CiteSeerX — Dissolved organic matter in oceanic waters

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Journal of Oceanography, Vol. 59, pp. 129 to 147, 2003

Review

Keywords:

⋅Dissolved organic matter (DOM),

⋅dissolved organic carbon (DOC),

⋅carbon cycle,

⋅molecular weight distribution,

⋅C:N ratio,

⋅chemical composition,

⋅biomolecules.

Dissolved Organic Matter in Oceanic Waters

HÎROSHI O^GAWA¹* and EÎICHIRO TÂNOUE²

1Ocean Research Institute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164-8639, Japan

2Graduate School of Environmental Studies, Nagoya University, Furo-cho, Chigusa-ku, Nagoya 464-8601, Japan

(Received 12 February 2002; in revised form 3 July 2002; accepted 3 July 2002)

The amount of information on oceanic dissolved organic matter (DOM) has increased dramatically in the last decade thanks to the advances in chemical characterization.

This information has supported the development of some novel and important ideas for DOM dynamics in the ocean. Consequently, we have a better understanding of the importance of DOM in oceanic biogeochemical cycles. Here we review studies published mainly during 1995–2001, synthesize them and discuss unsolved problems and future challenges. The measurement, distribution and turnover of dissolved or- ganic carbon (DOC) are presented as the bulk dynamics of the oceanic DOM. The size spectrum, elemental composition, and chemical compositions at molecular and functional group levels are described. The mechanisms proposed for the survival of biomolecules in DOM are discussed.

organic carbon occurs as non-living DOC (e.g., Cauwet, 1979). In contrast to the organic reservoirs on land, the processes by which DOM has been formed are unclear, and actual sources and the chemical nature of DOM are not well known.

DOM is still the least understood organic reservoir on the earth, but our knowledge of DOM has been rapidly increasing. Recent advancements in DOM study have been covered by reviews of the results of various approaches, trying to understand the dynamics and chemical nature of DOM (Trumbore and Druffel, 1995; Guo and Santschi, 1997; Nagata, 2000; Williams, 2000;

Kepkay, 2000; Myklestad, 2000; Ogawa, 2000; Benner, 2002; Hansell and Carlson, 2002; Hedges, 2002). Some fundamental new insights and observations have been booked recently, which we have summarized in this review. We thus focus here on papers published since 1995, although references to earlier fundamental work are included.

DOM has various functions and plays important roles in chemical, biological and even physical oceanography.

For example, DOM interacts with trace metals or radionuclides and controls their dynamics, it fuels the microbial loop, generates gases (CO, CO₂) and nutrients with biological and photochemical reactions, absorbs and extinguishes light, and affects satellite images, etc. The terrestrial input of DOM is also an important topic in the 1. Introduction

Dissolved organic matter (DOM) in the sea is one of the largest reservoirs of organic matter on the earth’s surface (others include soil organic matter and plant biomass on land), holding approximately as much carbon as is available in atmospheric carbon dioxide (Hedges, 1992).

The fact that DOM is a huge organic reservoir on the earth’s surface has continued to impel ocean scientists to investigate what DOM is, in terms of its source, chemical nature and function in marine environments, from early in the 20th century.

Since homeostatic feedback among the reservoirs of bioelements has controlled the past global environments over the geological time scale (e.g., Berner, 1989), the role and dynamics of dissolved organic carbon (DOC) have become of greater interest in the present global carbon cycle (e.g., Siegenthaler and Sarmiento, 1993). Rec- ognition of the importance of the microbial loop has also has given us new insight into the role of DOM in marine ecosystems (e.g., Pomeroy, 1974; Azam et al., 1983).

Primary production is the ultimate source of organic matter in the sea, but living biomass forms less than 1% of total organic carbon in seawater, while more than 90% of

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global carbon budget as well as carbon dynamics in coastal environments. In this review, however, we focus on recent advances in our understanding of the dynamics and chemical characterization of oceanic DOM. Functions of DOM in the environment are not described.

2. What is DOM?

The first difficulty that one must confront in any definition of DOM is to define the term “dissolved”. It usually has an operational definition as any material that passes through a given filter is termed “dissolved”. Glass fiber filters (Whatman GF/F, with a nominal pore size of 0.7 µm) are widely used for the collection and analysis of organic substances. However, vastly different filter types have been used for study purposes. In reality, a spectrum of material sizes exists; some material, such as colloidal matter, does not fit neatly into either category.

Small-sized plankton pass through the filter; for exam- ple, 22–38% of the total bacterial biomass (Lee et al., 1995) and 100% of viruses, if they occur alone, are not retained on the GF/F filter. We have to be aware that “dissolved organic matter” does not exactly indicate the true

“dissolved phase” of organic matter in the sea.

3. Distribution and Turnover Time of DOC in the Ocean

3.1 Methodology of DOC measurement

The abundance of DOM has generally been determined as dissolved organic carbon (DOC), which is a major element of organic matter. A controversy about the measurement of DOC concentrations in seawater was current during the late 1980s and into the early 1990s.

The high temperature (catalytic) combustion (HTC) method introduced by Sugimura and Suzuki (1988) led to an argument about the existence of a much larger reservoir of DOC in the ocean than had been estimated by the conventional method based on wet chemical oxidation (WCO, Menzel and Vaccaro, 1964). This problem excited many ocean scientists in the world because it had the potential to drastically change our concept of the oceanic carbon cycle (Williams, 1992). After 1993, the controversy calmed through further examination of the HTC technique by a few communities (Hedges et al., 1993;

Sharp et al., 1993, 1995) and independent groups (Ogawa and Ogura, 1992; Tanoue, 1992; Cauwet, 1994). It was found that the HTC measurement included a potentially high system blank relative to the DOC level in seawater (e.g., Williams, 1992; Benner and Strom, 1993). In conclusion, the DOC values obtained from the HTC method in the early stage were retracted (Suzuki, 1993) and the concept of “new” DOC that might have represented a huge oceanic carbon reservoir, missed by the WCO method, was rejected.

Thereafter, the HTC method was reassessed, improved, and reborn as a highly precise method, suitable for measuring the seawater DOC (Sharp, 1997). In addition, the international distribution of certified reference materials (CRMs) for DOC analyses, including low carbon water and deep seawater which were distributed by Dr. Jonathan Sharp in 1997 and Dr. Dennis Hansell since 1999, means that it has become possible to more correctly compare DOC values measured by different workers.

Consequently, both the accuracy and the precision of DOC data have been improved. Details of DOC measurement can be found in the recent review by Sharp (2002).

A typical range of error of the DOC measurement by the recent HTC technique is 1–2% as a relative value, i.e., coefficient of variation (c.v.), or 0.5–1 µM as an ab- solute value, i.e., standard deviation (s.d.) (Qian and Mopper, 1996; Hansell et al., 1997b; Ogawa et al., 1999).

This is approximately 1/10 the error of the traditional WCO method. The improved precision of the DOC method contributed greatly to the resolution of small differences in DOC concentrations in seawater in time and space, although the previous method had been able to detect part of them qualitatively. This improvement was essential for the advancement of oceanic DOC study since we had to resolve the active but small pool of DOC relative to the major part of DOC that was fairly refractory in seawater (see below). The introduction of this improved HTC method supplied quantitative and detailed information for an understanding of the role of DOC in the oceanic carbon cycle.

