Soil Microbial Composition and NitrogenCycling in a Disturbed Wet PrairieRestoration (Wisconsin)Jenny Kao-Knifjin and ieri C. Baiser (Dept of Soil Science,University of Wisconsin—Madison, 1525 Observatory Dr,Madison, WI53706-1299 USA. 608/345-3674, tcbalser@wisc.edu)

Replanted almost entirely by the botanist HenryGreene in the iy40s and 1950s, Lower Greene Prai-

rie is among the oldest wet-mesic prairie restoration sitesin the United States. Subsequent urbanization and high-way construction since the 1970s have led to an influxof stormwater, sediments, nutrient inputs, and invasiveplants. Approximately one-third of the prairie (> 3 ha) isnow dominated by reed canarygrass {Phalaris arundina-cea). The historical significance of the site now extendsto its present use as an adaptive restoration project thatintegrates research with restoration (Werner and Zedler2002, Zedler 2005), as researchers at the University ofWisconsin (under the supervision of Joy Zedler) plan todivert stormwater around the restored prairie.

Our objective was to determine diiferences in soil func-tion within the native restored prairie in comparison to thehighly disturbed section dominated by reed canar)'grass.Such measurements can provide a useful index to assesshow ecosystem function changes in relation to the differ-ent adaptive phases tjsed in restoration projects. We canthen assess whether the diversion of stormwater runoffaway from the site will alter soil function to resemble theundisturbed native plant community. Here we report tlieImpacts of the multiple disturbances on soil function bymeasuring gross nitrogen (N) mineralization rates and soilmicrobial community structure.

Lower Greene Prairie is a restored wet-mesic prairielocated in Madison, Wisconsin (43"r40" N, 89''26'15" E),and managed by the University of Wisconsin Arboretum(Figure 1). In July 2004, we sampled from replicated plots(« = 11) located widiin three zones of the restoration site:1) sites receiving heavy stormwater inputs and dominatedby a monoctilture of reed canarygrass (Invaded community);2) sites not receiving stormwater inputs and dominated byrestored native wet prairie plants (Native community); and3) sites in the transitional zone between reed canarygrassand the native wet prairie community (Mixed community).

One of the key functional indices was gross nitrogenmineralization rate, determined using the '^N isotope dilu-tion method (Hart et al. 1994). We used this process-basedindex to assess nitrogen cycling ín relation to microbialbreakdown of organic matter into ammonium. In eachplot, we pounded tour PVC cores (9 cm long and 5 cmdiameter) into the soil and removed these to label the soilwith 99% ^̂ N as (i^NHj2SO4- The stable isotope '^Noccurs at very low levels in soil (less than half a percent),

Figure 1. LowerGreene Prairie in Madison, Wisconsin. The wet-mesicprairie restoration (approximately 9 ha) was planted with a diversecommunity of native plants starting in the 1940s (top, foreground). Thesouthern portion (> Ï ha) of the site has received stormwater Inputssince the 1970s and is now dominated by reed canarygrass (top. back-ground). Plots (bottom) were located in the zones of reed canarygrass(RCG), the transitional zone (hetween reed canarygrass and restoredprairie), and native restored prairie. Photos by ). iiao-Kniffin (top) andcourtesy of TerraServer (bottom)

which makes it a useful tracer of nitrogen transformationprocesses. We placed two of the cores back into the groundfor 24 hours, while the other two cores were processedseparately in the lab for initial nitrogen readings then aver-aged together. Gross mineralization rates are determined bymeasuring the changes in the ^^N:'^N ratio when microor-ganisms mineralize the more abundant form of organic '^Ninto ''*NH4 after '^N enrichment. The second pair of coreswas retrieved the next day and processed the same as tbeinitial samples. Dilution of the '^N label in the recoveredsoil (24 h later) indicates nitrogen consumptive processes,which include microbial immobilization or assimilationinto plants. Thus gross nitrogen mineralization rates differfrom net nitrogen mineralization rates by accounting forthe various nitrogen consumptive processes.

