Biocatalytic quantification of α‐glucan in marine particulate organic matter

Abstract Marine algae drive the marine carbon cycle, converting carbon dioxide into organic material. A major component of this produced biomass is a variety of glycans. Marine α‐glucans include a range of storage glycans from red and green algae, bacteria, fungi, and animals. Although these compounds are likely to account for a high amount of the carbon stored in the oceans they have not been quantified in marine samples so far. Here we present a method to extract and quantify α‐glucans (and compare it with the β‐glucan laminarin) in particulate organic matter from algal cultures and environmental samples using sequential physicochemical extraction and enzymes as α‐glucan‐specific probes. This enzymatic assay is more specific and less susceptible to side reactions than chemical hydrolysis. Using HPAEC‐PAD to detect the hydrolysis products allows for a glycan quantification in particulate marine samples down to a concentration of ≈2 µg/L. We measured glucans in three cultured microalgae as well as in marine particulate organic matter from the North Sea and western North Atlantic Ocean. While the β‐glucan laminarin from diatoms and brown algae is an essential component of marine carbon turnover, our results further indicate the significant contribution of starch‐like α‐glucans to marine particulate organic matter. Henceforth, the combination of glycan‐linkage‐specific enzymes and chromatographic hydrolysis product detection can provide a powerful tool in the exploration of marine glycans and their role in the global carbon cycle.

constitute the majority of organic matter produced by algae (Myklestad, 1974), and are therefore a central part of the complex marine food web. Characterization and quantification of algal glycans represent an important task to understand microbial carbon cycling and sequestration in the marine environment (Engel et al., 2004;Hedges et al., 2001).
Glycans are complex and often nonlinear polymers built from numerous possible monosaccharides and chemical modifications (Laine, 1994). This complexity makes the structural analysis of glycans challenging and the analysis of glycans in marine dissolved or particulate organic matter even more difficult as the glycans are present in complex mixtures and at often low individual concentrations. The lack of structure-specific analytical tools for marine glycan samples leads to these glycans being commonly identified and quantified by their monosaccharide content after chemical lysis (Engel & Händel, 2011;Panagiotopoulos & Sempéré, 2005). This nonselective acid hydrolysis destroys information about glycosidic linkages and some chemical modifications. It is therefore not suitable to unequivocally identify different types of glycans. While there are derivatization methods in combination with mass spectrometry to distinguish sugar linkages (Galermo et al., 2018;Price, 2007), there is still a fundamental lack of knowledge about the structures and functions of marine polysaccharides and the marine carbon cycle (Hedges et al., 2001).
Glucans are glycans derived from D-glucose residues. Some glucans like starch, glycogen, and laminarin are energy storage glycans while other glucans like cellulose are structural polymers (Painter, 1983;Suzuki & Suzuki, 2013). Acid hydrolysis makes these glycans of different biological origins indistinguishable and obscures insight into their roles in the marine carbon cycle.
α-Glucans include a range of different types of polysaccharides, most of them containing α-1,4 and α-1,6-glycosidic linkages. Starch is a mixture of the linear α-1,4-linked amylose and the branched amylopectin composed of α-1,4and α-1,6-glycosidic bonds (Imberty et al., 1991). Starch is commonly known as a storage glycan for terrestrial plants but it is also found in green algae. Floridean starch is a storage α-glucan in red algae similar to amylopectin but with a higher degree of α-1,6-branching (Yu et al., 2002). Glycogen is another highly α-1,6-branched storage α-glucan found in animals, fungi, and bacteria (Ball & Morell, 2003). Although the amount of αglucans in the ocean is unknown, analysis of marine bacterial polysaccharide degradation pathways (Fang et al., 2019;Kappelmann et al., 2019) indicates that α-glucans are an important carbohydrate source for marine bacteria.
Glycan-specific glycoside hydrolases (GHs) can be applied to identify and quantify α-glucans in the ocean. There are several wellcharacterized starch-specific glycoside hydrolases (α-amylases and amyloglucosidases) that have been used to quantify starch in food (McCleary et al., 1994(McCleary et al., , 2002, yet a more sensitive method for starch quantification in marine environmental samples is missing. In this study, a set of commercial enzymes was adapted into an assay for the quantification of α-glucans in different types of microalgae and unprocessed marine environmental samples. This assay was tested in parallel with a previously developed laminarin assay (Becker et al., 2017) on different types of α-glucans and other polysaccharides to explore possible side reactions and compare the enzymatic hydrolysis (EH) with the commonly used acid hydrolysis (AH). Additionally, the detection range of the assay was tested using two types of glucose detection, the spectrophotometric PAHBAH assay, and HPAEC-PAD. Furthermore, the extraction of glucans from different types of microalgae was optimized. The α-glucan and laminarin content of these microalgae was tested on different days of algal growth and compared to the total particulate organic carbon (POC) in these samples. Finally, the assay was used on two sets of particulate organic matter (POM) samples from the North Sea (spring 2020) and the western North Atlantic Ocean (spring 2019) to demonstrate that this method allows for a quantification of low concentrations of α-glucans alongside laminarin in crude marine samples.