3.2 Distribution, accumulation and export of DOC in the upper water column

Since the WCO method was established (Menzel and Vaccaro, 1964), it has been consistently observed that elevated concentrations of DOC appear in the upper water column, decreasing with increasing depth, followed by a low, uniform value in the deep water (e.g., Barber, 1968; Ogura, 1970). This finding suggests that DOC is supplied by biological production in the surface ocean and consumed by microbial respiration, and the refractory component resistant to microbial degradation remains in the deep water (Barber, 1968). In addition, recent observations using the high-precision HTC method were successful in detecting the detailed distribution and fluctuation of DOC within the surface water column. The observed changes in the surface DOC appear to be mainly caused by hydrological transport combined with biological production since they were observed over different productive sites along with vertical convection or hori- zontal advection of seawater (Copin-Montégut and Avril, 1993; Carlson et al., 1994; Peltzer and Hayward, 1996;

Hansell and Carlson, 2001a, b). The apparent turnover of DOC that accumulated and disappeared in the surface

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Fig. 1. Typical vertical distributions of DOC in the ocean according to a: conceptual classification of biological reactivity, b: size distribution and c: chemical composition. At each depth the concentration of bulk DOC is representative of the oceanic value reported after 1994, mainly including equatorial, subtropical and temperate zones (see Table 1), without the Antarctic and the Arctic oceans. In a, the semi-labile DOC is expressed as the surface DOC in excess of that in deep water, i.e., the refractory DOC (=~40 µM), as proposed by Carlson and Ducklow (1995). The depth profile of fluorescence of humic-like materials (Ex:

320 or 350 nm, Em: 450 nm) simply indicates its relative pattern in typical oceanic sites of the Sargasso Sea and the North Pacific (Mopper et al., 1991; Chen and Bada, 1992). In b, DOC is separated into the three size classes; very high molecular weight (VHMW; >10 kDa), high molecular weight (HMW; 1–10 kDa) and low molecular weight (LMW; <1 kDa), obtained by the ultrafiltration method (Ogawa and Ogura, 1992; Benner et al., 1997). Here, the typical bulk DOC concentrations of the oceanic water column are the same as shown in a and the mean size distribution at six depths (<20, 100, 150–200, 400, 750–

1000, 4000 m) from the Pacific Ocean, the Atlantic Ocean and Gulf of Mexico are shown (Ogawa and Ogura, 1992; Guo et al., 1995; Benner et al., 1997; Ogawa, unpublished data). In c, DOC is classified into the three chemical components; total hydrolyzable amino acids (THAA) determined by HPLC method (Lindroth and Mopper, 1979), after acid hydrolysis with 6N HCl (Robertson et al., 1987), or a vapor phase hydrolysis (Tsugita et al., 1987), total hydrolyzable neutral sugars (THNS) determined by HPLC method after acid hydrolysis with 1.2M H₂SO₄ (Skoog and Benner, 1997), and total carbohydrate by the MBTH method (MBTH-TCHO), which is a colorimetric technique after hydrolysis with 1.2M H₂SO₄ (Pakulski and Benner, 1992). The TCHO component other than THNS is illustrated here because THNS should be included in TCHO. The concen- trations as carbon in these components were estimated by multiplying the bulk DOC concentrations as shown in a by their mean yields of DOC, which were obtained from different depths of the Pacific and Atlantic Oceans, i.e., THAA-C/DOC (Druffel et al., 1992; Ogawa, unpublished data), THNS-C/DOC (Skoog and Benner, 1997) and TCHO-C/DOC (Pakulski and Benner, 1994). The remainder, given by subtracting (TCHO+THAA)-C from the bulk DOC concentrations, is called the

“uncharacterized” component of DOC here. Thus, note that the bulk DOC concentrations, the size distribution in b and the chemical composition in c simply indicate their representative values and profiles in the ocean.

water column was occurring on time scales of months to a year (Carlson et al., 1994). This turnover time is longer than that of “labile DOC” including free amino acids, sugars and organic acids which turns over in minutes to days through rapid bacterial uptake, while it is shorter than that of “refractory DOC” in the deep ocean, which is quite resistant to microbial oxidation, with average ages of hundreds to thousands of years. Therefore, this fraction of DOC is called “semi-labile DOC” as distinguished from the labile and refractory DOC (Kirchman et al.,

1993; Carlson and Ducklow, 1995; Cherrier et al., 1996).

Assuming that the uniform concentration of the refractory DOC in deep water is extended to the whole water column, the concentrations of the semi-labile DOC would be given by the excess of surface DOC over that in deep water (Fig. 1a). According to recent reports, the concentration of deep water DOC is uniformly about 40 µM, while DOC in surface waters is 60–80 µM in a large area of the ocean, except for the Antarctic Ocean where DOC was restrained in low values (<ca. 60 µM, Table 1). There-

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Table 1. Oceanic DOC (TOC) concentrations measured by the HTC method after 1994.

(a)Surface mixed layer or <100 m. The data from coastal areas were excluded.

(b)>1000 m, * exceptionally high values were excluded.

(c)1. Hansell and Carlson, 1998a; 2. Kähler and Koeve, 2001; 3. Carlson et al., 1994; 4. Carlson et al., 1998; 5. Bates and Hansell, 1999; 6. Guo et al., 1995; 7. Guo et al., 1996; 8. Thomas et al., 1995; 9. Sharp et al., 1995; 10. Carlson and Ducklow, 1995; 11. Peltzer and Hayward, 1996; 12. Skoog and Benner, 1997; 13. Hansell et al., 1997b; 14. Doval and Hansell, 2000;

15. Hansell and Waterhouse, 1997; 16. Hansell and Peltzer, 1998; 17. Wedborg et al., 1998; 18. Carlson et al., 2000; 19. Ogawa et al., 1999; 20. Wiebinga and de Baar, 1998; 21. Kähler et al., 1997; 22. Opsahl et al., 1999; 23. Bussmann and Kattner, 2000;

24. Wheeler et al., 1997; 25. Wheeler et al., 1998.

Study area Latitude DOC (µM) Literature^(c) Note

Surface^(a) Deep^{(b )} Atlantic Ocean

Greenland Sea 75°N — 48 (av.) 1

13/48°W 48°N — 45 (av.) 1

northeast, 20°W 33–60°N 50–70 44 2

Sargasso Sea (Bermuda) 32°N 64–71 44 1, 3, 4

Sargasso Sea (Bermuda) 33–36°N 68–71 — 5 TOC

Middle Atlantic Bight 36°N 70–80 46–48 6, 7

equatorial 1°S–3°N 65–97 33–59* 8

Pacific Ocean

equatorial 60–70 35–40 9

central equatorial, 140°W 0° 63–67 39.5 (av.) 10 TOC

central equatorial, 140°W 12°S–12°N 60–80 36 (av.) 11 TOC

central equatorial, 140/135°W 12/2°S 72–82 44–45 12

western equatorial, 170°E–150°W 12°S–5°N 65–70 — 13 TOC

southwest, 170°W 42°S–0° 65–90 43 (av.) 1, 14 TOC

eastern, 103/110°W 67°S–23°N 40–85 — 15 TOC

northern North 23/58°N — 34–39 (av.) 1

Indian Ocean & Arabian Sea

central, 80/90°E 43°S–5°N 55–80 43 (av.) 1, 14 TOC

Arabian Sea 10–20°N 65–95 43 (av.) 1, 16 TOC

Antarctic Ocean

Weddell Sea 71–63°S 45–60 40–60 17 TOC

Ross Sea 77°S 43–55 (av.) — 4, 18

Australian sector 65–56°S 45–55 40–45 19

Indian sector, 62°E 66–49°S 52–63 42 (av.) 20 TOC

Antarctic Pacific 170°E/170°W 67–42°S 45–75 42 (av.) 1, 14

6°W 60–47°S 38–55 34–38 21

Arctic Ocean

Greenland 74–81°N 66–80 49–54 22

central 80–85°N 78 (av.) 53 (av.) 23

central 70–85°N 63–139 — 24 TOC

central 70–85°N 34–107 (av.) — 25

fore, 20–40 µM of DOC is estimated as making up the semi-labile fraction accumulating in the surface waters.