Another important functional index was change insoil microbial communit)' structure, determined using acombination of phosphoUpid fatty acid analysis and fattyacid mediyl ester analysis (PLFA/FAME). Individual fattyacids are found in the membranes of nearly all microorgan-isms, but because the relative amount of each facty acid

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varies among organism groups, lipid profiles are useful indetermining the presence and relative abundance of gen-eral groups of organisms in soil. We collected three (5 cmdiameter) soil cores from each plot, and pooled these as acomposite sample after plant debris was removed. We keptthe soil frozen at -20°C until the cores were freeze-dried,then we extracted lipids from 3 g of soil using die methodsdescribed by Kao-Kniffin and Baiser (2007).

For multivariate analysis of microbial lipid data, we pet-formed nonmetric multidimensional scaling (NMS) onthe arcsine-transformed mole fractions of individual lipidsusing PC-ORD (vets. 5. MJM Software, Gleneden OR). Wecalculated a matrix of Sorensen (Bray-Curtis) dissimilaritiesofthe mole fractions and subjected these to NMS. Startingfrom random configurations, 50 runs were performed withthe real data and checked against the runs carried out onrandomized data. The model suggested a two-dimensionalsolution as optimal, and NMS was rerun with two dimen-sions and the best starting configuration. Ihe final stressvalue for this solution was 3.3. We analyzed the NMS scoresand nitrogen data using a randomized complete block one-way ANOVA to evaluate the effects of vegetation/disturbancezone on the dependent variables using JMP software (vers.5, SAS Institute, Cary NC). We performed Fishers LSDpost hoc means comparisons test on data with significantprobability values of/) < 0.05. Microbial relative abundancerefers to the proportion of an indicator lipid relative to thesum of all microbiai lipids in a sample.

Nonmetric multidimensional scaling of microbial lipidsrevealed that the overall soil community structure underthe native plant community zone differed significantly(axis \:p < 0.05) from the invaded and mixed plant zones(Figute 2a). Axes 1 and 2 explained 98% ofthe variationin the dataset. Axis 1 was largely influenced by saturated,branched chain, and fliiigal lipids. In other words, micro-organisms indicative of fungi, gram-negative bacteria,and gram-positive bacteria drove much of the separationamong the plant community zones. Axis 2 showed nosignificant differences in microbial community structureamong plant community zones. Further analysis of lipidbiomarkers indicated that the ratio of gram-positive togram-negative bacterial indicators increased with greaterlevels of disturbance [p < 0.01) (Figure 2b). However, totalmicrobial biomass did not differ significandy among theplant community and disturbance zones.

In this study, stormwater runoff combined with the inva-sion of reed canarygrass inro the native wet prairie restora-tion site decreased gross nitrogen mineralization rates incomparison to the less disturbed sites in the restored nativeplant community and transitional zones (p < 0.05) (Figure2c). If slower rates of nitrogen mineralization in disturbedareas are sustained over time, the amount of extractablenitrogen could decline in the reed canarygrass and storm-water plots. The lower rate of gross nitrogen mineraliza-tion found in reed canarygrass plots was consistent with

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Figure 2. Soil microbial community structure and nitrogen (N) cycling:A) nonmetric multidimensional scaling of microbial lipid mole fractions;B) mean (± SE) relative abundance of gram-posilive to gram-negativebacterial lipid indicators; and C) mean (t SE) gross nitrogen mineraliza-tion in soils collected from invaded. Mixed, and Native plant communi-ties using '=N isotope dilution. Bars showing different letters indicatesignificantly different means at p < 0.05 (Fisher's LSD).

the shifts in the relative abundance of gram-positive andgram-negative bacterial indicators (Figures 2b, 2c). This isin agreement with other work showing that gram-negativebacterial abundance is positively associated with high ratesof nitrogen mineralization (Fraterrigo et al. 2006). Gram-negative bacteria tend to be "colonizers" and rhizospherespecialists capable of rapid growth and turnover (Schlegel1992). In contrast, gram-positive bacteria tend to be stresstolerant, slowly growing, and potentially associated withlower rates of nitrogen cycling (Balser 2005).

March 2010 ECOLOGICAL RESTORATION 28;1 I» 21

The ecological significance of this shift toward anincrease in gram-positive bacteria and decrease in gram-negatives may lead ro slower nitrogen and carbon cyclingin sites receiving stormwater inputs and dominated byreed canarygrass. In practical terms, slower rates of grossnitrogen mineralization indicate lower microbial activityand possibly lower quality substrates for microbial use.Over time, the shift towards slower nitrogen and carboncycling could lead to greater carbon accretion in the wet-lands receiving stormwater inputs. It also suggests thatnitrogen availability may eventually decline in the sitesreceiving stormwater and dominated by reed canarygrass.