| Algal cultures
To test different extraction protocols and enzymatic hydrolysis of algal glucans, three types of microalgae were used as model organisms for green and red algae and diatoms, respectively: The green microalga Ostreococcus tauri (Ral et al., 2004) was grown in L1 medium (Guillard, 1983;Guillard & Hargraves, 1993), and the red microalgae Porphyridium purpureum and the diatom Thalassiosira weissflogii were grown in NEPCC medium (Harrison et al., 1980 For the αand β-glucan quantifications algae were cultivated in triplicate batch cultures. A total volume of 250 ml in T75 suspension cell culture flasks was inoculated with 5 ml of a 25-ml culture that had been grown for 7 days. Ten to twenty-five milliliters of each culture were taken over 20 days and filtered as described above.

| Extraction of glucans from particulate algae material
The filters were cut into equally sized pieces and subjected to extraction tests. One filter piece was kept as a nonextracted reference. Each extraction was tested using filter-part triplicates of three different filters. Afterward, the extracted and non-extracted filter parts were put under AH conditions and the glucose content of the acid extract was tested using HPAEC-PAD. The extraction conditions tested include water or 1M NaOH extractions at 99°C or in a sonication bath, different extraction times, and sequential extractions.

| Acid hydrolysis
Glycan samples in solution or algal samples on glass fiber filters were incubated in 1 M HCl for 24 h at 100°C in sealed glass ampoules (Engel & Händel, 2011) to chemically hydrolyze glycans. Afterward, 100 μl of each acid solution was evaporated using a speed-vac (RVC 2-18 CDplus HCl resistant; Christ) and resuspended in 100 μl buffer or Milli-Q water.
One unit of α-amylase is the amount of enzyme required to release 1 μmole of p-nitrophenol from blocked p-nitrophenylmaltoheptaoside per minute (in the presence of excess αglucosidase), pH 5.4 at 40°C (Megazyme, 2022). One unit of amyloglucosidase activity is defined as 1.0 mg of glucose hydrolyzed from starch in 3 min at pH 4.5 at 55°C (Merck, 2022 Subsamples used for laminarin quantification (90 μl) were hydrolyzed using 90 μl aqueous glycan solution, 10 μl 500 mM MOPS buffer (pH 7.0), and laminarinases as previously described (Becker et al., 2017).

| PAHBAH reducing sugar assay
Photometric quantification of polysaccharide hydrolysis products was performed using the PAHBAH reducing sugar assay (Lever, 1972).
Hydrolyzed and non-hydrolyzed EH samples were used without further cleaning. After incubation for 5 min at 99°C, the samples were cooled on ice and the absorbance at 410 nm was measured using 10 mm pathlength semi-micro cuvets and a BioSpectrometer (Eppendorf). PAHBAH reaction mix A/B (9:1, v/v) was used as a blank.
Glucose was used as a quantification standard. The glucose standards used for quantification were treated the same way as the respective samples. This corrects for absorbance changes in the assay caused by the addition of specific buffers and enzymes, or by AH ( Figure A1).

