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Introduction

Organic matter (OM) in aquatic ecosystems consists in a mixture of products of synthesis, degradation and re-synthesis originated from biotic and abiotic mechanisms (Libes 2009). When OM reaches the bottom, its composition will reflect the combined effect of several processes, reflecting changes in its input rate, transport, degradation and preservation (Libes 2009). Thus, marine sediments contain an archive of past climatic and environmental settings influencing primary production and sedimentation dynamics (Libes 2009; Poleto and Charlesworth 2010). The preservation in the sedimentary column of substances or compositional patterns characteristic for primary producers (pigments, determined amino acids and amino sugars), zooplankton (chitin) or bacterial biomass (muramic acid and D- amino acids), together with more general parameters such as particulate organic C (POC) and N (PON), C:N ratios, or stable C and N isotopes can allow the identification of periods with higher or lower water fertility and the corresponding variations in biological activity (Jeuniaux and Voss-Foucart 1991; Emerson and Hedges 2008).

Amino sugars are important  HYPERLINK "http://en.wikipedia.org/wiki/Monosaccharide" \o "Monosaccharide" monosaccharides in living organisms (Horton and Wander 1980). For example, glucosamine is part of the structure of the polysaccharides  HYPERLINK "http://en.wikipedia.org/wiki/Chitosan" \o "Chitosan" chitosan and  HYPERLINK "http://en.wikipedia.org/wiki/Chitin" \o "Chitin" chitin, which compose the exoskeletons of crustaceans and other  HYPERLINK "http://en.wikipedia.org/wiki/Arthropods" \o "Arthropods" arthropods, cell walls in  HYPERLINK "http://en.wikipedia.org/wiki/Fungi" \o "Fungi" fungi and several higher organisms, being one of the main indicators in biogeochemical cycles (Emerson and Hedges 2008). Amino acids are one of the most abundant forms of organic nitrogen in planktonic organisms, in which they have essential metabolic and structural functions (Haake et al. 1993). The amino acid composition of OM in seawater is relatively similar to that of living organisms, yet with higher proportions of glicine, alanine and others indicative of biodegradation (Emerson and Hedges 2008). The relative abundance of the individual amino acids is changed during OM decomposition following well-known patterns, and is used to describe its diagenetic state (Dauwe and Middleburg 1998; Dauwe et al. 1999; Yamashita and Tanoue 2003).

The determination of amino acids and amino sugars in sediments is often carried out by HPLC by monomer pre-column derivatization after hydrolysis of the polymers in OM. A large variation in the conditions for treatment and hydrolysis can be found in the literature, depending on the type of sample material and the specific analytes. Several types of hydrolysis have been proposed for each of these compound classes (Hubberten et al. 1994; Zhang and Amelung 1996; Haake et al. 1993; Kaiser and Benner 2000), with different degrees of recovery. The hydrolysis times for soils and sediments range from 4.5 to 18 hours. Conditions that can be at an optimum for the determination of amino acids in e.g. bacteria (6 N HCl, 140�C, 70 min), are destructive for amino sugars (Kaiser and Benner 2000). For soils samples, Zhang and Amelung (1996) proposed 6 N HCl at 105�C for 8 hours for amino sugar determination. Other authors (Haake et al. 1993) used the same conditions for the determination of amino acids and amino sugars (6N HCl, 110�C, 22 hs ) while for other sugars they used different conditions (2 N HCl, 100�C, 3.5 hours).

The situation described above has typically led to separate hydrolysis of replicate sediment samples, with a duplication of the analytical effort. In the present work it was aimed to establish the optimum hydrolysis conditions that could allow a common protocol for both amino acids and amino sugars, allowing a simultaneous determination. For this purpose, ten different combinations of temperature and duration of acid hydrolysis were tested on natural marine sediment samples.


