Biol Fert Soils (1998) 7:173-178 © Springer-Verlag 1988 LL &8< == + °° C. Freeman « G. B. Nevison « S, Hughes B. Reynolds - J. Hudson Enzymic involvement in the biogeochemical responses of a Welsh peatland to a rainfall enhancement manipulation Received: 29 May 1997 Abstract Microbial enzyme activities were followed ‘during a field-based experimental simulation of the ef- fects of higher rainfall in a Welsh peatland. The treat ‘ment id not significantly affeet the activities ofthe ear- bbon eyeling enzymes, f-alucosidase, esterase or xylosi- dase. In contrast the activity of the enzyme sulphatase decreased by 44% (P<0,001) in response to the wetter conditions. The manipulation suggests that should cl ‘mate change cause conditions 10 become wetter in peatlands, then (with the exception of sulphatase) cur- rent levels of wetness may be sufficient to limit decom position processes, and thus any further increase in wetness is unlikely to induce a further decrease in de- composition rates. Correlations were found between the esterase activity and both nitrous oxide flux (r= 044, P<0.05), and methane release (r=053, <0.) Likewise, there was a correlation between xy losidase activity and both carbon dioxide emission (r=052, P-<0.01) and aluminium concentration (r=058, P<0.01). All of the enzymes correlated posi tively with dissolved organic carbon (range r=0.53, P<0.01 sulphatase to r=0.61, P<0.001 glucosidase), Together, the correlations iend support to recent hypo- theses suggesting that enzymes exert an influence Over ‘wetland biogeochemical properties, Key words. Wetland » Peat cchange + Increased rainfall Enzyme + Climate C Freeman (€3) ~ G.B, Nevison School of Bilogial Sconces, Universiy of Wales, Bangor, Gurynead, LLS? 2UW, United Kingdon Tals 41248382353, Fans +44 19355365, male frveman@bangor acuk S. Hughes - B, Reynolds Institute of Terestal Ecology, University of Wakes, Bangor, (Gwynedd, L137 2UW, United Kingdom Ji Hudson Insite of Hydrology, Stale, Lianbrynmir, Powys, SYI9 740, UR Introduction ‘The cool, wet climate of the northern latitudes has sup- ported the development of vast areas of peat-accumt- lating wetland [c. 346 million hectares (Gorham 1991)]. Recent global circulation models (Manabe and Wea. ‘herald 1986; Mitchell and Warrilow 1987; Rowntree ct al. 1989) suggest that the rainfall patterns that have al- lowed that development are likely 10 change as a conse- ‘quence of an enhanced greenhouse effect. Those con- ‘cerns have led to a number of studies being carried out to investigate the potential impacts of the changes on vegetation (Laine ct al. 1995), trace gas emissions (Moore and Knowles 1989; Moore and Dalva. 1993; Freeman et al. 1993a), hydrochemistry (Freeman et al 19936; VanHaesebroeck et al. 1997) and enzymic de- composition processes (Freeman et al. 1996) in a varie- ty of wetland soils. However, the studies to date have focused almost exclusively on the likely effects of drought, due to the perceived dependence of wetlands fon waterlogging for their stability and persistence. It hhas generally been overlooked that while many areas are predicted to become drier (and to experience more frequent droughts), others are likely to become far wet- ter (Amell 1992). Moreover, those areas include north- fern Canada, Scandinavia (Wellburn 1994) and the north of Britain (Cooper and McGechan 1996; Mansell 1997), where some of the Earth's greatest peatland re- sources are located. The following study aims to deter- ‘mine whether the potentially higher rainfall could im- pact upon the biogeochemistry of those peatland soils. ‘This study focuses on soil enzyme activities, due to their pivotal role in wetland biogeochemical cycling processes (Freeman et al. 1995). Esterase, feglucosi- dase and xylosidaso, enzymes participating in organic matter decomposition and hence carbon cycling, were studied by following the rate of hydrolysis of the model substrates methylumbelliferyl (MUF)-acctate, MUF-B- Deglucoside and MUF-6-D-xyloside, respectively. It hhas also been recognised that in wetland soils, organicmatter decomposition can proceed through a pathway which involves both carbon and sulphur cycles, the fer- mentation:sulphate reduction pathway (Howes et al 1984). As such the enzyme sulphatase was also studied, and was assayed by following the hydrolysis of MUF- sulphate. Finally, correlations were sought between those enzyme activities and biogeochemical properties which reflect decomposition-produet release from the soil, These include: (1) hydrochemistry, since enzymic degradation can release inorganic nutrients sequestered within the peat matrix (Freeman et al. 1995), and (2) trace gas emission, since the rate of enzymic cleavage of labile organic substrates (energy sources) from the pre- dominantly recalcitrant