| HPAEC-PAD
All samples for HPAEC-PAD (high-performance anionexchange chromatography with pulsed amperometric detection) were filtered using a 0.2 μm Spin-X filter and transferred into an HPLC vial with a micro-insert. The monosaccharide (and specifically glucose) content was determined using HPAEC-PAD with a Dionex CarboPac PA10 column (Thermo Scientific) and monosaccharide mixes as standards for calibration. The oligosaccharide content was determined using Dionex CarboPac PA200 and PA100 columns (Thermo Scientific) and appropriate malto-and laminarin oligosaccharides (Megazyme).

| Glucan quantification
Glucans in extracted samples were quantified as glucose equivalents based on a calibration curve generated with glucose standards.
The glucose signals were inferred from PAHBAH absorbance readings or integrated peak areas from HPAEC-PAD chromatograms.
Each hydrolyzed sample or non-hydrolyzed control was measured in triplicates. To correct for background signals in the enzymatically STEINKE ET AL.
| 3 of 14 digested samples, the glucose values detected for non-hydrolyzed samples were subtracted. To measure the total glucose or reducing sugar concentration after AH no background correction with nonhydrolyzed samples was applied.
The glucan concentrations in filter extracts were normalized by extraction volume, filter size, and filtration volume.

| Particulate organic carbon quantification
Particulate organic matter from cultured algae or environmental samples were both filtered on glass fiber filters. Defined pieces of these filters were punched out in triplicates and subjected to an acidic atmosphere with concentrated HCl for 24 h in a desiccator to remove inorganic carbon. Afterward, the filters were dried for 24 h at 60°C and wrapped in combusted tin foil. The carbon quantification was performed by an elemental analyzer (vario MICRO cube; Elementar Analysensysteme) using sulfanilamide as a calibration standard.

| Optimal conditions for enzymatic α-glucan hydrolysis
Based on an assay for amylose and amylopectin quantification in cereal starches and flours (Megazyme, 2018), a similar assay for total α-glucan quantification (without amylose/amylopectin separation) was developed and optimized for a sample volume of 100 μl, a starch concentration of 100 μg/ml (from corn; Sigma-Aldrich) and hydrolysis product detection using the PAHBAH reducing sugar assay (Lever, 1972). Optimal EH was achieved by incubating a 100 μl starch sample in 100 mM sodium acetate buffer (pH 4.5) for 35 min at 50°C in the presence of 0.4 U of the endo-acting α-amylase (from Aspergillus oryzae, Megazyme) and 0.9 U of the exo-acting amyloglucosidase (from A. niger; Merck). Under these conditions, PAHBAH reducing sugar signals were maximal ( Figures A2, A3). Subsequently, the complete hydrolysis of different α-glucan polysaccharides, namely starch, glycogen, amylose, or amylopectin down to glucose monosaccharides was confirmed by HPAEC-PAD ( Figure A4).

| Enzymatic hydrolysis of α-glucans is more effective than acid hydrolysis
The enzymatic α-glucan hydrolysis was tested on different starch concentrations and compared to AH using the PAHBAH reducing sugar assay as a glucose detection method ( Figure 1). The limit of quantification was found to be ≈11 μg/ml starch, both for AH, and for EH in combination with the PAHBAH assay. However, it was found that the EH of starch (1-1000 μg/ml) generated a higher photometric signal than AH suggesting that more complete polysaccharide hydrolysis was achieved or that EH is less prone to generate side products (Cai et al., 2014). It should be noted that for these measurements, the glucose calibration curve was prepared in water. Under AH conditions the glucose signals in the calibration curve were (≈−20%) lower ( Figure A1). These data indicate that lower glucose signals in acid hydrolyzed starch samples could partially be due to acid-catalyzed conversion of glucose. But this loss does not account for the total difference in the signal of acid and enzymatic starch hydrolysis and our results also indicate that AH of starch is less complete than EH.
Overall, these results show that AH-the traditional quantification method-may underestimate the true glycan content in environmental samples due to incomplete glycan hydrolysis and possible side reactions reducing the detectable monosaccharide content.