Globally applying the representative DOC distribution from tropical, subtropical and temperate zones (Fig. 1a based on the data of literatures given in Table 1), the glo-

bal carbon stock of the bulk DOC in the ocean would be approximately 700 Gt. This is close to the estimate given by Hansell and Carlson (680 Gt; 1998a). It consists of 650 and 50 Gt for the refractory and the semi-labile DOC, respectively. The semi-labile DOC is a quantitatively

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minor portion (<10% of the bulk DOC) of the carbon stock in the whole ocean, but it is important for carbon flux.

Hansell and Carlson (1998b) estimated that the global net production of semi-labile DOC was 17% of global new production (=1.2 Gt y^–1).

The hydrological current in the ocean is accompa- nied by the transportation of dissolved substances in seawater. The movement of surface water mass, which is caused by the seasonal vertical convection or the hori- zontal advection driven by winds, in particular results in the export of the semi-labile DOC which is accumulated due to the vertical stability. Most previous studies on the export of primary production from the euphotic zone, i.e., export production, have focused on the pathway due to the settling of large particles (POC) using sediment traps (Eppley and Peterson, 1979). By contrast, it was difficult to evaluate the role of DOC due to its dilute concentrations in seawater. Quantitative evaluations of the export production of DOC were keenly pursued in the late 1990s.

Consequently, some studies estimated a substantial portion of the export production in the ocean would be occurring as DOC, comparable to or exceeding that for sinking particles (Copin-Montégut and Avril, 1993; Carlson et al., 1994; Murray et al., 1994; Peltzer and Hayward, 1996; Libby and Wheeler, 1997; Emerson et al., 1997).

This finding was accepted positively because it was consistent with the hypothesis that DOC export would be responsible for the carbon imbalance between the export production by sinking particles and new production based on nitrate consumption. However, recent studies, espe- cially from the equatorial Pacific, indicated the opposite case that the vertical flux of DOC was unlikely to ac- count for significant export production, and POC flux should be more important (Murray et al., 1996; Hansell et al., 1997a; Hansell and Peltzer, 1998; Doval and Hansell, 2000). The relative importance of DOC and POC fluxes appears to depend largely upon both physical and biological features in the study sites. Not only highly precise measurements of DOC but also more detailed field observation would therefore lead to more realistic evaluations of the role of DOC export in the oceanic carbon cycle. The latest knowledge on DOC export in the ocean is discussed by Hansell and Carlson (2001a, b) and Hansell et al. (2002).

Recent studies have also provided the distribution of DOC in seawater on the global ocean scale. As summarized in Table 1, a number of reports of DOC distributions from a wide variety of regions in the world oceans were concentrated in a relatively brief period after the establishment of the improved HTC method (approximately after 1994). Some of these studies measured TOC instead of DOC when POC concentrations were insignifi- cant, so that the potential contamination of the filtration procedure could be avoided. A large variation of DOC

was observed in the surface waters (generally within the range of 45–90 µM) compared with that in the deep waters (35–50 µM), indicating the regional difference in the accumulation of semi-labile DOC. In general, the surface concentrations of DOC changed more depending on the latitudinal than the meridional direction, in the order of the Arctic (70–100 µM) > subtropical (~80 µM) > tropical (equatorial) and temperate (60–70 µM) > subarctic and subantarctic (50–60 µM) > Antarctic (40–60 µM). It is necessary to consider that some of the high concentrations of DOC in the Arctic Ocean are exceptional for the oceanic environments because they might be influenced by riverine inputs (Bussmann and Kattner, 2000), even though in situ biological production might also contrib- ute to this (Wheeler et al., 1997). With the exception of the Arctic Ocean, the hydrological properties appeared to be a major factor controlling the regional difference in the surface DOC concentrations in the ocean (Hansell and Waterhouse, 1997; Doval and Hansell, 2000; Hansell and Carlson, 2001a, b). In the equatorial area and the high latitude including the Antarctic Ocean where the deep water penetrates into the surface by vertical mixing and upwelling, the DOC concentrations are maintained at a relatively low level (ca. 40–60 µM). By contrast, elevated concentrations of DOC (~80 µM) prevail in the subtropical area where the strong vertical stratification restricts the vertical mixing of the surface water with the deep water. However, this latitudinal pattern suggests that in situ biological productivity would not be a unique factor controlling the DOC concentrations in the surface waters; the subtropical area, a typical oligotrophic ocean, shows higher concentrations of DOC than the productive regions including the high latitude and the equatorial area.

In the case of the equator this was explained by the advection model that some portion of DOC produced in the equatorial upwelling area is exported toward the north and south by advection, and accumulates in the surface waters of the subtropical system (Peltzer and Hayward, 1996; Archer et al., 1997). In the case of the high lati- tude, high productivity area, however, little is known about the fate of DOC produced in the euphotic layers.

Downward export, followed by accumulation in the sub- surface is a plausible interpretation, but there seems to be little evidence. Some recent studies have shown consistently that DOC in the surface waters of the Antarctic Ocean remains at considerably low concentrations, close to the deep water value (~40 µM, Table 1) (Kähler et al., 1997; Wiebinga and de Baar, 1998; Carlson et al., 1998, 2000; Wedborg et al., 1998; Ogawa et al., 1999). Deep vertical convection possibly restricts the accumulation of DOC within the surface water, despite high productivity (Ogawa et al., 1999). Carlson et al. (1998) showed that the biological production in the Antarctic Ocean is preferentially partitioned as POC rather than DOC in the sur-

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face water, while major portion of the primary production accumulates as DOC in the temperate area of the Atlantic Ocean. This could also possibly be attributed to the relatively low concentrations of DOC in the high lati- tude area. According to Carlson et al.’s findings, the ac- cumulation of the surface DOC enriched with the semi- labile DOC in the ocean, may be closely correlated with the partitioning of produced organic carbon (POC or DOC), which may be controlled by plankton community structure and food-web dynamics (Carlson et al., 2000;

Sweeney et al., 2000).

3.3 DOC in deep water

As argued above, the DOC in the deep ocean is the largest pool of fixed carbon in the ocean (~650 Gt), approximately equivalent to the carbon stock of atmospheric CO₂ (~750 Gt) or terrestrial biomass (~600 Gt) (Hedges, 1992). In addition, this reservoir contains remarkably unreactive carbon, resistant to microbial oxidation (Barber, 1968). The apparent average ¹⁴C age of 4,000–

6,000 ybp (Williams and Druffel, 1987) is indicative of this biologically refractory oceanic DOC, which exceeds the circulation time of the whole ocean (~1,000 yr). Thus, the refractory DOC appears to make a relatively minor contribution to the active sites in the global carbon cycle.

