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Soil Biology & Biochemistry 39 (2007) 3081–3092

www.elsevier.com/locate/soilbio

Free amino sugar reactions in soil in relation tosoil carbon and nitrogen cycling

Paula Robertsa, Roland Bolb, Davey L. Jonesa,�

aSchool of the Environment and Natural Resources, University of Wales, Bangor, Gwynedd LL57 2UW, UKbCross Institute Programme for Sustainable Soil Function, Institute of Grassland and Environmental Research (IGER),

North Wyke Research Station, Okehampton, Devon EX20 2SB, UK

Received 30 January 2007; received in revised form 3 July 2007; accepted 5 July 2007

Available online 7 August 2007

Abstract

Amino sugars represent a major constituent of microbial cell walls and hydrolyzed soil organic matter. Despite their potential

importance in soil nitrogen cycling, comparatively little is known about their dynamics in soil. The aim of this study was therefore to

quantify the behaviour of glucosamine in two contrasting grassland soil profiles. Our results show that both free amino sugars and amino

acids represented only a small proportion of dissolved organic N and C pool in soil. Based upon our findings we hypothesize that the low

concentrations of free amino sugars found in soils is due to rapid removal from the soil solution rather than slow rates of production.

Further, we showed that glucosamine removal from solution was a predominantly biotic process and that its half-life in soil solution

ranged from 1 to 3 h. The rates of turnover were similar to those of glucose at low substrate concentrations, however, at higher

glucosamine concentrations its microbial use was much less than for glucose. We hypothesized that this was due to the lack of expression

of a low affinity transport systems in the microbial community. Glucosamine was only weakly sorbed to the soil’s solid phase

(Kd ¼ 6.471.0) and our results suggest that this did not limit its bioavailability in soil. Here we showed that glucosamine addition to soil

resulted in rapid N mineralization and subsequent NO3� production. In contrast to some previous reports, our results suggest that free

amino sugars turn over rapidly in soil and provide a suitable substrate for both microbial respiration and new biomass formation.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Amino acid; Biodegradation; Carbon cycling; Dissolved organic nitrogen; Hexosamine; Mineralization; Nitrogen cycling

1. Introduction

Most of the nitrogen (N) contained in agricultural soilsexists in an organic form as solid organic matter (SOM;Stevenson, 1982). Its breakdown plays a critical role in soilfertility and the supply of nutrients to both plants and soilmicroorganisms. Acid hydrolysis of soil organic matter hasrevealed that approximately 5–10% of soil N is present asamino sugars while for comparison 40–60% may bepresent as amino acids (Stevenson, 1982; Zhang andAmelung, 1996; Mengel, 1996). While the biotic andabiotic reactions of free amino acids in soil have been wellcharacterised (Jones et al., 2005), the short-term fate of

e front matter r 2007 Elsevier Ltd. All rights reserved.

ilbio.2007.07.001

ing author. Tel.: +441248 382579; fax: +44 1248 354997.

ess: d.jones@bangor.ac.uk (D.L. Jones).

monomeric, free amino sugars in soil remains poorlyunderstood.As plants do not synthesize significant amounts of amino

sugars (e.g. glucosamine, galactosamine), the majority ofamino sugars in soil are assumed to originate from theturnover of the soil microbial community (Dai et al., 2002;Appuhn and Joergensen, 2006). In a polymeric form (e.g.cross-linked N-acetyl-b-D-glucosamine and b-D-glucosa-mine), amino sugars represent major component of thecell walls of both fungi (e.g. chitin, chitosan) and bacteria(e.g. muramic acid; Atlas and Bartha, 1993; Appuhn andJoergensen, 2006). Small quantities of amino sugars mayalso be present in soil within the exoskeletons of soilinvertebrates, earthworm gut linings, nematode eggs,mollusc polysaccharides and snail mucilage (Agudeloet al., 2004; Marxen et al., 1998). Unlike other commonlyfound low molecular weight compounds in soil (e.g. amino

ARTICLE IN PRESSP. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–30923082

acids and organic acids), the exudative loss of amino sugarsfrom living organisms is believed to be extremely small andtherefore most amino sugars can be expected to enter soilupon organismal death (Glaser and Gross, 2005).

Amino sugars have been hypothesized to play twoprimary roles in soil. Firstly, as 1–10% of the dry weight ofgram positive bacteria and 5–10% of fungi may becomposed of amino sugar polymers (Knorr, 1991; Blacket al., 1994; Schnabel and White, 2001; Appuhn andJoergensen, 2006), they may represent a significant sourceof N for both plants and microorganisms. Secondly, theymay also contribute significantly to the polysaccharidemediated binding of soil particles leading to the formationof stable aggregates (Stevenson, 1982). Amino acidpolymers may also have a role in metal binding reactionsin some soils (Guibal, 2004). Extracellular enzymes arethought to be primarily responsible for breaking downamino sugar polymers in soil (e.g. chitosanases, EC3.2.1.132; chitin deacetylase, EC 3.5.1.41; chitinases, EC3.2.1.14; b-N-acetylglucosaminidases, EC 3.2.1.52; Saitoet al., 1999; Parham and Deng, 2000; Shrestha et al., 2004;Mayer et al., 2006). Upon release of the amino sugarmonomers into the soil solution they can undergo anumber of possible fates in soil including: (1) uptake bythe soil microbial biomass, (2) uptake by plant roots,(3) abiotic mineralization, (4) sorption to the soil’s solidphase, (5) formation of stable humic polymers, and(5) leaching down the soil profile (Bondietti et al., 1972;Zhang et al., 1998, 1999; Amelung et al., 2002; Schnier,1987). Quantitative information of the relative importanceof these individual amino sugar reactions in either rhizo-sphere or bulk soil, however, is lacking.