| Enzymatic hydrolysis of α-glucans is specific
To explore the specificity of the enzymatic α-glucan assay compared with AH, 14 commercially available polysaccharides were tested. The enzymatic laminarin assay was included as a control. Figure 2 shows F I G U R E 1 Enzymatic hydrolysis is more effective than acid hydrolysis for starch: Enzymatic hydrolysis (EH) products of starch, acid hydrolysis (AH) products of starch, nondigested starch, and glucose as calibration standards were measured in α-glucan assay buffer (100 mM sodium acetate buffer, pH 4.5) using the PAHBAH reducing sugar assay. A 410nm signals above 1.9 were measured in diluted samples. Error bars denote standard deviation (n = 3, technical replicates). Solid lines represent regression lines. A plot zoomed-in on starch concentrations <100 μg/ml can be found in the appendix ( Figure A5).
the reducing sugar assay results for the eight polysaccharides that could be hydrolyzed by either of the enzymatic assays compared to AH. Additionally tested polysaccharides that could only be hydrolyzed by acid are listed in Table A1.
The reducing sugar signal of enzymatically hydrolyzed amylopectin and glycogen equals approximately 100 μg/ml glucose, indicating complete hydrolysis to glucose. Amylose is poorly soluble in water, and therefore, only 60 μg/ml glucose equivalents, presumably produced by shorter-chain amylose, could be measured using EH.
Similar to the results in Figure 1 the concentration of glucose hydrolysis products is often higher for the α-glucan enzymatic assay than for the AH with the notable exception of pullulan, which is less effectively hydrolyzed by the enzymatic assay than by AH. Pullulan is comprised of α-1-4-maltotriose units linked by α-1-6-linkages and should-in theory-be completely hydrolyzed by the applied endo-1-4-α-amylase and exo-1,4/1,6-α-amyloglucosidase. However, as the observed reducing sugar signal is only slightly increased by the application of these enzymes, it can be assumed that this α-amylase needs a longer uninterrupted α-1-4-linked glucose chain to be more active. Thus, the α-glucan assay can mainly be used to identify and quantify starch-like α-glucans with 1-4 chains and optional 1-6 branches.
While the laminarin assay is effective on both laminarins tested and the amount of released glucose is similar and slightly higher than for AH, the only other polysaccharide being partially hydrolyzed by this assay is lichenan. As lichenan is comprised of β-1-3 and β-1-4 linked glucose units and some of the applied laminarinases have a β-1-3 activity (Becker et al., 2017), this small side reaction is to be expected. However, the results show that the laminarin assay cannot detect β-glucan from barley and any of the tested α-glucans.
Overall, these results demonstrate that a combination of the enzymatic assays can distinguish the important storage glucans; starch-like α-1,4/1,6-glucans and laminarin-something that cannot be achieved using traditional chemical hydrolysis.

| Microalgal glucans can be extracted using hot water and sonication
The use of enzymatic assays to quantify glucans in marine POM requires that the glucans can be extracted from environmental samples. Particulate microalgal material was used to test different extraction protocols. The extraction efficiency was determined by measuring the ratio of glucose detected by HPAEC-PAD on extracted and nonextracted filter pieces after AH conditions, assuming that these conditions are sufficient for a complete glucan extraction and hydrolysis. For the extraction of glucans from filters, we tested different combinations of water and NaOH incubations, with and without sonication. F I G U R E 2 Enzymatic hydrolysis of α-glucans is specific and more effective than acid hydrolysis: Hydrolysis efficiency of different polysaccharides (100 μg/ml) using acid hydrolysis (AH) compared to enzymatic hydrolysis (EH) using the α-glucan or laminarin assay. All tested polysaccharides are listed in Table A1. Only the results of polysaccharides that could be hydrolyzed by either of the enzymatic assays are depicted. Data points represent individual samples, and error bars denote standard deviation (n = 3, technical replicates). STEINKE ET AL.
| 5 of 14 The extraction was slightly improved when using 1 h sonication in 1 M NaOH followed by 1 h at 99°C. This treatment resulted in residual glucose values of 3%, 20%, and 25% in T. weissflogii, O. tauri, and P. purpureum, respectively ( Figure 3d). However, this additional alkaline extraction did not substantially increase the efficiency of the extraction protocol. Therefore, POM samples were extracted using hot water (1 h, sonication; 1 h, 99°C) in the following sections. The integrity of laminarin and starch was also tested and confirmed by EH of starch and laminarin standards that had been subjected to extraction conditions compared to nonsubjected samples.
These results demonstrate that glucans in O. tauri and P.
purpureum are more resistant to aqueous extraction methods than glucans in T. weissflogii. This result is probably due to different glucans present in these species. While the material of the diatom T.
weissflogii should contain laminarin (Becker et al., 2017), the green microalgae O. tauri and the red microalgae P. purpureum should contain starch and Floridean starch, respectively (Sheath et al., 1979;Sorokina et al., 2011). Laminarin is highly soluble in water, whereas α-glucans vary in their water solubilities; branched α-glucans like amylopectin and glycogen have a high solubility and linear amylose has a low solubility. The differences in extraction efficiency should be taken into account with regard to glucan quantifications as the true content might be higher.