On the other hand, it plays a potential role for the seques- tration of fixed carbon in the ocean, suggesting its importance in understanding of the mechanism of global warming. Here, assuming a steady state for the refractory DOC pool in the ocean, its global turnover rate would be approximately 0.1 Gt y^–1 by dividing the stock (~650 Gt) by the average age (4,000–6,000 ybp). Although this value is much lower than that of the semi-labile DOC (~1.2 Gt y^–1; Hansell and Carlson, 1998b), it is comparable to the global carbon flux of sediment burial in the ocean (~0.1 Gt y^–1; Berner, 1989). However, since this estimate is based only on the average ¹⁴C age, it has been pointed out that the estimated flux would increase greatly if the deep DOC is a composite pool of which DOC is older and younger than the average age (Mantoura and Woodward, 1983). These authors proposed that a significant contribution of riverine DOC to the oceanic DOC pool would be consistent with the existence of these different DOC ages. However, a few later studies measuring δ¹³C or lignin contents of DOM as indicators of the terrestrial origin demonstrated that most of the oceanic reservoir of DOC was of marine origin, and the terregenious source made a minor contribution (Meyers- Schulte and Hedges, 1986; Williams and Druffel, 1987;

Opsahl and Benner, 1997).

In the 1990s, deep DOC studies focused on its pho- tochemical degradation (Kieber et al., 1989; Mopper et al., 1991; Moran and Zepp, 1997; Benner and Biddanda, 1998). These studies showed that the refractory DOC in

deep water was photochemically reactive, and degraded by sunlight into low molecular weight, biologically labile, organic compounds and carbon monoxide. The conclusion was that photochemical degradation is an important pathway for removing refractory DOC in the ocean.

Moreover, the estimated turnover time of the oceanic reservoir of refractory DOC (500–2100 yr), based on the photochemical removal rate, was less than the apparent average ¹⁴C age of DOC in the deep ocean (Mopper et al., 1991). Cherrier et al. (1999) measured the natural ¹⁴C in the nucleic acids of marine bacteria from the surface waters of the North Pacific. Interestingly, their results gave direct evidence that marine bacteria utilized ¹⁴C- depleted old carbon, i.e., the refractory DOC, probably through photochemical transformation into biologically labile DOC (Kieber et al., 1989). On the basis of the bac- terial utilization rate of old carbon, the turnover time of the oceanic reservoir of the refractory DOC is estimated as ~1,100 yr, which is also significantly shorter than the average ¹⁴C age of the deep DOC. These findings demonstrate that some portion of refractory DOC is more dynamic than estimated for the bulk pool, and support previous suggestions about the existence of the different age components in the refractory DOC pool in the deep ocean (Mantoura and Woodward, 1983).

Sedimentary organic matter is a possible source of the older component in the deep DOC. Bauer and Druffel (1998) found elevated DOC concentrations with a more

14C-depleted, “older”, signature in the continental slope and rise waters than those from the adjacent North Atlan- tic and North Pacific central gyres. This strongly suggests older DOC inputs from ocean margins to the open ocean interior, although a recent study pointed out that their interpretation of the DOC age gradient may be wrong (Hansell et al., 2002). Burdige et al. (1999) estimated a significant DOC flux from marine sediments (~0.2 Gt C y^–1), roughly equivalent to riverine DOC input, using in situ measurements of benthic flux of DOC from continental margin sediments. This estimate emphasizes the importance of marine sediment as a source of refractory DOC in the oceans. Detailed profiles of DOC concentrations and ¹⁴C contents in the water column above sediments in continental margin would give a further test of this hypothesis.

The DOC concentrations in the deep ocean have long been considered to be quite uniform, which is consistent with the previous concept that the DOC in deep water would be a kind of residue of microbial process that is resistant to further degradation (Barber, 1968). Recent studies with the high precision HTC method, however, revealed clear signals for DOC concentration gradients from the global survey of deep ocean DOC (Hansell and Carlson, 1998a). These authors observed that DOC concentrations in deep waters decreased by 14 µM from the

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North Atlantic Ocean, that is the region of deep water formation, to the terminal of the deep water circulation in the North Pacific Ocean, suggesting the occurrence of long-term decomposition of DOC along the global deep water current. Their findings are basically consistent with the fact that the apparent ¹⁴C age of the deep water DOC in the Sargasso Sea is ~2,000 yr younger than that in the north central Pacific (Bauer et al., 1992). On the other hand, their results also indicated small increases in the deep water DOC concentrations in the mid-latitudes of the Southern Hemisphere, suggesting inputs of DOC to the deep ocean.

Moreover, in the 1990s there were some important discoveries concerning the photochemical process of DOM in the ocean. As described above, photochemical degradation was found to be an important pathway for the removal of biologically refractory DOC in the ocean.

Interestingly, exposure to the sunlight in the surface water is also likely to induce the production of refractory DOC from bioavailable substrates (Kieber et al., 1997;

Benner and Biddanda, 1998), implying a broad role for the photochemical process in the DOC dynamics in the ocean. A photochemically reactive component in natural DOC is generally known as a humic-like substance hav- ing fluorescent properties inherent in this structure. The refractory DOC in the deep ocean is representative of this humic type of fluorescent organic matter. The vertical distribution of this fluorescence is characterized by a dras- tic decrease in the upper water column with decreasing depth (Fig. 1a). The profile displays evidence for the photodegradation of fluorescence of DOM in the surface ocean (Mopper et al., 1991; Chen and Bada, 1992). Al- though it is difficult to directly convert the humic fluorescence to its DOC concentration, the refractory DOC in surface waters is possibly less than in deep waters. The semi-labile DOC in surface waters, when it is estimated on the basis of a uniform concentration of the refractory DOC in the whole water column, might therefore be un- derestimated (Fig. 1a). These findings suggest that DOC in the surface ocean may be involved in more dynamic carbon cycling than was previously considered.

The mass balance assessment for the oceanic reservoir of DOC indicates that the riverine input (~0.2 Gt y^–1; Meybeck, 1982) is roughly equivalent to the turnover of the refractory DOC in the ocean (~0.1 Gt y^–1), but the signature of terrestrial origin remains insignifi- cant in the oceanic pool of DOC (Meyers-Schulte and Hedges, 1986; Williams and Druffel, 1987; Opsahl and Benner, 1997). The riverine DOC is known to be typical of humic-like substances and relatively resistant to microbial degradation, which is similar to the deep ocean DOC (Mantoura and Woodward, 1983). In addition, recent studies indicate that riverine DOC is generally characterized by enhanced fluorescence and photochemical

reactivity. The above discrepancy (i.e., what is the fate of riverine DOC?), therefore, is perhaps explained by its rapid removal through photodegradation prior to enter- ing the oceanic DOC pool (Kieber et al., 1990; Coble, 1996; Opsahl and Benner, 1997).

4. Characterization of DOM in Seawater

4.1 Size distribution

Molecular size spectrum is one of the important char- acters of DOM. It was found that the results of earlier reports were not necessarily consistent with each other.