peat matrix can regulate the availability of substrates for microbial production of trace gases such as CH,, CO; and NO (Freeman etal 1997). Methods ‘Tae experimental simulations of enbianced precipitation were car- ried out at Cerri yr- Wyn, Piynimon, mid’ Wales (UK Nat. Ref. SN 820 866) on a site that consists of a discontinuous series of peat-accumulating, wetlands (c. 30'S m) which are domi nated by Sphagnum and Juncus species and which lie along the base of a small gully (Fig. 1). The natural dcontinuities allow isolation of various sub-sections of the wetland, such thatthe site can be used as a natura laboratory where diferent manipulations an be applied tothe various subunits. Since 1991, experiments at the site have studied the effects of drought, while the present ‘manipulation commenced in 1995, and has involved increasing water inflow to a subsection of the wetland by diverting waters from a parallel watershed, through a system of pipes, into the ex: ind (Fig. 1). Over the course of the study, the in the control site ranged between 69.6% and 92.4%, while inthe wetted ste, values ranged between 74.4% and 96.5%! Samples of peat and pore water were collected at 2-m tervals long a transect through the centre of the two bogs. P sol samples were collected 10 em" cubes, while pore waters were Collected from soil solution samplers placed at 10 em depth (Frec- ‘man et al. 1993). Temperature and pH were measured in the field Using conventional clectrometic techniques, All other analyses were carried out in the laboratory using previously described methods (Freeman et al. 1935). The gases CHy COs and NO ‘were analysed using an Ai Cainbridge model 92 gas ehromato: raph with twin Porapak QS columns and Mame fonisation and leciron capture detectors, Nitrogen was used as the carrier pas at flow rate of 70 cm’ min” for analysis of nitrous oxide and 13, fem’ min for methane and carbon dioxide. Before hydrochem cal analysis, all samples were passed through a DIONEX On- Guard-P, simple pre-treatment cartridge prior to analysis with Dionex 2000i5p lon Chromatograph with « conductivity detector sand auto selfzegencrating suppression. Nitrate, chloride and sul hate were determined using an AS4A column, while calium, ‘magnesium and aramonium used a CS12 column Aluminium was analysed by atomic absorption, while dissolved organic carbon, (DOC) was analysed by Skala auto analyser, Paired tests on the mean of five replicates from each welland were adopted to deter. mine whether differences between the two treatments were stati cally significant over time ‘Enzyme activities were assayed through » modified fuorogen: ic subsirate assay for peatland soils (Freeman et al, 1995). The assay uses HPLC to avoid the problems quenching interfer fences (Freeman 1997), but doos not require an expensive fuori ‘metric detector (Kang and Freeman 1997), A mixed substrate s0- lution was prepared to contain 100 wM each of methyiumbellfe ty sulphate (MUF-SO,),-methylumbetifery-p-D-glucoside ig. 1 Plan ofthe peatland water table manipulation site il ing the by-pass pipe through which waters are diverted into i experimentally “wetter” bog, and the position of the site relative to earlier drought experiments (MUF- lu), methylumbelifery@-D-xyloside (MUP-xyl) and me Uylumbeliferyl acetate (MUF-est). Each substrate was pred solved in 2 mi methyl cellosolve (Hoppe 1983) prior to incorpon. tion into the mixture, One hundred ml of substrate mixture wa transferred 0 cach of five replicate stomachor homozenisalcn bags (Seward Colworth) containi-g 10:mi wetland sediment. AL ter 1,5 and 180 min, 25 mil aliquot was removed from each bag. transferred to a centrifuge tube, and 2-5 ml of methanol added inate the reaction. Alter centrifugation at 7200%¢ for {min the supemnant was removed and filtered through a 0% ym fier ‘The concentration of each MUF substrate was estimated without delay using UV detection following separation ofthe constituents by HPLC. The HPLC (Dionex 2000), was set for isocrat tion with a 50% methanol eluent flowing at 0:3 ml mit system used a VDML2 detector sotto 320 nm and a 20.em Sper isorb SSP pheny] colurnn (Phase Separations). Hydrolysis wat lin fear for 48 for MUF-glu, MUF-SO, & MUF-xi, but only 10 min for MUF-est.As such, the rate of enzymic hyrolysis was clea lated from the fall in substrate concentration between I min std three h for MUF-gu, MUF-SO, & MUF-xy, and between 1 nin vnd 5 min for MUF-ace Results The imposition of wetter conditions made little impact on the pH of *he wetland soil waters (Fig. 2a). Howev-er, itis interesting to note that the higher water content resulted in soil temperatures becoming less variable ‘over time (Fig. 2b). For example, in the summer ‘months of July and August, the wetted soil was less sus- ceptible to