile from BIOTRANS area (n=6) , 2O i MUF (nM h-l) d 20 40 60 80 100 completely different, showing a subsurface m a x i m u m in 2 cm or 3 cm s e d i m e n t depth (Fig. 5). The decrease with sediment depth was slower than that found for the other hydrolases. I n f l u e n c e of m a c r o f a u n a l s t r u c t u r e s Groups of vertical macroscopic burrows, stretching from 1 cm to 10 cm s e d i m e n t depth, were found in the sediments of several multiple corer samples from the BIOTRANS area. Even after mixing the s e d i m e n t layers of 3-5 different cores, a clear difference from the usual type of profiles could be found in the distribution of enzymatic activity. A significant increase in activity was measured, especially at the bottom of burrows, l e a d i n g to "reversed" profiles (Figs5, 6, 7). No impact on the activity of a m i n o p e p t i d a s e was found. DISCUSSION Activities of specific hydrolases The potential activities of specific hydrolases indicate the microbial reaction to the pool of organic matter (Hoppe, 1983), which consists of a variety of compounds. In d e e p - s e a sediments from the BIOTRANS area, a m i n o p e p t i d a s e had the highest activity of all i n v e s t i g a t e d enzymes. The r a n k i n g of the activity of the investigated MCA (uM h - l ) 0 , MUF (nM h-l) i 4O I I 80 I i 120 I i 160 I i Fig. 7. Hydrolysis of MUF-~-glucoside and MUF-stearate. Sediment profile from BIOTRANS area, 27.3. 1992. In the three mixed cores several macrofaunal burrows were observed. A polychaete was recovered from one of the burrows saccharidases (chitobiase > ~-glucosidase > > ec-glucosidase) matches the availability of their respective substrates in d e e p - s e a sediments. Chitin is the most a b u n d a n t polysaccharide in m a r i n e e n v i r o n m e n t s with little resources of fresh plant detritus (Smucker & Kim, 1991). Food deposits in the d e e p - s e a are limited a n d low in energetic quality. Easily digestable organic materials like o~-glucosidic c o m p o u n d s a n d fresh proteins are rare, since they are already d e g r a d e d d u r i n g transportation through the water c o l u m n (Karl et al., 1988). D i s t r i b u t i o n of h y d r o l y t i c a c t i v i t i e s w i t h s e d i m e n t d e p t h Chitobiase, ~-glucosidase a n d iipase activities were found to decrease e x p o n e n t i a l l y with s e d i m e n t depth. These profiles r e s e m b l e gradients of the distribution of easily d e g r a d a b l e s u b s t a n c e s (Westrich & Berner, 1984) . A m i n o p e p t i d a s e activity d e c r e a s e d slowly with s e d i m e n t depth, after showing a subsurface m a x i m u m in cm 2 or 3 which, according to our observations, seems typical for this enzyme. This fits with the distribution of proteinaceous materials in the BIOTRANS area (Pfannkuche, u n p u b l , data), which also decrease slowly with s e d i m e n t depth, h a v i n g their m a x i m a b e t w e e n cm 2 a n d 4. A significant part of these proteinaceous c o m p o u n d s can be refractory material (Rice, 1982). Profiles of bacterial n u m b e r s (Loc hte & Rheinheimer, 1990 ) a n d phospholipids (Boetius, 1992 ) from the BIOTRANS are show a distribution typical for sediments: the highest values were found at the s e d i m e n t surface, decreasing slowly with s e d i m e n t depth. C o m p a r e d with profiles of e n z y m e activities, it can be c o n c l u d e d that hydrolytic e n z y m e activities are rather related to their respective organic substrates, than to microbial biomass. A n impact of biogenic structures on the distribution of hydrolytic activity was observed in the sediments from the BIOTRANS area. W h e n m a c r o f a u n a l tubes were present, higher hydrolytic activities were m e a s u r e d in d e e p e r s e d i m e n t layers. O n top of some cores, detritus particles were found, consisting of a g g r e g a t e d diatoms a n d coccolithophores. The same material was recovered from the gut of a p o l y c h a e t e (Hemleben, pers. comm.). It is k n o w n that microbial activity is e n h a n c e d at m a c r o f a u n a l burrows, b e c a u s e of local a c c u m u l a t i o n of organic material (Ailer & Yingst, 1978; Aller & Aller, 1986; Kbster & Meyer-Reil, 1991) . Due to bioturbation a n d feeding habits of the infauna, organic particles are mixed into the s e d i m e n t column. This may explain the