In the 1990s, however, figures about the size distribution of DOM determined using the ultrafiltration technique were compiled (see the review by Ogawa, 2000). The significant finding is that the low molecular weight (LMW) fraction is the major size fraction of DOM throughout the whole water column in the ocean. The LMW fraction (less than 1 kDa) accounts for approximately 65–80% of the bulk DOC, while the high molecular weight (HMW) fractions are minor portions of DOC (ca. 20–35% for >1 kDa and 2–7% for >10 kDa; Fig. 1b). In addition, it was found that the HMW fraction was abundant in the surface (ca. 30–35% for >1 kDa and 5–7% for >10 kDa) compared with the deep water (20–25% for >1 kDa and 2–4% for >10 kDa). Thus, most old DOM in the deep ocean is LMW-DOM (75–80% of the bulk DOC). This finding suggests that the HMW-DOM is relatively reactive while the LMW-DOM is the major form of refractory DOM in the ocean. The results of degradation experiments (Amon and Benner, 1994, 1996) and ¹⁴C-age measurements (Guo et al., 1996) of different size classes of DOM support this concept. However, we have no ap- propriate explanation for why the LMW-DOM is resistant to microbial oxidation, so that it forms a large organic reservoir in the ocean. This remains one of the big- gest questions for the future marine DOM research. Since the ultrafiltration technique allows us to obtain sufficient HMW-DOM sample for various chemical analyses, our knowledge of the chemical nature of the HMW fraction has been increasing rapidly (see below). However, the LMW-DOM is the least known fraction of DOM because of the lack of a method for concentrating and desalting this fraction. Methodological improvement and further characterization of the LMW-DOM is needed for better understanding of the entire DOM nature.

4.2 Elemental composition

Elemental information, the C:N ratio in particular, is essential to understand the origin and processing of natural organic matter. The C:N ratio of particulate organic matter (POM) retained on a glass fiber filter has been directly determined with a multiple elemental analyzer, typically a CHN analyzer. It is widely known

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that the C:N ratio of POM ranges from approximately 5 to 15 in oceanic waters, and the value in surface waters is close to that of marine phytoplankton (6–8). However, fewer studies have focused on the C:N ratio of DOM in seawater, essentially due to the difficulty of precisely determining this parameter, which is generally obtained from separate measurements of DOC and dissolved organic nitrogen (DON). The concentration of DON is in- directly determined by subtracting the concentration of dissolved inorganic nitrogen (DIN) from total dissolved nitrogen (TDN). Therefore, the precision of DON data depends upon measurements of DIN (=(nitrate + nitrite)

+ ammonium) and TDN. In particular, this becomes a criti- cal problem in deep water samples in which most TDN consists of nitrate (up to ca. 45 µM) while DON (~2 µM) is a relatively minor portion of TDN. If both TDN and DIN analyses are performed with normal precision (2–

3% of c.v.), it would give an approximately 50% error of DON value in deep waters. Finally, it would lead to approximately ±10 of the C:N value (as s.d.), even if precise DOC data are used.

Recently, two approaches have been used to determine carefully the C:N ratio of DOM in seawater. One is the development of the highly precise method of TDN Table 2. The C:N ratio of dissolved organic matter in oceanic seawater.

Study area C:N atom ratio Literature

Bulk DOM (or TOM indicated by *)

*Sargasso Sea (Burmuda) 10–14 (surface) Bates and Hansell, 1999 14 ± 2 (surface) Hansell and Carlson, 2001b 14–15 (deep)

North Atlantic (33–60°N) 8–12 (surface) Kähler and Koeve, 2001

*Eastern Pacific: equatorial 11–14 (surface) Hansell and Waterhouse, 1997 subtropical 17–18 (surface)

high latitude 8–12 (surface)

*Equatorial Pacific 7–9 (surface) Libby and Wheeler, 1997

Southern Ocean 5–8 (surface) Kähler et al., 1997

4–5 (deep)

Southern Ocean 5–16 (surface) Ogawa et al., 1999

11–22 (deep)

Ross Sea 8–23 (<150 m) Carlson et al., 2000

14–20 (150–600 m)

Arctic Ocean 9–25 (surface) Wheeler et al., 1998

HMW-DOM (>1 kDa) concentrated by ultrafiltration

North Pacific, Equatorial Pacific, 13–18 (surface) McCarthy et al., 1993, 1996, 1997, 1998;

Gulf of Mexico, Sargasso Sea 18–22 (deep) Benner et al., 1997

Pacific 16–17 (surface) Clark et al., 1998

17–18 (deep)

Middle Atlantic Bight 11–16 (surface) Guo et al., 1996 14–24 (deep)

Humic-like matter collected using XAD resins (type 2 or 8)

North Pacific, Sargasso Sea 39–57 Druffel et al., 1992

North Pacific, Equatorial Pacific 36–47 Hedges et al., 1992

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measurement combined with the HTC system for DOC analysis (Hansell, 1993; Kähler et al., 1997; Alvarez- Salgado and Miller, 1998; Ogawa et al., 1999). The con- troversy about the DON (TDN) measurement as well as DOC was current in the latter 1980s, started by the report of the Japanese workers that the HTC technique gave much higher DON concentrations in oceanic seawaters than the conventional WCO method (Suzuki et al., 1985).

However, after further examination by other workers (Walsh, 1989; Maita and Yanada, 1990; Hedges et al., 1993; Koike and Tupas, 1993; Hansell, 1993), their results were found to have little reproducibility, and the DON values obtained by the HTC method in the early stage was finally retracted (Suzuki, 1993). Ogawa et al.

(1999) improved a commercial HTC unit of DOC analysis for the simultaneous and highly precise measurement of DOC and TDN, to which a chemiluminescence detec- tor for nitric monoxide (NO) produced from TDN compounds was connected. They reported a mean error of ±1 for the C:N value with this modified HTC system. How- ever, the high precision HTC method for TDN measurements does not yet compare with DOC for general use.

The other approach is to concentrate DOM in seawater, followed by the same elemental analysis as used for POM. The ultrafiltration technique is generally used for this purpose, which largely reduces the error of C:N measurements of DOM by the effect of concentration with desalting, which includes the removal of DIN. The data obtained by this approach, however, is limited to the HMW-DOM fraction which is concentrated by ultrafiltration, and it is not applicable to the bulk and the LMW- DOM.

The recently reported C:N ratio values of oceanic DOM are summarized in Table 2. The observed C:N ratios of DOM (mostly >8) were higher than the typical value of POM, although a few studies reported relatively lower values (<10). Considering that the source of oceanic DOM is marine organisms that have a lower C:N ratio (mostly <8), carbon-rich materials would be selectively supplied and/or preserved as DOM. The HMW- DOM (>1 kDa) have a relatively higher and uniform value of C:N, mostly between 13 and 20, compared with the bulk DOM, suggesting a difference in the elemental composition between the HMW- and the LMW-DOM. The HMW-DOM consists of relatively carbon-rich materials, as revealed in recent findings by NMR, which show that carbohydrates containing no nitrogen are the principal functional group of the HMW-DOM in oceanic waters (Benner et al., 1992; Aluwihare et al., 1997). However, we have little direct evidence for lower C:N ratio of LMW- DOM because of difficulty in the precise measurement of DON in the unconcentrated LMW fraction of DOM in sweater. Further methodological improvement is needed to determine the C:N ratio of the LMW-DOM.

Interestingly, the humic-like substance that was collected and concentrated using XAD resins (nonionic macroporous resins) from acidified seawater samples showed much higher C:N ratios (36–57, Druffel et al., 1992; Hedges et al., 1992), indicating that the majority is considerably carbon rich. This implies the possible occurrence of nitrogen-rich materials in non-humic-like substance of DOM to compensate for the bulk C:N ratio (ca. 8–20). It is difficult to specify chemical species of these substances because they are only operationally de- fined. However, this finding demonstrates that DOM consists of various fractions that have different C:N ratios.