| α-Glucans and laminarin can be quantified in parallel in particulate matter extracts from cultivated microalgae
To investigate the proportion of αand β-glucans in the aforementioned three microalgae, enzymatic α-glucan and laminarin hydrolysis assays have to be used. Three non-axenic cultures of each of the microalgae O. tauri, P. purpureum, and T. weissflogii were maintained at a constant temperature of 15°C, with a 12-h/12-h light/dark cycle, without stirring. Samples from each culture were taken over 20 days and filtered onto glass fiber filters.
Glycans were extracted from these filters using sonication and hot water (as described above) and hydrolyzed by AH or EH. Figure 4 shows the glucose equivalents as determined by the reducing sugar assay over the course of 20 days of algal growth.
Laminarin was detectable in T. weissflogii diatom cultures after 6 days, but could not be detected in cultures of green microalgae O. tauri and red microalgae P. purpureum over the 20 days incubation period. Conversely, α-glucans were detected in O. tauri and P.
purpureum from Day 4 or 3, respectively. Overall, these findings match the polysaccharide profiles of these three microalgae (Becker et al., 2017;Sheath et al., 1979;Sorokina et al., 2011). However, a small amount of α-glucans could also be measured in two of the three T. weissflogii cultures on Days 10 and 20. As the algal cultures were nonaxenic, it is possible that these α-glucans originate from bacteria.
Overall, AH produces a similar total glucose signal as both enzymatic assays combined. Since the PAHBAH assay would be reactive to all reducing sugars released by AH this indicates that α-glucans and laminarin (a β-glucan) are the predominant glycans in these algal species and under the culture conditions and duration used here. Figure 5 shows the glucan quantification on Day 20 in relation to the total POC. It should be noted that POC was quantified directly off glass fiber filters without prior extraction, whereas for glucan quantification the filters were extracted by sonication and hot water.
As this extraction might not be complete, the true glucan (especially α-glucan-) content might be higher than depicted.  April 2020. In both cases, POM in surface water was filtered onto 0.7 μm glass fiber filters. The POC content was quantified directly from nonextracted filter-circle-cutouts, while the glucan content was determined using AH or EH of hot water extracts of quarter filters.
The glucose content in the samples was found to be too low for quantification by the photometric reducing sugar assay. Therefore, HPAEC-PAD was used to determine the glucose content in hydrolyzed and non-hydrolyzed extract samples. With HPAEC-PAD, as little as 0.3 and 0.2 μg/ml glucose could be detected following AH or EH, respectively, of a starch standard sample. In comparison, the quantification limit for the reducing sugar PAHBAH assay was 11 μg/ml.
HPAEC-PAD analysis of enzymatically hydrolyzed α-glucans and laminarin shows that the only product is glucose ( Figure A4).
Therefore glucose can be used as a standard to quantify and compare α-glucans and laminarin. Similar chromatograms of hydrolyzed and nonhydrolyzed extracts from algal cultures and environmental samples are depicted in Figure A6. F I G U R E 4 α-Glucans can be quantified alongside laminarin in microalgal extracts using glucan-specific enzymatic hydrolysis: glucan quantification in POM from Thalassiosira weissflogii, Ostreococcus tauri, and Porphyridium purpureum laboratory cultures expressed as glucose equivalents. Laminarin and α-glucans were quantified in microalgal POM extracts using enzymatic hydrolysis and subsequent PAHBAH reducing sugar assay (EH, blue and green symbols). Total glucan was quantified by acid hydrolysis and subsequent PAHBAH reducing sugar assay (AH, magenta symbols). The amount of glucose equivalents in each sample was calculated using glucose calibration curves. Error bars denote standard deviation (n = 3, biological replicates). AH, acid hydrolysis; POM, particulate organic matter.
F I G U R E 5 α-Glucans account for a substantial amount of the total POC in red and green algae: α-Glucan and laminarin quantification of microalgal POM from Thalassiosira weissflogii, Ostreococcus tauri, and Porphyridium purpureum laboratory cultures after 20 days of growth. Laminarin and α-glucans were quantified in microalgal POM extracts using enzymatic hydrolysis and subsequent PAHBAH reducing sugar assay (EH, blue and green). Total glucan was quantified by acid hydrolysis and subsequent PAHBAH reducing sugar assay (AH, magenta). The amount of glucose equivalents in each sample was calculated using glucose calibration curves. α-Glucan quantities are shown as glucose equivalent carbon relative to the total POC. Error bars denote standard deviation (n = 3, biological replicates). AH, acid hydrolysis; EH, enzymatic hydrolysis; POC, particulate organic carbon. and 1-4 μg/L, North Sea). The combined glucose value for laminarin and α-glucan, determined with EH, is similar to the total glucose value determined with AH. This observation is consistent with the results shown above (Figures 4 and 5). This result further indicates that most glucose-containing polysaccharides are detected by one of the two enzymatic assays. Laminarin content in POM appears to correlate significantly with increasing total POC (R 2 = 77.5%, p < 0.001)-in accordance with previous studies (Becker et al., 2020)-, whereas αglucan content is lower but apparently noncorrelating with total POC (R 2 = 9.4%, p > 0.360). It should be noted that these α-glucans may originate not only from starch-producing red and green algae but also from glycogen-producing bacteria.