Dissolved organic nitrogen (DON) has been found to bea major N loss pathway in many terrestrial ecosystems(Christou et al., 2005; Lipson and Nasholm, 2001). Theexact chemical nature of this DON, however, remainslargely undefined and this is severely hampering ourunderstanding of the functional significance of DON interrestrial ecosystems (Jones et al., 2005). Evidence suggeststhat in comparison to sugars (e.g. glucose), amino sugarpolymers and monomers are relatively recalcitrant in somefreshwater and marine environments (Nagata et al., 2003;Kawasaki and Benner, 2006). If amino sugars are similarlyrecalcitrant in soil, their loss to surface and ground watercould represent a major DON loss pathway. Recentevidence has also suggested that plant uptake of DONmay be a major regulator of ecosystem productivityparticularly in N limiting environments. Whether plantscan take up amino sugars from soil remains unknown,however, knowledge about their reactions in soil will aidour understanding of plant–microbial competition for Nresources.

It is clear form the above discussion that amino sugarsrepresent a significant N pool in soil. If we are to designsustainable agricultural systems and reduce N pollution itis imperative that we have a good mechanistic under-standing of the factors regulating the dynamics of these

major forms of organic N in soil. Consequently, the initialobjective of this study was to determine the quantitativesignificance of free amino sugars to the soil DON pool. Thesecond objective was to quantify the sorption and C and Nmineralization reactions of one major amino sugar in soil(glucosamine) and to compare these results with those ofother common low molecular weight substrates found insoil (e.g. glucose and amino acids).

2. Materials and methods

2.1. Soil samples

Two sheep grazed agricultural grassland soils locatedalong a catena sequence in Abergwyngregyn, Gwynedd,UK (531140N41010W) were used in the study. Soil 1 (Eutriccambisol) is a lowland (15m altitude), freely draining,heavily sheep-grazed grassland which receives regularfertilization (120 kgN, 60 kgK and 10 kgP yr�1) andsupports a sward consisting predominantly of perennialryegrass (Lolium perenne L.), clover (Trifolium repens L.)and crested dog’s tail (Cynosurus cristatus L.). Soil 2(Dystric gleysol) is poorly drained, sheep grazed grasslandthat received no fertilization and supports a sward ofperennial ryegrass and clover. These two soils represent thedominant soil types in animal production systems in theWestern UK. For both soils, the Ah horizon was sampledin triplicate from a depth of 0–10 cm. Additionally, subsoilswere sampled in triplicate from the Bw horizon in theEutric cambisol (20–30 cm) and from the Bg horizon inthe Dystric gleysol (30–40 cm). The main characteristics ofthe soil are presented in Tables 1 and 2.

2.2. Chemical analyses

pH and electrical conductivity (1:1 w/w soil-to-distilledwater) were determined with standard electrodes. Moisturecontent was determined by drying at 105 1C for 24 h. TotalC and N was determined using a CHN2000 analyzer (LecoCorp, St. Joseph, MI). Exchangeable cations were ex-tracted using 0.5MBaCl2 at a 1:10 w/v ratio of soil-to-0.5M BaCl2. The samples were extracted by shaking for 1 hat 250 revmin�1, centrifuged for 10min at 13000g and thesupernatant recovered for analysis and frozen untilanalysed using an Ultrace ICP-OES (Horiba Jobin YvonCorp, Edison, NJ). Dissolved organic C (DOC) and totaldissolved N (TDN) were determined using a ShimadzuTOCV-TN analyzer (Shimadzu Corp., Kyoto, Japan).NO3� and NH4

+ were determined with a Skalar segmentedflow autoanalyser (Skalar Inc., Norcorss, GA) andmethods described in Downes (1978) and Mulvaney(1996). DON was calculated as the difference betweenTDN and the sum of (NH4

++NO3�). Free amino sugars

were analysed by an on-column, fluorescent derivatization,reverse phase HPLC procedure according to Jones et al.(2005). Briefly, the amino sugars were derivitized witho-phthaldialdehyde and 2-mercaptoethanol and the samples

ARTICLE IN PRESS

Table 1

Characteristics of the topsoil and subsoil of two contrasting agricultural grassland soils (Eutric cambisol and Dystric gleysol)

pH Exchangeable cations Total C

(g kg�1)

Total N

(g kg�1)

C-to-N

ratio

Moisture

content (g

kg�1)

EC

(mS cm�1)Soil respiration

(mgCO2 kg�1 h�1)

Na

(mgkg�1)

K

(mgkg�1)

Ca

(mgkg�1)

Mg

(mgkg�1)

Al

(mgkg�1)

Eutric cambisol

0–10 cm 6.170.1 3476 11576 883768 84719 1272 3.0170.16 0.2370.01 13.070.2 516769 6876 4.570.6

30–40 cm 6.370.2 1572 6674 679718 8076 1172 1.4670.05 0.1170.01 13.070.2 237732 4773 2.170.2

Dystric gleysol

0–10 cm 5.870.1 5379 138721 9287172 215735 1472 11.271.23 0.9670.11 11.870.2 12087195 5574 11.172.2

20–30 cm 5.770.1 3272 7075 409747 2471 1973 3.7770.37 0.3570.03 10.870.2 530733 4173 2.670.2

P value

(topsoil)

NS NS NS NS * NS *** *** ** NS NS *

P value

(subsoil)

* ** ** NS *** NS ** *** ** * NS NS

Values represent means7SEM (n ¼ 3). WHC indicates water holding capacity and EC indicates electrical conductivity. Significant differences between the

two soil types are indicated by *, ** and *** which represent differences at the Po0.05, Po0.01 and Po0.001 level respectively and where NS indicates

P40.05.