Initial extractable NH^'-N and NOj-N indicated thatsoils under the different plant community and disturbancezones had similar levels of available N. Soils with identi-cal levels of available nitrogen can have differing rates ofnitrogen mineralization because nitrogen transformationprocesses can vary significandy across biological and envi-ronmental parameters. Extractable NH4'-N and NO3 -Ndo not indicate biological- or physicochemical-based ratechanges in nitrogen availability. Extractable nitrogen issimply a measure oí inorganic nitrogen available for plantand microbial uptake, whereas gross nitrogen mineraliza-tion indicates the total amotint of nitrogen released fromthe microbial conversion of organic nitrogen to NH4\Research projects that document shifts in soil function,therefore, need process-based measurements as a biologicalindex of change. However, repeated sampling of the plotsover several years or after subsequent phases of restora-tion are implemented could show changes in the levels ofextractable nitrogen across the plant community zones.Seasonal or multiple sampling points weithin a year mayalso reveal differences in extracrable nitrogen levels that oursingle time point method did not find. As many restora-tion projects are limited by rhe financial costs of replicatedand repeated sampling, we chose to use a single time point(summer) of soil functional measurements to keep labanalysis costs to a minimum. Satnpling in the summercaptures high levels of microbial activity. Consistent resam-pling at the same time points should provide an adequatemeasurement of functional changes as a basic index.

However, nitrogen and microbial community measure-ments alone do not give a sufficient understanding of thetrajectory of ecological processes ai a restoration site. Basedon our results, we suggest measuring Utter turnover, soiltotal organic carbon, and (physical) carbon fractions asadditional indices of soil function that highlight carbondynamics. These inexpensive techniques could provide amore comprehensive view of both nitrogen and carbon

cycling changes at wetland restoration sites in the context ofglobal change. Taken together, these soil functional indicescan help elucidate how different restoration or land man-agement techniques modify biogeochemical componentsof a werland throughout the sire and over time.

Our initial measurements indicare that multiple dis-turbances brought about by stormwater runoff can alterecosystem function by slowing down nitrogen cycling rates,with concurrent changes in soil microbial communitystructure. New sets of hypotheses can be tested after theplanned stormwater diversions to determine if and whensoil function can revert to ptedisturbance levels. Thuscontinued measurements of biogeochemical parametersthroughout adaptive phases could be tiseful in evaluatinghow restoration efforts influence ecosystem fiinctioningacross space and time.

AcknowledgmentsWe thank Filip Litwinuik and Katherine Fau.st with help ¡n the fieldand labotatory. This study was supported by grants from the Depart-ment of Energy's National Institute for Climate Change Reseatchand the Univetsity of Wisconsin-Madison Graduate School.

ReferencesBaiser, T.C. 2005. Humification. Pages Í95-207 in D. Hillel,

j . Hatfield, D. Powlson and C. Rosenzweig (eds).Encyclopedia of Soils in the Environment., vol. 2. Oxford:Elsevier.

Fraterrigo, j.M.,T.C. Baiser and M.G.Turner. 2006. Microbialcommunity variation and its relationship with nitrogentnineralization in historically altered forests. Ecology87:570-579.

Hart, S.C., J.M. Stark, E.A. Davidson and M.K. Firestone. 1994.Nitrogen mineralization, immobilization, and nitrification.Pages 985-1018 in A.L, Page (ed), Methods of Soil Analysis,Part 2: Microbiological and Biochemical Properties. MadisonWI: Soil Science Society of America.

Kao-Kniffin, J. and T.C. Baiser. 2007. Elevated CO2 difFerentiallyalters belowground plant and soil microbial communitystructure in teed canary grass-invaded experimental wetlands.Soil Biology & Biochemistry 39:517-525.

Schlegel, H.G. 1992. General Microbiology. Cambridge:Cambridge University Press.

Werner, K.J. and J.B. Zcdier. 2002. How sedge meadow soils,microtopography, and vegetation respond to sedimentation.

Zedler, J.B. 2005. Restoring wetland plant diversity: Acomparison of existing and adaptive approaches. WetlandsEcology and Management 13:5-14.

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