| DISCUSSION
A method for the detection and quantification of starch-like α-glucans in the ocean has been developed and applied to microalgal cultures and environmental marine POM. Using this method enabled the parallel quantification of α-glucan and laminarin (Becker et al., 2017) in marine POM for the first time. While there are other methods to analyze the structure of glycans based on derivatization (Price, 2007), partial chemical lysis combined with mass spectrometry (Amicucci et al., 2020), or spectroscopy (Battistel et al., 2014), their glycan-specific quantification in environmental samples at low concentrations is difficult (Lang et al., 2014).
This method is highly specific and robust. However, the assay cannot distinguish between the different types of α-1,4/1,6-glucans and is therefore not suitable for determining the origin of the αglucans detected. To further investigate this question, the degree of α-1,6-branching could also be determined by adding an endo-acting α-1,6-isoamylase to the assay ( Figure A2). Knowing the average degree of α-1,6-branching in POM glucans does not unequivocally identify the type of α-glucans detected, but it might allow for an estimate if the main α-glucan is the highly branched glycogen.
Another approach to identify α-glucan types could be the application of separation techniques based on different solubilities. This might allow for determining the origin of α-glucans as starch from red (Yu et al., 2002) and green algae (Busi et al., 2014) or as bacterial storage glycogen (Ball & Morell, 2003).
Hot water extraction from POM is probably not complete (El Halal & Kringel, 2019) for all types of α-glucans, and thus the true α-glucan content of the environmental POM samples may be higher than the one detected. However, the extraction approach used in our method allows the parallel detection of other polysaccharides, such as laminarin (Becker et al., 2020), by enzymatic assays.
This study has highlighted problems in quantifying glucans after chemical lysis, as AH consistently produced fewer glucose products from polysaccharide standards than enzymatic hydrolysis. It is known that different hydrolytic conditions result in different hydrolysis yields for distinct polymers within a sample (Zhu et al., 2020). The hydrolysis efficiency depends on the hydrolysis method as well as the carbohydrate types and even after harsh hydrolysis conditions some polysaccharides do not hydrolyze into monosaccharides (Panagiotopoulos & Sempéré, 2005;Zhu et al., 2020). Despite this problem, the information about the monosaccharide composition provided by non-specific glycan acid hydrolysis is still very valuable for complex samples (Engel & Händel, 2011).
α-Glucan concentrations in marine surface water POM in our samples were found to be lower than laminarin but at constant levels, independent of increased POC concentrations during the 2020 North Sea seasonal algal bloom. To determine the relevance of α-glucans in the marine carbon cycle more glycan-specific data need to be collected. As bacterial storage glycogen, α-glucans could be an important indicator of bacterial carbon turnover (Kappelmann et al., 2019). In addition, higher α-glucan concentrations are expected near coasts and estuaries due to the input of terrestrial plants and locations with distinct green and red algal growth. However, it is likely that in marine glycan samples, α-glucan concentrations are usually lower than laminarin concentrations (Becker et al., 2020). This low concentration of specific types of glycans in the marine carbon pool is a major obstacle to the analysis of marine glycans by EH and requires the use of appropriate detection methods with low detection limits, such as HPAEC-PAD (Lo-Guidice & Lhermitte, 1996), mass spectrometry (Hofmann & Pagel, 2017), or fluorometric methods (Mutuyimana et al., 2018;Tran et al., 2021) for glucose quantification.
The lack of knowledge about the specific carbohydrate structures that are present on the sea surface and the ones that are exported to the deep sea remains a serious hindrance to our understanding of the role of glycans in the marine carbon cycle.
We need to analyze marine samples with methods that allow high F I G U R E 6 α-Glucans can be quantified alongside laminarin in marine POM samples using glucan-specific enzymatic hydrolysis: POM samples on 0.7 μm glass fiber filters were extracted and hydrolyzed using acid hydrolysis (AH) (magenta symbols) or enzymatic hydrolysis (EH) (blue and green symbols). Square symbols represent samples from the western North Atlantic Ocean (May 2019). Triangle symbols represent samples from the North Sea (April 2020). Error bars denote standard deviation (n = 3, technical replicates). POM, particulate organic matter.
structural resolution. The method described in this study allows the specific identification of α-glucans. We envisage that this method will be one of many glycan quantification methods based on EH for environmental samples.