warming (P<0.05) while in the winter months between December and March, those soils were less cooled (P<0,001). However, that reduction in temporal variability was only apparent in one of the enzymes assayed, namely xylosidase (Fig. 2c). One en- zyme, sulphatase, exhibited consistently lower activities (on introduction of the wetter conditions (P<0.001; Fi 3a), while the three carbon-cycling enzymes glucosi- dase, xylosidase and esterase were not significantly af- fected (Fig. 3b-d). A two-way analysis of variance re- vealed significant temporal variation in the enzyme ac- tivities and a 2-factor interaction between the season of sampling and the enzymes (P<0.01). But while distinct seasonal changes in activity were apparent, only sulpha- tase showed maximum activities at exactly the same time as the maximum temperatures were observed. Maximum activities in the three carbon-cycling en- zyines were delayed until after the summer tempera- tures had started to decline in late autumn/early winter. Likewise, the minimum activities occurred substantially later than the temperature minimum, in the spring. ‘A Spearman Rank correlation analysis revealed @ number of correlations between biogeochemical prop- erties and the enzyme activities. When trace gas emis- sions were considered, for example, esterase activity correlated inversely with nitrous oxide fluxes (Fig. 4a: = -0.4, P<0.05), and yet positively with methane re- lease (Fig. 4b: r=0.53, P<0.01). Likewise, there was a strong positive correlation between xylosidase activity and carbon dioxide flux (Fig, 4c: r=0.52, P<0.01). We were unable to detect a negative correlation between sulphatase activity and methane emission. Upon con- sideration of the soil solutes, xylosidase correlated with aluminium concentration (Fig. 4d: r=058, P<0.01). Al of the enzymes correlated positively with DOC [range r=0.53, P<0.01 sulphatase to r=0.61, P<0.001 aa} 9 6 ee 2 fF eoo F suite in pa ‘mont Fig.24,b The pH (a) ond temperature (b) st 10 em depth in the soll of control (fled circles) and experimentally wetter (open cr les) wellands. Error bars indicate = 5¢ Aes ultats 9°.) Act Mois gm) ‘Activity (utols bg * dm ‘Atty ukals ng dm gs a mont Fig. 3o-d_ Activities of sulphatase (a), f-glucosidase (b), xylost- ‘dase (6) and esterase (@) in control led circles) and experimen tally Wetter (open circles) wetlands. Error bars indicate '8.¢ slucosidase (Fig. 4e)]. However, none of the enzymes correlated with either Ca, Mg, Cl, NOs, NH, or pH. Discussion ‘The type of experiment carried out in this study can be considered the opposite of the ficld-based drought si- mulation experiments that have been carried out in re~ cent years (Freeman et al. 1996). The responses to the wetting manipulation might therefore be expected 10 represent an exact opposite of those seen in the drought experiments. There are examples where this has been observed to be the case. For example, drought conditions have been found to allow summer soil tem- peratures to rise to higher levels than were seen in a (control) permanently wet site (Freeman et al. 1993). In the present study, the introduction of wetter conditions induced the opposite response ~ a lowering of the max- imum summer soil temperatures observed (Fig. 2b). Laboratory and field studies indicate that warmer, drier conditions stimulate mineralisation as indicated by: (i) an increase in the release of inorganic nutrients into peatland drainage waters (Heathwaite 1990; Freeman et al. 1993b; Freeman et al, 1994), (i) subsidence of the peat surface (Schothorst 1977), or (ii) stimulation of enzymic decomposition (Freeman et al. 1996). As such,‘ety (ukols bg.) Activity (Mois hg" am) N,0 cna mn?) ‘Act (uMols 1° 9° rm) ‘ty (aMols 9 am) "909 2009, S000 6000 CO, (mg m6) Atv (tals gm) OG mgt" Fig. 4e-e Relationships between (i) esterase activity and nitrous fnide (a) and methane (b) Mux, (i) xylesidase activity and carbon Gioxide Mux (6) and aluminium concentration (@), and (ii) frgha- ‘osidase activity and dissolved organic carbon concentration (¢) it might be predicted that the introduction of wetter conditions would have the opposite effect and suppress mineralisation processes. However, with only onc en- zyme, sulphatase, has this proven to be the case. Over the year as a whole, 44% (P<0.001) less sulphatase ac~ tivity was detected under the wetter conditions (Fig. 3a), and there was significantly less (41%, P<0.001) ‘sulphate in the soil pore waters. The observed reduc tion in sulphatase activity is of a similar order to that observed by Pulford and Tabatabai (1988) in mineral soils (—5% to —31%). Work by Press ct al (1985) sug- gests that sulphate in acid rain may reduce peat sulpha- tasc activities. The greater flux of sulphate through the site (due to the inereased throughput