Table 2

Soil solution concentrations of N and C from the topsoil and subsoil of two agricultural grassland soils (Eutric cambisol and Dystric gleysol)

NO3�

(mgN l�1)

NH4+

(mgN l�1)

DIN

(mgN l�1)

DON

(mgN l�1)

Free amino acids

(mgN l�1)

Free amino sugars

(mgN l�1)

DOC

(mgC l�1)

DOC:DON

ratio

Eutric cambisol

0–10 cm 0.9470.13 4.0471.00 5.070.7 2.9670.70 0.7870.02 o0.01 59.074.3 21.374.1

30–40 cm 0.9470.54 2.9470.53 3.970.8 2.5670.55 0.7470.02 ND 57.475.2 23.172.1

Dystric gleysol

0–10 cm 0.1270.03 2.2270.61 2.370.5 0.8970.27 0.8670.04 o0.01 22.771.4 28.676.5

20–30 cm 0.5370.27 1.5370.04 2.170.2 0.5770.13 0.9270.03 ND 14.970.8 27.573.6

P value

(topsoil)

* NS * * NS *** NS

P value

(subsoil)

NS * NS * NS *** NS

Values represent means7SEM (n ¼ 3). DIN indicated total dissolved inorganic N, DON indicates dissolved organic N, DOC indicates dissolved organic

C and ND indicates not determined. Significant differences between the two soil types are indicated by *, ** and *** which represent differences at the

Po0.05, Po0.01 and Po0.001 level respectively and where NS indicates P40.05.

P. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–3092 3083

analysed with a Varian Prostar HPLC (Varian Inc, PaloAlto, CA) equipped with a Varian Prostar 310 fluorescencedetector (Ex. 340 nm, Em. 445 nm) and a Varian StarChromatographic Integrator. The HPLC column was anODS stationary phase (4mm i.d.� 250mm; 5 mm particlesize; Phenomenex) running with a 0.05M sodium acetate,methanol and tetrahydrofuran mobile phase. Free aminoacid concentrations were determined using an EZ:faastanalysis kit (Phenomenex Corp., Torrance, CA) and aVarian CP3380 gas chromatograph with flame ionizationdetector and a 10m� 0.25mm Zebron ZB-AAA column(Varian Inc, Palo Alto, CA). Purification of extracts,derivatization and analysis of the amino compounds wereperformed according to the manufacturer instructions.Measurements of soil respiration at quasi steady state weremade on 40 g of soil at 20 1C using an automatedmultichannel SR1-IRGA soil respirometer (PP Systems

Inc., Hitchin, UK) immediately after soil collection fromthe field.

2.3. Amino sugar mineralization to CO2

Soil (10 g) was weighed into a polypropylene tube(50 cm3) and 0.5ml of a 14C-labeled glucosamine (D-Glucosamine-1-14C-hydrochloride; 370MBqmmol�1; Sig-ma Chem Co., St. Louis, MO) or 14C-labeled glucose(9990MBqmmol�1; Sigma Chem Co.) solution was addedto each soil type at concentrations ranging from 1 mM to10mM (90 kBq kg�1 soil). To trap any 14CO2 evolved fromthe soil, a polypropylene vial containing 1ml of 1M NaOHwas placed above the soil and the sample tubes hermeti-cally sealed with a rubber stopper and maintained at 20 1C.The incubation temperature reflects that experienced in thesoil during the summer months when plant growth is

ARTICLE IN PRESSP. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–30923084

optimal. To quantify respired 14CO2, the traps wereremoved after 1, 3, 6, 24, 48 or 168 h after 14C labeladdition. After removal, the amount of 14CO2 trapped inthe NaOH was determined by liquid scintillation countingusing OptiPhase Hi-Safe3 scintillation cocktail (PerkinEl-mer Life and Analytical Sciences Inc., Wellesley, MA) anda Wallac-1409 scintillation counter (PerkinElmer Life andAnalytical Sciences Inc.). After incubating for 7 d, the soilwas shaken with 12.5ml 0.5M K2SO4 for 10min at200 revmin�1 to recover any 14C label remaining insolution or on the exchangeable phase (Kuzyakov andJones, 2006). The extracts were then centrifuged (13000g,5min) and the amount of 14C in the extract determined byliquid scintillation counting as described above. Substratehalf lives were estimated using double first-order exponen-tial decay equation (Boddy et al., 2007), fitted by a leastsquares optimization routine to the mineralization datausing Sigmaplot 8.0 (Systat Software UK Ltd., London,UK).

2.4. Microbial inhibitors

To determine whether free amino sugar breakdown ispredominantly a biological process, glucosamine miner-alization in the Ah horizons was investigated in thepresence of microbial toxins of after soil sterilization.Incubations were carried out with 14C-labeled glucosamine(10mM) as described above (92.5 kBq kg�1 soil) and 14CO2

evolution measured over 3 h. The individual treatmentsincluded: (1) untreated soil (control); (2) autoclaved soil(1 h, 121 1C); (3) fumigated soil (CHCl3, 24 h; Joergensenand Mueller, 1996); (4) 100mM HgCl2; (5) 100mM NaN3.

The heat and CHCl3 sterilization treatments were appliedimmediately before the glucosamine addition. In the case ofHgCl2 and NaN3, the cytotoxins were added to the soilalongside the glucosamine.