CONFLICTS OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
All data are provided in full in this paper.

ETHICS STATEMENT
None required. F I G U R E A1 PAHBAH signal of glucose: Different concentrations of glucose either directly dissolved in water or subjected to AH conditions. AH, acid hydrolysis.
F I G U R E A3 Optimal conditions for enzymatic α-glucan hydrolysis: enzymatic activity rates were measured using the PAHBAH reducing sugar assay with 100 μg/ml μ 100 starch. The greatest activity observed was set as the 100% reference value for each parameter tested. F I G U R E A5 Figure 1, zoomed-in on lower starch concentrations: enzymatic hydrolysis (EH) products of starch, acid hydrolysis (AH) products of starch, nondigested starch, and glucose as calibration standard were measured in α-glucan assay buffer (100 mM sodium acetate buffer, pH 4.5) using the PAHBAH reducing sugar assay. A 410nm signals above 1.9 were measured in diluted samples. Error bars denote standard deviation (n = 3, technical replicates). Solid lines represent regression lines.
F I G U R E A6 Enzymatic hydrolysis assays for aqueous extracts produce glucose: HPAEC-PAD chromatograms (PA100 column) of extracts from (top) environmental POM samples, (bottom) algal cultures. Chromatograms of hydrolyzed samples were moved by 2, 4, and 6 to improve clarity. AH, acid hydrolysis; EH, enzymatic hydrolysis; POM, particulate organic matter.