It appears that there are some regional differences in the C:N ratio of DOM in the ocean. Ogawa et al. (1999) and Carlson et al. (2000) showed that the C:N ratios of the bulk DOM were higher in deep waters than in the surface ocean, suggesting that nitrogenous compounds would be selectively decomposed during the downward transport of DOM. Similarly, relatively higher C:N ratios of the HMW-DOM were found in deep waters than at the surface (Guo et al., 1996; Benner et al., 1997). Further- more, Clark et al. (1998) determined the C:N:P ratio of the HMW-DOM in waters from different depths in the Pacific Ocean. Their results showed that the C:P ratio increased dramatically with depth from 247 at the surface to 539 at 4000m, with significant increases in both C:N and N:P ratio, suggesting that phosphorus in DOM would be most preferentially remineralized in the oceanic water column, followed by nitrogen, and carbon was relatively preserved.

It was observed that the C:N ratios of the bulk DOM in the surface of the Antarctica Ocean and the subarctic area of the Atlantic and the South Pacific, were relatively lower (~10) than those from other oceanic sites including subtropical and temperate areas (Hansell and Waterhouse, 1997; Ogawa et al., 1999; Carlson et al., 2000; Kähler and Koeve, 2001). It is possible that the nutritional condition would control the C:N ratio of DOM in the surface ocean (Williams, 1995).

4.3 Chemical composition at molecular level

Chemical analyses of marine organic matter at the molecular level have typically involved degradation (e.g., acid or alkaline hydrolyses, oxidation or pyrolysis) for the release of compounds at monomer level that can be determined by colorimetry, or identified and quantified after chromatographic separations. The chemical analysis of DOM is not exceptional. From the results of this approach, it has long been stated that most DOM in seawater is uncharacterized at the molecular level (Williams and Druffel, 1988; Hedges et al., 2000). This conclusion remains essentially unchanged today. Even considering the analytical uncertainties and lack of data as mentioned below, the contribution of the major bio-

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chemical constituents, i.e., amino acids, carbohydrates and lipids, may not exceed 30% of bulk DOM. This figure is in striking contrast to particulate organic matter (POM) of which more than 80% of organic carbon comprises these three organic constituents (e.g., Wakeham et al., 2000).

Amino acids and neutral sugars are generally determined to be major biochemical components that are identifiable at the molecular level. The total concentrations of amino acids and sugars, as measured by chromatographic techniques after hydrolysis of seawaters, amount to only 1–3% and 2–4% of DOC, respectively (Fig. 1c).

Although a few modifications of the hydrolysis method were proposed to improve the yields of amino acids (Robertson et al., 1987; Keil and Kirchman, 1991) and neutral sugars (Skoog and Benner, 1997), their contribution to the bulk DOC did not change considerably. Lipids are one of the most well documented organic compounds in POM and sedimentary organic matter, whereas only a few analyses have been done on dissolved lipids (e.g., Parrish, 1988; Liu et al., 1998; Muhlebach and Weber, 1998; Mannino and Harvey, 1999; Harvey and Mannino, 2001). Dissolved lipids in the Delaware Estuary com- prised up to 0.3% and 1.6% of 1–30 kDa and 30 kDa–0.2 µm fractions of HMW-DOC, respectively, and values were several times lower than those of carbohydrates and amino acids in these fractions (Mannino and Harvey, 1999, 2000;

Harvey and Mannino, 2001). The major components of dissolved lipids are fatty acids, with other lipids, including sterols, hydrocarbons etc., representing a minor con- tribution (Parrish, 1988; Liu et al., 1998; Muhlebach and Weber, 1998; Mannino and Harvey, 1999). A systematic survey of dissolved lipids in oceanic water is required, since lipids are the major biochemical constituent in organisms with proteins and carbohydrates and could play an important role in the formation of colloids in situ in seawater.

A modified spectrophotometric determination with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) was proposed for measuring the total carbohydrate (TCHO) concentrations in seawater (Pakulski and Benner, 1992). The carbon concentrations in TCHO accounted for approximately 15–35% of DOC in the surface and 5–10% in deep waters of various oceanic areas, indicating that carbohydrates are the most abundant component measurable in marine DOM (Pakulski and Benner, 1994) (Fig. 1c). Although the MBTH method is sensitive to sugar-derived aldehydes, the method is colorimetric and does not directly determine the sugar molecules.

These results demonstrate that a significant portion of DOC occurs as carbohydrate-like compounds that are identifiable as the functional group (aldehydes) but uni- dentified by the typical chromatographic technique. In fact, only 7–20% of TCHO in marine DOM was identi-

fied as neutral sugars, which are major constituents of carbohydrates in marine organism (Skoog and Benner, 1997).

Total dissolved amino acids (TDAA) that are quantified after acid hydrolysis represent the largest well-de- fined molecules of DON. Amino acid nitrogen accounts for approximately 10% or less of bulk DON (Huberten et al., 1995; Dittmar et al., 2001) and <10% in HMW-DON (>1 kDa fraction) (McCarthy et al., 1997). The dominant amino acid in TDAA is glycine, irrespective of the temporal and spatial differences in observations, followed by alanine, aspartic acid or serine (Hubberten et al., 1994, 1995; Keil and Kirchman, 1999; Dittmar et al., 2001).

Fractionation of DOM using XAD resin into the hydrophobic “humic” and hydrophylic compounds revealed that glycine and other dominant amino acids in TDAA are enriched in the hydrophobic fraction (Hubberten et al., 1994, 1995). Since the concentration of amino acids in the hydrophilic fraction correlated with chlorophyll, but no relationship was found between the hydrophobic fraction and chlorophyll, it was concluded that amino acids are selectively utilized by bacteria and that amino acids in the hydrophobic fraction occur as a background TDAA, forming part of recalcitrant DOM (Hubberten et al., 1994, 1995). Enrichment in glycine was also found in fluvial riverine DOM (Dittmar et al., 2001). The preferential accumulation of glycine was suggested along the progres- sive degradation from fresh phytoplankton to deep-sea sediment (Dauwe et al., 1999).

The occurrence of microbial-derived D-enantiomers of amino acids has given us an invaluable clue to the con- tribution of microbes to DOM (McCarthy et al., 1998;

Dittmar et al., 2001). In the HMW-DOM (>1 kDa) frac- tion from entire water columns in the central Pacific and the Gulf of Mexico, alanine showed the most pronounced enrichment, with D/L ratios near 0.5, whereas ratios of aspartic acid, glutamic acid and serine fell between 0.2 and 0.4, and no other D-amino acid was detected at sig- nificant concentrations (McCarthy et al., 1998). Highly selective patterns of D-amino acid enrichment are a strong indication that D-amino acids did not derive from abiotic racemization but represent a constituent of peptidogly- can in the bacterial outer membrane. A systematic survey of amino acids and their D-enantiomers in the bulk DOM and POM also demonstrated the major microbial contribution to the bulk DON in seawater in the Arctic Ocean (Dittmar et al., 2001). Contributions of TDAA nitrogen to DON were almost constant and accounted for approximately 10% in the water column from the near-shore to open-sea areas. The D-enantiomers of alanine, aspartic acid, glutamic acid and serine were determined in the TDAA, and the proportions of all D-enantiomers increased with depth, with D-alanine comprising 44% of total alanine in deep water. The similarity of amino acid

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composition and high D/L ratios of TDAA, exceeding the values of bacterioplankton (McCarthy et al., 1998; Amon et al., 2001), in wide areas of the ocean indicates that phytoplankton-derived amino acids were replaced by microbial organic matter during the process of decomposition, and the repeated replacements might shape the chemical composition of both TDAA and DOM (Meon and Kirchman, 2001; Amon et al., 2001; Dittmar et al., 2001).