2.5. Amino sugar mineralization to mineral N

Soil (10 g) was placed into a polypropylene tube asdescribed above and glucosamine or glucose added to thesoil at rates of 0, 0.05 0.5, 5, 50, and 500 mmol kg�1. Thesoils were then incubated at 20 1C for 7 d. After this time,the soil was extracted with 0.5M K2SO4 and the amount ofNH4

+ and NO3� in the extract solution undertaken as

described above.

2.6. Amino sugar sorption

Amino sugar sorption to the soil’s solid phase wasdetermined using CHCl3—fumigated soil (Joergensen andMueller, 1996) to prevent microbial mineralization of theamino sugar during the sorption assay (Kuzyakov andJones, 2006). We assume that CHCl3—fumigation does notsignificantly change the sorption properties of hydrophiliclow molecular weight compounds to the soil’s exchangephase (Kuzyakov and Jones, 2006). For comparison, we

also studied the sorption of the amino acid, lysine, which isknown to adsorb strongly to soil (Vinolas et al., 2001).Briefly, different concentrations (0–200 mM) of 14C-labeledglucosamine or L-lysine (8880MBqmmol�1; Sigma Chem.Co.) were prepared in a background solution of 0.01MCaCl2. Briefly, the

14C-labeled solutions (5ml) containingeither lysine or glucosamine were added to 1 g of fumigatedsoil from the Ah horizons contained in a 15ml centrifugetube (92.5 kBq kg�1 soil). The soil and sorption solutionswere then shaken together for 15min at 200 revmin�1.Immediately after shaking, the soil suspension wascentrifuged at 13000g for 5min and the supernatantrecovered for 14C determination as described above. AFreundlich sorption isotherm was then fitted to theexperimental data where

S ¼ a� ESCb, (1)

and where ESC is the equilibrium solution concentration atthe end of the experiment (mmol l�1), S is the amount ofsolid phase sorption (mmol kg�1) and a and b areempirically derived parameters. Partition coefficients (Kd)were calculated as follows:

Kd ¼ S=ESC: (2)

2.7. Statistical analysis

Statistical analysis, (t-test, Anova with tukey pairwisecomparison) were performed using SPSS 14 (SPSS Inc.,Chicago, IL) and Minitab 14 (Minitab Inc., State College,PA) with significance defined at the Po0.05 unlessotherwise stated.

3. Results

3.1. Soil characteristics

The two grassland soil used in the experiments differedsignificantly in most of their measured general soilproperties reflecting the different soil forming factorsoperating at each site (Tables 1 and 2). The soluble Npool in both soils was dominated by inorganic N andparticularly NH4

+ in both soils. DON represented only4172% of the total dissolved N (TDN) in the Eutriccambisol and 2473% of the TDN in the Dystric gleysol. Inthe top soils, free amino sugars represented o1% of thetotal DON whilst total free amino acids represented2477% of the DON in the Eutric cambisol and 2378%of the DON in the Dystric gleysol. Free amino sugarsrepresented o0.2% of the DOC in both soils whilst totalfree amino acids represented 771% of the DOC in theEutric cambisol and 571% of the DOC in the Dystricgleysol. The concentration of individual amino sugars insoil solution (e.g. glucosamine, mannosamine, galactosa-mine) was extremely low and below the HPLC detec-tion limits (o150 nM). Similarly, the concentration of

ARTICLE IN PRESSP. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–3092 3085

individual free amino acids was also extremely low rangingfrom o1 to 258 nM (data not presented).

3.2. Mineralization

The substrate-dependent mineralization of 14C-labeledglucosamine and glucose to 14CO2 is shown in Figs. 1and 2. At substrate concentrations p100 mM for glucosa-mine and p10mM for glucose, both substrates exhibitedan initial rapid linear phase of mineralization (0–6 h; linearregression r2 ¼ 0.68–0.99) followed by a secondary slowerphase of mineralization. In contrast, at high glucosamineconcentrations (1–10mM), and particularly in the subsoils,this initial linear pattern was not apparent. In some cases, ashort lag phase in glucosamine mineralization was ob-served (0–48 h) after which the rate of mineralizationincreased. The total amount of glucosamine mineralized toCO2 in the Eutric cambisol (23–45% of the total) wassignificantly greater than that in the Dystric gleysol(11–22% of the total; Po0.05). In contrast to glucose,there were significant differences in the total amount andrate of the 14C-glucosamine mineralization for the differentsubstrate addition rates. Overall, depth appeared to be lessimportant than either the substrate type or addition rate inregulating the rate of mineralization. Only small amountsof 14C could be recovered in a K2SO4 extract at the end ofthe experimental period. Typically this represented o0.5%of the total 14C initially added for the glucose treatmentsand o2% for the glucosamine treatments suggesting thatonly very small amounts of the substrates remained in soilsolution or on the exchangeable phase after 7 d.

a

N

14C

O2 e

vo

lutio

n (

% o

f to

tal

14C

-sub

str

ate

applie

d)

0

5

10

15

20

25

0 24 48 72 96 120 144 1680

5

10

15

20

Time after subs

b}a

Fig. 1. Time dependent mineralization of different concentrations of 14C-labe

topsoil (0–10 cm; A, B) and subsoil (20–30 cm; C, D) of a Dystric gleysol grass

statistically significant differences between the treatments at the Po0.05 level.