of sulphate-laden rainfall), may have thus induced the lower sulphatase activities noted in the present study. In contrast, the wetting treatment made no signifi cant impact of the carbon-cycling enzymes. It could be speculated that this may have been duc to the more fa- Yourable temperature regime following the introduc tion of wetter conditions (Freeman etal. 1993). The ob servation of greater thermal stability under wetter con- ditions has positive implications for the enzymes of the system. It could potentially increase the period of time fover which enzymes are synthesised by the micro-or anisms by avoiding microbial inactivation through de Siccation in summer (West et al. 1992) or extreme cold in winter (MeClaugherty and Linkins 1990). The intro duction of wetter conditions also promotes DOC re- lease (Forsberg, 1992; Mitchell and McDonald 1992; Hughes et al. 1996), which would increase substrate abundance for microbial metabolism and would sup- port the synthesis of new enzymes (Kiister 1993). Thus, the combination of a more benign temperature regime ‘against the anticipated detrimental effects of the more ‘waterlogged environment. A degree of support for the proposed benefits of increased substrate supply can be found in our observation that all of the enzymes corre- lated positively with DOC abundance (cg, Fig. 4). Relationships were not exclusively associated with DOC, however. When trace gas emissions were consi cered for example, a positive correlation was found be- ‘tween esterase activity and methane release (Fig. 4b: 7=053, P<0.01). Such a finding is not unexpected, as esterase can release acctate into solution (Sakai et al {1996), a compound which many studies have found stimulatory to methane emissions (Sorrell and Boon 1992). However, that same enzyme correlated inversely with nitrous oxide fluxes (Fig. 4a: r= ~0.44, P<0.05). ‘This may simply reflect the highly negative redox po- tential in wetland sediments, which would mean that any additional labile organics entering the soil (cz. through the action of esterase) would benefit the me- thanogens (redox —250 to —350 mv) to a greater ex- tent than the (N.O-forming) denitrifiers (redox 250 my). Likewise, there was a strong positive correlation between xylosidase activity and carbon dioxide flux (Fig. 4c: r=0.52, P<0.01), which may have been antici- pated on the grounds that carbon dioxide is an end Product of microbial metabolism, and enzymes such as xylosidase produce products which are readily utilised by micro-organisms (Chrost and Rai 1993). We were tunable to detect a negative correlation between sulpina- tase activity and methane emission, which seemed sur- prising in that it has been proposed that sulphatase ean release sulphate which inhibits methanogencsis (Free- man et al. 1997). Xylosidase correlated with aluminium concentration (Fig, 4d: r=0.58, P<0.01). It is interest- ing to note that other studies have suggested that alu- ‘minium hydroxide can promote enzyme activities by encouraging the formation of more active enzymatic complexes (Gianfreda ct al, 1995). However, none of the enzymes correlated with either Ca, Mg, Cl, NOs, NH, or pH, vhich contrasts with other studies whichLe] suggest that Ca, Mg, and pH in particular, can have a ‘marked impact on enzyme activities (Wetzel 1993; Pind et al, 1994) The introduction of wetter conditions may suppress the activities of certain mineralising enzymes (eg, sul- phatase); however, it would appear that for the carbon- cycling enzymes, even present levels of waterlogging are capable of limiting their activites. Thus, the erea- tion of still wetter conditions has had no further detri- ‘mental impact on those enzymes. Another study re- cently reported an increased mobilisation of ions such as bromide in response to wetter conditions (Hughes et al. 1996). The authors proposed that the release was due to # stimulation of organic matter breakdown, However, the absence ofa stimulation in the activity of ‘enzymes associated with decomposition in the present study, suggests that the increased bromide release is ‘more likely to be due to physical leaching processes than to the decomposition of organics. ‘Clearly these studies of the effects of a manipulative experiment on wetland enzyme activities have aided ‘our interpretation of the biogeochemical responses 10 hydrological changes that may be induced by global ‘warming. However, the methods used in this study do not allow us to comment on whether the changes in ac- tivity were due to shifts in the rate of enzyme synthesis as a consequence of changes in the composition of the ‘microflora, or whether the changes were due to edaphic control of enzymes immobilised on soil colloids (Nanni- pieriet al, 1990). 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