increase in e n z y m e activity in deeper s e d i m e n t layers where burrows occurred. Phospholipid m e a s u r e m e n t s from the same mixtures of s e d i m e n t layers did not show a n impact of the b i o g e n i c structures on microbial biomass (Boetius, u n p u b l , data). Investigations of e n z y m e production of different bacteria strains {Priest, 1984) or mixed populations (Chrost, 1991) show that the production of specific hydrolytic enzymes is i n d u c i b l e by their substrates a n d repressable by their end-products. The c h a n g e s in the hydrolytic activity per cell were d e t e r m i n e d by the availability of organic substrates. Up to now, data on the distribution of different organic c o m p o u n d s w i t h i n deep-sea sediments are rare. S e d i m e n t e d particulate organic material first a c c u m u l a t e s at the s e d i m e n t surface, w h e r e it is used as food by b e n t h i c organisms. By b i o t u r b a t i o n it is mixed into d e e p e r s e d i m e n t layers. D e p e n d i n g on the velocity of this process, as well as on the d e g r a d a t i o n rate, each organic c o m p o u n d has its typical c o n c e n t r a t i o n profile (Berner, 1980; Emerson et al., 1985). Concentrations of labile c o m p o u n d s decrease quickly with s e d i m e n t depth after their m a x i m a at the s e d i m e n t surface. Profiles of refractory material are m a i n l y d e t e r m i n e d by bioturbation rates, which also decrease with s e d i m e n t depth. If the production of hydrolytic e n z y m e s is induced by their respective orgamc substrates, the potential e n z y m e activities are related to the substrate c o n c e n t r a t i o n . Therefore, activity profiles should mirror the distribution of their substrates with s e d i m e n t depth. From the results of this investigation, the idea of a close correlation b e t w e e n potential e n z y m e activities a n d the availability of orgamc substances in d e e p - s e a sedim e n t s can be supported. C o m p a r i s o n of h y d r o l y t i c a c t i v i t i e s of d e e p - s e a s e d i m e n t s w i t h t h o s e of s e d i m e n t s f r o m o t h e r m a r i n e e n v i r o n m e n t s The d e g r a d a t i o n of organic material By production of hydrolytic e n z y m e s is a c o m m o n feature of heterotrophic bacteria. Therefore, data from different e n v i r o n m e n t s c a n be c o m p a r e d w h e n e n z y m e activity is normalized with microbial biomass. Potential activities of specific hydrolases in shallow-water e n v i r o n m e n t s show differences from those of d e e p - s e a e n v i r o n m e n t s , p r e s u m a b l y reflecting the availability of their respective substrates {Table 1). This can be s e e n in the a m o u n t of specific activity as well as in the r a n k i n g of different hydrolases. T h e s p e c i f i c h y d r o l y t i c a c t i v i t i e s w i t h i n all t h r e e e n v i r o n m e n t s differ b y a f a c t o r of 1000. In d e e p - s e a s e d i m e n t s f r o m t h e B I O T R A N S area, a - g l u c o s i d a s e w a s o n l y 0 . 1 % of t h e s p e c i f i c a c t i v i t y as m e a s u r e d in Kiel f j o r d s u r f a c e w a t e r s ( H o p p e , 1983). T h e v a l u e s for [~-glucosidase a n d c h i t o b i a s e s p e c i f i c a c t i v i t i e s in t h e t w o e n v i r o n m e n t s a r e c o m p a r a b l e . In s h a l l o w - w a t e r s e d i m e n t s t h e s p e c i f i c a c t i v i t i e s of [~-glucosidase, a n d p a r t i c u l a r l y c h i t o b i a s e , w e r e s u b s t a n t i a l l y h i g h e r t h a n in d e e p - s e a s e d i m e n t s (King, 1986). For a m i n o p e p t i d a s e a h i g h e r s p e c i f i c a c t i v i t y w a s f o u n d in d e e p s e a s e d i m e n t s f r o m t h e B I O T R A N S a r e a t h a n in t h e Kiel fjord. A m i n o p e p t i d a s e a c t i v i t y in s e d i m e n t s f r o m t h e B I O T R A N S a r e a w a s also h i g h e r t h a n in s e d i m e n t s of Kiel b i g h t , i n v e s t i g a t e d b y M e y e r Reil (1987). S i n c e a d i f f e r e n t i n c u b a t i o n t e c h n i q u e ( i n j e c t i o n of s u b s t r a t e into u n d i s t u r b e d cores) w a s u s e d , this r e s u l t c a n b e a m e t h o d o l o g i c a l effect. A g a i n , t h e s p e c i a l role of a m i n o p e p t i d a s e in d e e p - s e a s e