3.3. Microbial biomass yield

The amount of glucose-C and glucosamine-C immobi-lized in the microbial biomass after the addition of a rangeof substrate concentrations is shown in Fig. 3. Generally,the majority of the substrate C was immobilized in themicrobial biomass with only a relatively small amountmineralized to CO2. The immobilization-to-mineralizationratio (microbial biomass yield) was greater for glucose(mean7SEM ¼ 0.8370.01) than for glucosamine acrossall the soils and substrate concentrations (mean7SEM ¼0.7670.02; Po0.001). Overall, in the glucose treatmentsthe microbial biomass yield was relatively insensitive tosubstrate concentration. In contrast, the microbial biomassyield for glucosamine significantly declined with increasingsubstrate addition particularly in the Eutric cambisol(Po0.05).

3.4. Substrate half-life

As expected from the 14C-labeled glucosamine andglucose mineralization profiles, the half-life of the sub-strates in both soils was concentration dependent (Fig. 4).At low substrate concentrations (1–10 mM) there was asmall but significant difference in the half-life of glucoseand glucosamine (Po0.01). Within this low concentrationrange and across all the soils and depths the mean half-lifewas 1.170.2 h for glucose and 3.070.7 h for glucosamine.At substrate concentrations X100 mM, the half-life ofglucosamine became progressively greater with increasingconcentration in comparison to that of glucose (Po0.05).

S

b

1 μM

10 μM

100 μM

1000 μM

10000 μM

trate addition (hours)

0 24 48 72 96 120 144 168

a

}b

b

}

}a

ab

ab

lled glucose (Panels A and C) and glucosamine (Panels B and D) in the

land soil. Values represent means7SEM (n ¼ 3). Different letters indicate

ARTICLE IN PRESS

NS

} a

b

}

c

b

}

b

14C

O2 e

volu

tion (

% o

f to

tal

14C

-substr

ate

applie

d)

0

10

20

30

40

50

60

1 μM

10 μM

100 μM

1000 μM

10000 μM

Time after substrate addition (hours)

0 24 48 72 96 120 144 1680

10

20

30

40

50

0 24 48 72 96 120 144 168

b

a

a

Fig. 2. Time dependent mineralization of different concentrations of 14C-labelled glucose (Panels A and C) and glucosamine (Panels B and D) in the

topsoil (0–10 cm; A, B) and subsoil (20–30 cm; C, D) of a Eutric cambisol grassland soil. Values represent means7SEM (n ¼ 3). Different letters indicate

statistically significant differences between the treatments at the Po0.05 level.

P. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–30923086

At the highest substrate concentration (10mM), the half-life of glucosamine in soil was 32710 fold greater than forglucose in the Eutric cambisol and 2177 fold greater thanfor glucose in the Dystric gleysol.

3.5. Net N mineralization

A significant change in soil inorganic N status wasseen when glucosamine was added to soil at rates of500mmol kg�1 (equivalent to 7mgNkg�1) whilst nosignificant changes in soil N were observed at substrateaddition rates p50mmol kg�1 (data not presented). In bothsoils, the addition of glucosamine at rates of 500mmol kg�1

caused an increase in both NH4+ and NO3

� although themagnitude of the net N mineralization response was bothsoil type and soil depth dependent (Fig. 5). The average rateof net N mineralization from the added glucosamine overthe 7 d incubation period was 0.1770.03mgNkg�1 d�1

indicating that the majority of the glucosamine-N remainedimmobilized in the biomass (8373% of the total added N).In contrast to glucosamine, the addition of glucose causedan immobilization of both NO3

� and NH4+ in three out of

four of the soils (Fig. 5). The average rate of net Nimmobilization for glucose was 0.1170.06mgNkg�1 d�1

across all the depths in the two soils.

3.6. Microbial cytotoxins

Cytotoxins were added to the soil to test whetherglucosamine loss from soil was a biotic or abiotic process.

All the microbial cytotoxins investigated here significantlyinhibited both the uptake and mineralization of glucosaminefrom both soils (Po0.05; Fig. 6). Generally, the two pre-treatments designed to completely eliminate the microbialpopulation (fumigation and autoclaving) caused the greatestinhibition with the glucosamine mineralization and uptakerates being o2% of that observed in the untreated (control)soils. The chemical toxins (HgCl2 and azide) added at thesame time as the glucosamine significantly reduced glucosa-mine uptake by 6075% and mineralization by 9073% incomparison to the control soils. Generally, there were nosignificant differences between the responses of the two soilsto the microbial toxins (P40.05).

3.7. Glucosamine sorption

At the solution pHs found in our soils (pH 5.7–6.3) wepredict that the majority of the glucosamine in solution willcarry a positive charge as a result of ionization of the aminogroup (pKa of glucosamine ¼ 7.75; Ng et al., 2006). Similarly,lysine will also carry a net positive charge of 1 at the pHs usedhere (Jones et al., 1994). The concentration of free lysine in thesoil solution ranged from o1 to 20nM (data not presented).The sorption of glucosamine to the solid phase was

concentration dependent and followed a similar pattern inboth soils (Fig. 7). Neither lysine nor glucosamine showedsaturating sorption tendencies over the concentration rangeinvestigated here. As the concentration of free lysine andglucosamine in soil were extremely low (Table 2) we did notcorrect the sorption isotherms to account for the lysine and

ARTICLE IN PRESS

NS NS NS ∗∗ ∗∗∗ NS NS NS ∗∗∗

∗∗

NSNS NS

∗∗ ∗∗

NSNS NS NS

∗

Substrate concentration (μM)

1 10 100 1000 10000

Mic

robia

l bio

mass y

ield

(μm

ol

bio

mass C

μm

ol substr

ate

C-1

)

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Glucose

Glucosamine

Substrate concentration (μM)

1 10 100 1000 10000

Substrate concentration (μM)