Material and Methods

During this work, sediments of significantly different organic matter content were used in order to compare yields at hydrolysis conditions assumed to be most favorable according to literature and preliminary experiments. Samples were obtained from the Argentinean continental shelf in 2009 and from the German Bight in 2010. The organic carbon content (%Corg) was determined by removing inorganic C by acidification with 1 N HCl, in order to calculate the necessary sample amount for the determination of amino acids and amino sugars, taking into account that ca. 0.5 mg of organic C/sample are necessary for a measurement under optimum conditions with the Fisons NA 2100 analizer. This was carried out by HPLC with o-phtaldialdehyde (OPA) pre-column derivatization and fluorescence detection (Hubberten et al. 1994).

In a first experimental setting named T1-T8, sediment from the Argentinean Shelf (%Corg =1.6) was divided in 8 duplicate aliquots of 30.5 mg that were put into 10 mL glass ampoules. To each of them, 4 mL of 6N HCl was added, the dead volume was purged with N2 and they were immediately flame sealed. Hydrolysis was carried out following 8 treatments (T1 to T8, Table 1) combining two temperatures (105� and 110�C) and four durations (6, 7, 8 and 22 hours). In a second setting named A-D, a sample from the German Bight (%Corg = 0.5) was divided in four duplicate aliquots of 100.6 mg, and hydrolyzed under 4 different treatments (A to D, Table 2, 105� and 110�C during 22 and 24 hours). 

After hydrolysis and cooling, 1 mL of the supernatant was withdrawn and evaporated to dryness (~2 hours) under low pressure with a Heidolph Synthesis 1 evaporator. After that, 2 mL of sodium citrate buffer (pH: 2.65) were added and the tube contents vortexed to dissolve and homogenize the hydrolysate. From that solution, two 800 �L aliquot duplicates were pippeted out, transferred to vials and kept at -18�C until analysis. 


Results and Discussion

Amino sugars

The T1-T8 set showed clearly different responses in the recovery of glucosamine and galactosamine from sediments (Figure 1). For both amino sugars, at 110�C there was a decrease of around 4% from 6 to 7 hours, keeping practically constant to 8 hours and decreasing ~30% after 22 hours (Fig. 1a, b). At 105�, from 6 to 22 hours there was a smooth increase of 18% for glucosamine and 9% for galactosamine (Fig. 1c, d). The lowest amount from both compounds was recovered after T8 treatment (22 hours at 110�C), which is usually used for the hydrolysis of combined amino acids, also in our laboratory, while T4 (22 hours at 105�C) yielded the highest amounts. The difference between T4 and T8 was 40% and 36% for glucosamine and galactosamine, respectively.

In the A-C array (Figure 2) the best yields for both amino sugars were also obtained with 22 hours at 105�C (treatment A), notably higher than those at 110�C, either for 22 or 24 hours. For galactosamine, the yield of treatment A was 39% higher than C, while compared to B the difference was only 2%. At 110�C, the yield was slightly enhanced (3%) for a hydrolysis duration of 22 hours as compare d to 24 hours.

Amino acids
In the hydrolysates of the T1-T8 array, the amounts of glutamine, glycine, alanine and serine were quantified (Figure 3) in order to evaluate the amino acid yields at the hydrolysis conditions used for amino sugars. These specific compounds were selected for representing a large percentage of the combined amino acids in marine organic matter (Hubberten et al. 1994). Comparing hydrolysis during 22 hours at 105�C (T8) and 110�C (T4), the amount of glutamine, glycine and alanine were respectively 3, 4 and 1% higher at 110�C, while serine was 9% lower. At 105�C recoveries increased with time from T1 to T4, while at 110�C the variation was lower, excepting for serine, which decreased 10% from T5 to T8.

The results from the A-C set (Figure 4) showed practically no differences for glycine and alanine between 22 and 24 hours at 110�C, and these yields were both equal as or only slightly higher (1.5%) than in treatments A and B at 105�C. In contrast, serine was about 10% lower in treatments C and D compared to A and B.