d i m e n t s is i n d i c a t e d , s i n c e it w a s t h e o n l y e n z y m e t h a t h a d a h i g h e r s p e c i f i c a c t i v i t y t h a n in t h e s h a l l o w w a t e r e n v i r o n m e n t s . T h e o t h e r i n v e s t i g a t e d e n z y m e s in d e e p - s e a s e d i m e n t s f r o m B I O T R A N S a r e a s h o w e d l o w e r activities; this w a s e x p e c t e d f r o m t h e l i m i t e d f o o d r e s o u r c e s in t h e abyss. P r e s u m a b l y , t h e d i f f e r e n c e s in h y d r o l y t i c a c t i v i t i e s p e r m i c r o b i a l b i o m a s s b e t w e e n d i f f e r e n t e n v i r o n m e n t s a r e r e l a t e d to t h e a v a i l a b i l i t y of o r g a n i c s u b s t r a t e s . A t y p i c a l r a n g e of p o t e n t i a l h y d r o l y t i c a c t i v i t y w a s f o u n d for e a c h e n z y m e . A m i n o p e p t i d a s e h a d t h e h i g h e s t activity, e x c e e d i n g t h a t of t h e o t h e r i n v e s t i g a t e d e n z y m e s b y a f a c t o r of 1000. C h i t o b i a s e w a s t h e m o s t a c t i v e s a c c h a r i d a s e in d e e p - s e a s e d i m e n t s f r o m t h e B I O T R A N S a r e a . C o m p a r e d to ~ - g l u c o s i d a s e a n d c~-glucosidase, t h e r e l a t i v e a c t i v i t i e s h a d a ratio of 5 . 1 : 0 . 1 . L i p a s e a c t i v i t y w a s as h i g h a s t h a t of [~g l u c o s i d a s e . T h e e n z y m a t i c p o t e n t i a l of t h e i n v e s t i g a t e d h y d r o l a s e s c a n b e r e l a t e d to t h e a v a i l a b i l i t y of t h e i r r e s p e c t i v e o r g a n i c s u b s t r a t e s . E a c h e n z y m e h a d its c h a r a c t e r i s t i c p r o f i l e of d i s t r i b u t i o n w i t h s e d i m e n t d e p t h : t h e s a c c h a r i d a s e a n d l i p a s e a c t i v i t i e s d e c r e a s e d e x p o n e n t i a l l y , w h e r e a s a m i n o p e p t i d a s e s h o w e d a s u b s u r f a c e m a x i m u m f o l l o w e d b y a m o r e l i n e a r d e c r e a s e of a c t i v i t y . T h e s e p r o f i l e s c a n b e r e l a t e d to t h e d i s t r i b u t i o n of e i t h e r l a b i l e or m o r e r e f r a c t o r y o r g a n i c c o m p o u n d s , r e s p e c t i v e l y , r e s e m b l i n g t h e i r g e o c h e m i c a l g r a d i e n t s as p r o p o s e d b y Gaffl a r d & R a b o u i l l e (1992). E x c e p t i o n s to t h e s e t y p i c a l g r a d i e n t s w e r e f o u n d w h e n m a c r o f a u n a l b u r r o w s o c c u r r e d in t h e s a m p l e s , r a i s i n g e n z y m a t i c a c t i v i t i e s in d e e p e r s e d i m e n t l a y e r s . T h i s c o u l d b e e x p l a i n e d b y l o c a l a c c u m u l a t i o n of o r g a n i c m a t e r i a l in a s s o c i a t i o n w i t h t h e b i o g e n i c s t r u c t u r e s , i n d i c a t i n g t h e i r i m p a c t o n m i c r o b i a l a c t i v i t y in s e d i m e n t s . A c o m p a r i s o n of h y d r o l y t i c a c t i v i t i e s p e r b a c t e r i a l cell in m a r i n e s h a l l o w - w a t e r e n v i r o n m e n t s s h o w s d i f f e r e n c e s in t h e r e l a t i v e a b u n d a n c e s of h y d r o l a s e s c o r r e s p o n d i n g to t h e a v a i l a b l e o r g a n i c pool. All e n z y m e s e x c e p t t h e p e p t i d a s e h a d l o w e r s p e c i f i c a c t i v i t i e s in t h e d e e p - s e a s e d i m e n t s . T h e r e s u l t s of this i n v e s t i g a t i o n s u g g e s t a c l o s e c o r r e l a t i o n b e t w e e n a v a i l a b i l i t y of o r g a n i c s u b s t r a t e s a n d m i c r o b i a l h y d r o l y t i c e n z y m e a c t i v i t y in d e e p - s e a s e d i m e n t s . Acknowledgements. I would like to thank the BIO-C-FLUX group and the crew of R. V. "Meteor" for their help with work at sea, also Dr. Karin Lochte for her helpful comments on the first draft of this manuscript. This investigation was supported by the Bundesministerium ffir Forschung und Technologie. This is BIOTRANS publication No. 27. L I T E R A T U R E C I T E D N e w York, 29 - 59 . Deming, J. W. & Yager , P. L. , 1992 . Natural bacterial assemblages in d e e p - s e a sediments: towards a Pariente . Kluwer, Dordrecht, 11 - 28 . 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A. Boetius. Microbial hydrolytic enzyme activities in deep-sea sediments, Helgoland Marine Research, 177, DOI: 10.1007/BF02368348