1 10 100 1000 10000

Mic

rob

ial b

iom

ass y

ield

(µ

mo

l

bio

mass C

µm

ol substr

ate

C-1

)

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Substrate concentration (μM)

1 10 100 1000 10000

Glucose

Glucosamine

Glucose

Glucosamine

Glucose

Glucsamine

Fig. 3. Microbial biomass C yield as a function of substrate concentration after the addition of either 14C-labeled glucose or glucosamine to the topsoil

(Panels A and C) and subsoil (Panels B and D) of two grassland soils (Eutric cambisol (A and B) and dystric gleysol (C and D)). Values represent

means7SEM (n ¼ 3). Significant differences between the glucose and glucosamine treatments for each concentration are indicated where *, ** and ***

indicate differences at the Po0.05, Po0.01 and Po0.001 level, respectively, and where NS indicates P40.05.

Substrate concentration (μM)

1 10 100 1000 10000

Substr

ate

half-life (

hours

)

0

5

10

15

20

25

30

35

0-10 cm

30-40 cm

Substrate concentration (μM)

1 10 100 1000 10000

Substr

ate

half-life (

hours

)

0

50

100

150

200

250

300

350

0-10 cm

30-40 cm

Substrate concentration (μM)

1 10 100 1000 10000

0-10 cm

20-30 cm

Substrate concentration (μM)

1 10 100 1000 10000

0-10 cm

20 30 cm

Fig. 4. Substrate half-life for a range of glucose (Panels A and B) or glucosamine (Panels C and D) concentrations after addition to the topsoil (A and C)

and subsoil (B and D) of two grassland soils (Eutric cambisol and dystric gleysol). Values represent means7SEM (n ¼ 3).

P. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–3092 3087

ARTICLE IN PRESS

Dystric gleysol

Glucosamine

0-10 cm 30-40 cm

Glucose

0-10 cm 30-40 cm

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

NO3-

-0.4

-0.2

0.0

0.2

0.4

Net N

min

era

lization (

mg k

g-1

d-1

)

Eutric cambisol

Net

N m

inera

lization (

mg k

g-1

d-1

) NH4+

Glucosamine

0-10 cm 30-40 cm

Glucose

0-10 cm 30-40 cm

NO3-

NH4+

Fig. 5. The net mineralization/immobilization of N after the addition of either glucose or glucosamine to the topsoil and subsoil of two grassland soils

(Eutric cambisol and dystric gleysol). The substrate addition rate to the soil was 0.5mmol kg�1 and the incubation period was 7 d.

P. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–30923088

glucosamine already present in the soil. The Freundlichsorption isotherm equation fitted well to the experimentalresults for both glucosamine (r2 ¼ 0.99270.005) and lysine(r2 ¼ 0.99970.001). The mean soil values for the twodimensionless Freundlich parameters a and b were0.7170.01 and 1.0170.02 for glucosamine respectively and4.9270.61 and 0.9370.01 for lysine respectively. The solidphase partition coefficients (Kd) showed some concentrationdependency but were always significantly greater for lysinethan glucosamine (Po0.001). Across both soils the mean Kd

for lysine was 6.471.0 while for glucosamine it was0.7270.03. Glucose showed no sorption in these soils (i.e.Kd ¼ 0; Kuzyakov and Jones, 2006).

4. Discussion

4.1. Factors regulating soil solution amino sugar

concentrations

The results presented here suggest that concentrations ofindividual free amino sugars and amino acids in grassland

soil solutions are very low and only represent a smallproportion of the total DON and DOC. This finding is inagreement with the general findings of Michalzik andMatzner (1999) for total amino sugars (free plus combined)in soil solutions extracted from coniferous forest soils(0–0.13mgN l�1). Our findings suggest therefore that eitherthe rate of free amino sugar production is very low or thattheir biotic or abiotic removal from solution occursrapidly. Previous work on these soils suggests that theycontain a diverse microbial community including substan-tial amounts of fungi (Bardgett et al., 2001) and that themicrobial community turns over relatively rapidly (Boddyet al., 2007). Taking a conservative estimate for totalcombined (i.e. polymeric) amino sugar concentrations intemperate grassland soils of 5–50mgNkg�1 (Stevenson,1982), a microbial biomass combined amino sugar contentof 1–6mgNkg�1 (assuming 10% of total microbial-N isamino sugar-N; Appuhn and Joergensen, 2006), and amicrobial turnover time of 30 d (Boddy et al., 2007), weestimate that the rate of free amino sugar flux through thesoil solution at steady state is probably in the region of

ARTICLE IN PRESS

Control HgCl2 Na-azide

14C

O2 e

volu

tion

(% o

f to

tal

14C

-glu

cosam

ine a

dded)

0

2

4

6

8

10

12

14Eutric cambisol

Dystric gleysol

Control Autoclaved Fumigated HgCl2 Na-azide

Glu

cosam

ine u

pta

ke r

ate

(μm

ol kg

-1 h

-1)

0

1

2

3

Eutric cambisol

Dystric gleysol

a

b

b

c

c

bb

c

a

b b

bc

c

aa

b bb

cc

Autoclaved Fumigated

Fig. 6. Effect of chemical sterilization (HgCl2, Na-azide or CHCl3fumigation) or heat treatment (autoclaving) on 14C-glucosmaine uptake

(Panel A) or mineralization (Panel B) in the topsoil of two grassland soils.

Values represent means7SEM (n ¼ 3). Significant differences between the

sterilization treatments and the control are indicated by *, ** and *** for

differences at the Po0.05, Po0.01 and Po0.001 levels, respectively.