4.4 Approaches to characterizing DOM at functional group and biomacromolecular levels

One of the goals of marine organic geochemistry is to identify organic compounds and to describe the in situ states of these materials in seawater. For bulk chemical characterization of DOM, a combination of two techniques, i.e., concentration of DOM with tangential (or cross) flow ultrafiltration and application of nuclear mag- netic resonance (NMR) to the concentrate, has given us some fundamental new insights into the chemical nature of DOM. The majority of functional groups in HMW-DOC (>1 kDa) determined by ¹³C and ¹H NMR spectroscopy indicate that carbohydrates are the major constituents of DOM (Benner et al., 1992; Aluwihare et al., 1997), par- ticularly photosynthetically derived acyl oligosaccharides (Aluwihare et al., 1997). The results of ¹⁵N and ³¹P NMR analyses of HMW-DOM (>1 kDa) indicated that approximately 70–90% of organic nitrogen is in the form of amide (McCarthy et al., 1997) and organic phosphorus is domi- nated by two major classes, phosphorus esters and phosphonates (Clark et al., 1998; Kolowith (née Clark) et al., 2001).

The NMR studies clearly demonstrate that most HMW-DOM (>1 kDa) is identifiable as functional groups common to biochemical constituents in organisms. A combination of two techniques, i.e., tangential (or cross) flow ultrafiltration and gel electrophoresis, provided direct evidence that specific protein molecules occur universally in HMW-DOM (>10 kDa), and some proteins were identified as bacterial outer membrane proteins, collectively termed “porin” (Tanoue, 1995, 2000; Tanoue et al., 1995, 1996; Suzuki et al., 1997; Yamada et al., 2000). These findings, as well as the occurrence of D-enantiomers as mentioned above, indicate that biomolecules, particularly bacterial membrane components, survive in DOM. In the culture experiments of marine bacteria, Ogawa et al.

(2001) found that DOM, including LMW-DOM (<10 kDa), rapidly accumulates (<48 h), indicating that bacterial processes are a possible pathway of production of even the molecularly uncharacterized component of DOM.

Interestingly, the spectra of ¹⁵N, ³¹P and ¹³C NMR, did not show any significant temporal and spatial vari- ability. Such a stable DOM fraction, a major component

of DOM, has been collectively designated “water humus”.

The humic substances are formed by condensation reactions that depend on intermolecular collisions of degradation products of photosynthetically produced biopolymers. These condensations include the Maillard reactions between carbohydrates and amino acids (or proteins) that form “melanoidins”, and such processes are termed “humification”. Although the occurrence of amino acids and carbohydrate condensation products has not been reported in marine environments, the mechanisms of the Maillard reactions have been investigated in the field of biochemistry because several diseases are caused by the Maillard reaction, indicating the reactions progress on a relatively short time scale.

In the biochemical literature (e.g., review by Njoroge and Monnier, 1989), glucose and other reducing sugars react with protein by non-enzymatic, post-translation modification process called non-enzymatic glycosylation or glycation, and the products of the reaction are termed advanced-glycation end-products (AGEs). The compound N-phenacylthiazolium bromide (PTB) was used to break down AGEs (Vasan et al., 1996). The results of applica- tions of PTB treatment to geochemical samples indicated that AGEs certainly formed in ca. 20,000 year old and ca.

4,000 year old sediments (Poiner et al., 1998; Nguyen and Harvey, 2001) and during the short time scale (3 months) plant litter degradation experiments (Fogel and Tuross, 1999). “Humification” does occur in the environments on a certain time scale. However it appears that there is no real evidence that such heteropolycondensation reactions occur in the water column, with extremely low levels of organic matter, low temperature and lack of catalytic agents (e.g., Tanoue, 2000). Occurrences of the pyridine-type N and/or imine-N signals in geochemical samples are thought to be evidence of AGEs resulted from the Maillard reaction (Evershed et al., 1997). However, no pyridine-type N and/or imine-N signals were found in the above-mentioned ¹⁵N NMR spectra of HMW-DOM (McCarthy et al., 1997). Knicker (2000) found only amide-N in peptide-like structures with little evidence of pyridine-type N and/or imine-N signals from the ¹⁵N NMR spectra of the algal residue after two months degradation. Amide and ester linkages and C-P bonds are also difficult to form via abiological condensation reactions (e.g., Hedges et al., 2000). The NMR spectra, particu- larly ¹⁵N and ³¹P NMR spectra (McCarthy et al., 1997;

Clark et al., 1998; Kolowith (née Clark) et al., 2001), imply that such humification is not a major process for the formation of stable DOM.

In marine environments, primary production by phytoplankton is the ultimate source of marine organic matter; however, growing evidence supports the idea that the phytoplankton-derived organic matter is decomposed rapidly by the microbial processes. During the process,

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certain organic compounds escape from rapid remineralization and form microbiologically recalcitrant organic molecules. Thus, degradation processes are more important than the formation of humic materials in controlling and maintaining the reactive pool of organic matter on the earth’s surface (e.g., see review by Volkman and Tanoue, 2002). Microbial processes probably control the molecular composition of DOM, not only identified molecules but also the molecularly uncharacterized fraction, a major part of DOM (Ogawa et al., 2001).

5. Survival of Biomolecules in the Environments The discussions reported above are persuasive enough to lead us to believe that some biomolecules, particularly proteins and peptides, survive in marine and terrestrial DOM. In the sediments, the materials that can be extracted from sediments with an alkaline solution or denaturing reagents were subjected to chemical digestion and cleaved by proteolytic enzymes (Pantoja and Lee, 1999; Nguyen and Harvey, 2001), indicating that proteinacous materials were preserved. The protein rem- nants were also identified from the insoluble geopolymers (humin and kerogen) by thermochemolysis with the te- tramethylammonium hydroxide (TMAH)/GC-MS tech- nique (Zang et al., 2000; Mongenot et al., 2001; Knicker et al., 2001). Direct and indirect evidence consistently indicates that proteins and proteinacous materials, including peptides and amino acids, are present in detrital organic pool in the environments. Such recognition is an encouragement to further explain and delineate the processes by which such materials survive and are preserved.

Hypotheses proposed in the fields of chemical oceanography, organic geochemistry and soil chemistry, are described briefly below.

(a) Geopolymerization: The concept is that the monomers and/or oligomers, particularly amino acids and reduced sugars, react with each other abiologically (i.e., Maillard reaction) to form biologically refractory humic substances and further altered to kerogen. This concept has been considered to be the major process for formation of uncharacterized DOM; however, recent studies suggest that this process is not responsible for the formation of refractory DOM as mentioned above.

(b) Sorption: The concept that organic matter sorbed onto a mineral surface or inside the micropore of a mineral is protected from biological degradation.