The obtained yields for glucosamine and galactosamine, indicate that in the range of used temperatures and HCl concentration, a difference of only 5�C could be critical in the process of chitin hydrolysis and decomposition of the produced glucosamine, as well as of other amino sugars present in the sedimentary organic matter. At 105� the acid cleavage of the �-1,4 glycosidic linkage of chitin and the deacetylation of N-acetyl-D-glucosamine produces glucosamine in amounts increasing with time from 6 to 22 hours. In contrast, hydrolysis at 110�C shows a maximum glucosamine yield at 6 hours (with a lower yield that at 105�C, 22 hours) and decreases to a minimum at 22 hours. This behaviour is likely attributable to the deamination of glucosamine, producing glucose, not detectable using the OPA-derivatization method, which reacts with primary amines. Oppositely, at the same hydrolysis conditions (110�C, 22 hours) slightly higher values of the amino acids glutamine, glycine and alanine were obtained than at 105�C. 

Zhang and Amelung (1996) reported a reduction in the glucosamine yields when hydrolyzing soil samples at 105�C for more than 8 hours and attributed this fact to presence of lignin phenols. Yet, in our case, since marine sediments contain much less lignin compound than terrestrial soils, (Emerson and Hedges 2008; Gardner and Menzel 1974), this is unlikely the reason for the decrease. Kaiser and Benner (2000) suggested that the optimum hydrolysis conditions for amino acid determination would probably be destructive for amino sugars, what is confirmed by our results.


Conclusions

Taking into account that (a) the amino acid yields after 22 hours of hydrolysis at 105� did not greatly nor consistently differ from those at 110�C (<4% lower for glutamine, glycine and alanine and ~10% higher for serine, (b) the treatment at 110�C produced a amino sugar yield of ~35% lower than at 105�C, and (c) the hydrolysis during 24 hours did not bring any obvious advantage for neither method; the hydrolysis at 22 h hydrolysis at 105� appears adequate for the processing of large sediment sample series in which both, amino acids and amino sugars must be determined. We believe this choice represents a satisfactory balance between analytic efficiency and quality, costs and time reduction, particularly adequate for regional, synoptical evaluation of marine sediment characteristics.

Acknowledgments
We sincerely acknowledge and thank for the excellent technical support from the Centre for Tropical Marine Ecology, particularly Christina Staschock and Dieter Peterke. To� Florencia Biancalana and� German Kopprio, Instituto Argentino de Oceanograf�a, for the corrections and suggestions that helped to increase the quality of this work.

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Emerson SR, Hedges JI (2008) Chemical Oceanography and the Marine Carbon Cycle. Cambridge University Press, New York

Gardner WS, Menzel DW (1974) Phenolic aldehydes as indicators of terrestrially derived organic matter in the sea. Geochimica et Cosmochimica acta 38: 813-822

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Yamashita Y, Tanoue E (2003) Distribution and alteration of amino acids in bulk DOM along a transect from bay to oceanic waters. Marine Chemistry 82: 145�160

Zhang X, Amelung W (1996) Gas chromatographic determination of muramic acid, glucosamine, mannosamine, and galactosamine in soils. Soil Biology and Biochemistry 28: 1201-1206.

Table Captions
Table 1: Different hydrolysis treatments for sediment samples from the Argentinean shelf in the T1-T8 setting.
Table 2: Different hydrolysis treatments for sediment samples from the German Bight in the A-D setting.

Figure Captions
Fig. 1: Release of glucosamine (GlcN) and galactosamine (GalN) from sediment after increasing hydrolysis times at 105�C and 110�C. 

Fig. 2: Release of glucosamine (GlcN) and galactosamine (GalN) from sediment after hydrolysis times of 22 and 24 hours at 105�C (treatments A, B) and 110�C (treatments C, D), respectively. 

Fig. 3: Release of glutamine (GLU), glycine (GLY), alanine (ALA) and serine (SER) from sediment after increasing hydrolysis times at 105�C and 110�C. 

Fig. 4: Release of glutamine (GLU), glycine (GLY), alanine (ALA) and serine (SER) from sediment after hydrolysis times of 22 and 24 hours at 105�C (treatments A, B) and 110�C (treatments C, D), respectively.

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