Solid

phase s

orp

tion (

mm

ol kg

-1)

0.0

0.2

0.4

0.6

0.8

Equilibrium solution concentration (mmol l-1)

0.00 0.04 0.08 0.12 0.16 0.200.0

0.2

0.4

0.6

Glucosamine

Lysine

Fig. 7. Solid phase sorption isotherms for 14C-labeled glucosamine or

lysine in the topsoils of two contrasting grassland soils (Panel A, Eutric

cambisol and Panel B, dystric gleysol). Values represent means7SEM

(n ¼ 3). Symbols represent experimental data points while curves represent

fits of the Freundlich isotherm to the experimental data.

P. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–3092 3089

0.03–0.20mgNkg�1 d�1 (ca. 0.1–0.7mgN l�1 in soil solu-tion). In comparison to the amount of DON in soil(0.5–3.0mgN l�1), this suggests that relatively largeamounts of free amino sugars may pass through theDON pool from the extracellular breakdown of aminosugar polymers such as chitin/chitosan and muramic acid.This calculation assumes that the microbial cells are brokendown in soil either by autolysis or via the action ofextracellular enzymes. It is known, however, that microbialcells can also be ingested and mineralized inside protozoaand mesofauna. In this case, the amino sugar monomerswould not pass through the soil solution. Estimates of theamount of microbial biomass that passes through protozoavaries widely (ca. 5–50% of the total) and appears to behighly dependent upon many soil factors (e.g. water status,texture, presence of a rhizosphere, etc. Kuikman et al.,1991; Rutherford and Juma, 1992; Vinten et al., 2002;Schroter et al., 2003). On balance, however, we hypothesizethat the observed low solution concentrations are duelargely to rapid removal from soil solution rather thanslow rates of production. This supports similar findings forfree amino acids where soil solution concentrations also

remain low due to rapid microbial removal (Jones et al.,2005).Previous studies have indicated that free amino acids and

organic acids can be abiotically mineralized via oxidationby minerals such as MnO2 (Wang and Ling, 1993). Ourresults employing microbial inhibitors, however, suggestthat the abiotically mediated mineralization of free aminosugars occurs extremely slowly and is much less importantthan biotically mediated mineralization. Studies by Bon-dietti et al. (1972) have demonstrated that amino sugarsmay also form humic-type polymers in soil, and that theseare relatively recalcitrant. Comparison of the loss ofglucosamine in the presence and absence of microbialinhibitors, alongside the results presented in Bondietti et al.(1972), however, suggest that this is a minor reactionpathway for amino sugar transformation in soil. Otherstudies into the biotic and abiotic mediated mineralizationof amino acids and organic acids in similar soils havereported comparable results (Jones et al., 2005). Ourfindings are also supported by Amelung et al. (2001) whofound that minerals did not greatly affect amino sugarturnover in soil.Typically, acid hydrolysis of amino sugar polymers is

undertaken prior to quantifying the soil’s amino sugar

ARTICLE IN PRESSP. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–30923090

content (Zhang and Amelung, 1996; Kandeler et al., 2000;Amelung, 2001; Glaser et al., 2006). While this provides auseful indicator for determining the source and character-istics of SOM it provides limited information on theirrelative bioavailability unless coupled with stable isotopepool dilution techniques (Glaser and Gross, 2005; Glaser etal., 2006). In this study we measured the free amino sugarsin soil solution, but failed to quantify other potentiallybioavailable forms of amino sugars. This includes aminosugars present in a polymeric form in solution, either in adissolved or particulate organic form, and amino sugarsheld on the exchange phase. Based upon our Kd values foramino sugars and those for DON (Jones and Willett, 2006),we conclude that free amino sugars held on the solid phasestill only represent a small proportion of the DON in soil(Jones and Willett, 2006). To our knowledge there are noreports on the relative amounts of free and polymericamino sugars present in soil solution and this certainlywarrants further investigation both in terms of quantifyingthe amount and nature (e.g. molecular weight, charge, etc.)of the material and its relative bioavailability. Based uponthe relative amounts of amino acids present in a polymericand free state in soil presented by Yu et al. (2002), it islikely that this polymeric amino sugars may represent asignificant proportion of the DON.

4.2. Amino sugar sorption

Previous studies have suggested that amino sugars maybe fixed on clay surfaces (Zhang et al., 1998, 1999;Amelung et al., 2002). Sorption to the soil’s solid phasecould also potentially remove significant amounts of freeamino sugars from solution as occurs for tricarboxylicacids (e.g. citrate; van Hees et al., 2003). While we did notdetermine the sites of sorption in our soils, our experimentsshowed that cationic glucosamine can readily bind to thesoil’s solid phase and that the saturation capacity may belarge (41.4mgNkg�1). However, the shape of thesorption isotherms and comparison of the Kd values witheither NH4

+ (Kd for NH4+¼ 5–25) or other cations suggest

that the strength of binding to the solid phase is weak(USEPA, 1999; Sanchez et al., 2002). Consequently, uponmicrobial depletion from soil solution it can be expectedthat the amino sugars will rapidly desorb back intosolution and become bioavailable (Schnier, 1987). This issimilar to a range of amino acids which are also weaklyheld on the soil’s exchange phase and which can be readilydesorbed using salts such as 2M KCl and 0.5M K2SO4

(Jones and Willett, 2006). However, this contrasts stronglywith other anionic components of the DOC (e.g. organicacids such as citrate3�) where the sorption process is notreversible in the short term, where the Kd values can bemuch greater and where microbial uptake is repressed bysolid phase sorption (Jones and Edwards, 1998). Basedupon the arguments presented above and our amino sugarmineralization results, we again conclude that soil solution

concentrations are primarily maintained at low levels dueto rapid uptake by the soil microbial community.