Simple sorption: It is well known that concentrations of sedimentary organic matter are related to the grain size, and in turn to the mineral surface area (e.g., Hedges and Keil, 1995, and references therein), and that sorption to solid surfaces has been shown to inhibit bacterial degradation of labile organic matter and proteins (Marshmann and Marshall, 1981; Taylor et al., 1994a, b; Keil et al., 1994; Hedges and Keil, 1995; Nagata and Kirchman,

1996).

Mesopore sorption: Based on Mayer’s (1994) study of the pore sizes of the coastal sediment, a globular macro- molecule, e.g., a 67 kDa protein with a Stokes radius of 3.6 nm, or a diameter of 7.2 nm (Creighton, 1993), is ca- pable of diffusing into mesopores of the sediments. Mol- ecules incorporated into the mesopores are protected from enzymatic as well as bacterial attack because enzymes cannot access the molecules.

(c) Selective preservation: The concept is that some or- ganic matter is preserved selectively because of its refractory nature. The idea originally came from studies on kerogens, i.e., the insoluble organic matter of sedimentary rocks that has been thought to be an origin of oil.

Chemical and microscopic examinations suggested that types of kerogens were principally associated with an

“ultralaminae” microstructure, being constituted with non-hydrolyzable and diagenetically resistant macromol- ecules based on a network of long, saturated hydrocar- bon chains, termed collectively “algaenans”. The algaenans are ubiquitous in freshwater green algae (e.g., de Leeuw and Largeau, 1993), as cutans in cuticles and suberans in periderm tissue of higher plants (e.g., Collinson et al., 1994), as tegmens in inner seed coats of freshwater plants (e.g., Hemsley et al., 1993), and as polycadinenes in resins (van Aarsssen et al., 1990).

Cyanobacteria and bacteria (as bacterans) and marine algae also synthesize algaenans (e.g., Largeau, 1995; Gelin et al., 1996, 1999).

(d) Encapsulation: The concept is that the proteinaceous materials are incorporated into a surrounding organic matrix and are protected from biological attack. The organic matrix could be different in different environments and time scales. In laboratory experiments, it has been demonstrated that spiked amino acids or proteins associated with existing natural DOM and in situ biological utilization rates decrease. The results imply that abiotic association with an organic matrix is potentially important for surviving labile organic compounds in seawater (Carlson et al., 1985; Keil and Kirchman, 1994).

Membrane encapsulation: Proteolytic digestion indicated that peptides, particularly phytoplankton membrane components, are preferentially enriched as compared with the cytosol proteins in estuarine POM (Laursen et al., 1996).

The occurrences of bacterial outer membrane components in HMW-DOM (e.g., Tanoue et al., 1995; McCarthy et al., 1998) suggest that the concept could be one of the possible mechanisms for survival of biomacromolecules in DOM.

Liposome encapsulation: Nagata and Kirchman (1992) found that one type of flagellate fecal pellets, consisting of phospholipids, enzymes and other biochemically labile substances, form liposome-like colloidal particles.

They proposed that liposome-like organic complexes pro-

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duced by flagellates could provide a microenvironment for geochemical modification of organic matter to pro- ceed (see also review by Nagata and Kirchman, 1997).

Network encapsulation: The concept has come from stud- ies of sedimentary organic matter. Proteinacous materials are incorporated within the above-mentioned refractory aliphatic macromolecular matrix and are sterically protected from bacterial hydrolysis (Knicher and Hatcher, 1997). The proteinacous materials extracted from the sediment were readily degradable by chemical and enzymatic procedures.

(e) Aggregation: Nguyen and Harvey (2001) showed in laboratory experiments that proteolytic digestable peptides were preserved in diatom detritus by the formation of large aggregates. They mentioned that non-cova- lent interactions, the conformation alteration and the hydrophobicity of the original polypeptides could be important for the preservation of protein and/or peptides.

Every hypothesis itemized above is applicable to the respective natural environments. Considering the conditions of the water column in the marine environments, the hypotheses of encapsulation or aggregation seem to be probable mechanisms for the survival of biomolecules.

The majority of DOM has a molecular mass of <1 kDa.

D-enantiomers of amino acids are also detected in the LMW-DOM, indicating that the hypotheses of membrane and network encapsulations may not be applicable to the entire DOM. At present, liposome encapsulation or aggregation is a likely mechanism for survival of biomolecules, assuming that such a macrostructure is formed reversibly from LMW-DOM, although the bound- ary conditions for association-dissociation equilibrium are unknown.

So far, the sources of the biomolecules that we have identified are very specific. For example, the source of the porin protein identified from DOM (e.g., Tanoue et al., 1995) was derived not from the bulk bacterial popu- lation but from a specific species of Gram-negative bac- terium. Biosynthesis of phosphonates has been reported from a few groups of organisms (e.g., Clark et al., 1999), although an actual source of phosphonates in DOM is unknown. The above hypotheses are based on the same idea, i.e., how biomolecules are protected from biological attack. However, the hypothesis does not explain why selective biomolecules actually survive in DOM. From this point of view, the physicochemical properties of the biomolecules or the process by which the biomolecules are transferred from source organisms to DOM might be other factors determining the survival of the biomolecules in DOM. In fact, synergism among different factors, pro- tection mechanisms, physicochemical properties or trans- fer processes, might control the survival of the biomolecules in the sea.

6. Concluding Remarks

The amount of information on oceanic DOM has increased dramatically in the last decade, supported by progress in the chemical approaches, including both the improvement of conventional methods and the introduction of new techniques. We are now looking at various aspects of DOM from different viewpoints, such as the bulk properties (i.e., DOC), size distribution, elemental, isotopic, molecular and functional group levels. In addition, we know that both biological and physicochemical processes contribute significantly to DOM dynamics in the ocean, including its production, transformation and decomposition. Some findings have had a great individual impact upon our concepts. However, the combination of different types of information allows us to better face various aspects of the oceanic DOM and the new problems that are challenges for future. From this point of view, four topics are given here although they are closely related with one another.

6.1 Semi-labile DOC

The high precision HTC method has been successful in detecting the detailed distribution and fluctuation of the semi-labile DOC in the ocean, and in quantifying the role of DOC in export production (Copin-Montégut and Avril, 1993; Carlson et al., 1994; Carlson and Ducklow, 1995; Peltzer and Hayward, 1996; Hansell and Carlson, 1998a, b, 2001a, b; Hansell et al., 2002). This semi-labile DOC, however, is simply a conceptual category of DOC based on vertical distributions of DOC (see Fig. 1a), but is supported by no evidence from chemical characterization. Moreover, little is known about the mechanisms of production and decomposition of the semi- labile DOC in the ocean. Thus, a future challenge should focus on its qualitative characterization as well as the quantitative evaluation.

6.2 LMW-DOM

Most of the oceanic DOM consists of the LMW (<1 kDa) components (Ogawa, 2000; see Fig. 1b). This is consistent with other information demonstrating that LMW-DOM is responsible for the major nature of bulk DOM in the ocean, which is biologically refractory and molecularly uncharacterized (Amon and Benner, 1994, 1996; Guo et al., 1996). However, there is little knowl- edge about its chemical features and processes of production, transformation and decomposition. In particular, the reason why these small molecules are apparently resistant to microbial utilization is the key to understanding them; what mechanism allows part of the biologically fixed carbon in the ocean to remain in seawater for a long period and to develop a huge carbon reservoir of DOM?