4.3. Microbial mineralization of amino sugars

Our results suggest that free glucosamine possesses a shorthalf-life in both top- and sub-soils (t1/2 ¼ 1–3 h) particularlywhen present at low solution concentrations. This contrastswith the findings of Nagata et al. (2003), Kawasaki andBenner (2006) and Praveen-Kumar et al. (2002) who havesuggested that free and combined amino sugars arerelatively recalcitrant in the environment. Our turnoverresults, however, are very similar to the t1/2 values reportedfor other common low MW organic solutes in soil (e.g.sugars, amino acids and organic acids; Boddy et al., 2007;Jones et al., 2005). This result also supports our argumentthat the low concentration of free amino sugars in soilsolution is due to rapid microbial removal. At high aminosugar concentrations the rate of mineralization was sig-nificantly slower than that for glucose. This tended to occurat higher rates of amino sugar addition (X0.7mgNkg�1)and also corresponded with a reduction in microbial yield(Fig. 3). This divergence in microbial yield between glucoseand glucosamine at high substrate concentrations providesevidence that the glucosamine-C and glucose-C followeddifferent metabolic pathways once inside the microbial cells.It also suggests that glucosamine was not extracelluarlydeaminated to glucose before its subsequent uptake and usein the microbial cell (as otherwise the microbial-C yieldwould have remained identical to glucose even if its uptakewas limited by external deaminase activity). The high valuesobtained for the immobilization-to-mineralization ratio forglucosamine were between those obtained for other lowMW substrates in these and other soils (e.g. aminoacids ¼ 0.65, organic acids ¼ 0.90; Jones et al., 2004).As the amount of glucosamine added to the soil

increased, we observed changes in the proportion ofglucosamine-C that was mineralized by the microbialbiomass. Based upon the concentration-dependent kineticsfound here (Figs. 1 and 2), this suggests to us that either theglucosamine uptake rate from the soil or the community’sinternal capacity to assimilate the substrate may limit itsutilization (Uldry et al., 2002). Whether the internalcellular accumulation of glucosamine exerts feedbackcontrol on the rate of membrane transport requires furtherstudy (Plumbridge et al., 1993). We also observed anapparent lag phase in mineralization at high glucosamineconcentrations. We hypothesize that this is due to either aglucosamine-induced stimulation of microbial growth orthat the community is upregulating its transport andassimilatory capacity. Based upon the size of the microbialcommunity in these soils (200–600mg biomass-C kg�1;Boddy et al., 2007) and the rates of substrate addition(ca. 30mgCkg�1) we speculate that this lag phase is notdue to microbial growth, but moreover to an initial lack oftransport capacity. This is also supported by the glucoseresponse that showed no lag-phase in mineralization.

ARTICLE IN PRESSP. Roberts et al. / Soil Biology & Biochemistry 39 (2007) 3081–3092 3091

4.4. Amino sugar derived N mineralization

It has been shown at both the molecular and physiolo-gical level that microorganisms possess a range of aminosugar-specific transporters for exogenous substrate capture(Brinkkotter et al., 2000; Meibom et al., 2004). Thissuggests that, as occurs for amino acids (Barraclough,1997), the glucosamine was taken up intact into the cell anddeaminated intracellularly with subsequent excretion ofNH4

+ into the soil. In our experiments we clearlydemonstrated that high levels of glucosamine addition tothe soil resulted in NH4

+ production and its subsequentconversion to NO3

�. Our results also support grasslandstudies suggesting that the breakdown of organic-Npolymers to their monomeric units and not their subse-quent ammonification or rate of nitrification are the ratelimiting steps in N mineralization (Bremner and Shaw,1954; Jones et al., 2004). Interestingly, the total amount ofamino sugar-N and amino sugar-C mineralized were alsogenerally similar (ca. 20% of the total added to the soil).Due to the high C-to-N ratio of the amino sugars (6:1) itwould be predicted that less N mineralization will occurthan for amino acids (C-to-N ratio mean7SEM ¼ 3.870.5, n ¼ 21). Clearly, further work usingstable isotopes and isotopic pool dilution techniques arerequired to elucidate the rate of amino sugar N miner-alization under more representative concentrations.

4.5. Influence of soil type and depth

Despite their differences in pedogenesis, overall itappears that both soils exhibited similar responses to freeamino sugar addition. It might be expected that differencesin soil O2 and water status might have resulted insignificant differences in microbial community structurebetween the two soils, particularly in the subsoils and thatwould have affected glucosamine-C partitioning, etc. Thelack of observed differences in glucosamine use, however,may suggest common metabolic pathways for free aminosugar use within the microbial community or a large degreeof functional redundancy within the population. This issupported to some extent by Stevenson (1982) who showedthat total amino sugar contents of soils across the world donot differ to a great extent (typically constituting 3–10% oftotal organic N). This study has highlighted a number ofdeficiencies in our current understanding of hexosaminecycling in soil. Further work is therefore required todetermine the kinetics of amino sugar uptake by themicrobial biomass and its relationship to available C and Nsupply. In addition, the direct and indirect (e.g. as apriming agent or in metabolic control) role of glucosaminein the cycling of polymers in soil organic matter (e.g. chitin,chitosan etc) warrants further investigation. The relativeimportance of biotic use of amino sugars versus theirinvolvement in humic substance formation is also neededespecially in view of current research focused on under-standing the factors regulating C storage in soils.

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