This is an author produced version of a paper published in Geomicrobiology Journal. This paper has been peer-reviewed but may not include the final publisher proof-corrections or pagination. Citation for the published paper: Amha,Yosef; Bohne, Heike; Alsanius, Beatrix. (2015) Patterns of Fungal and Bacterial Carbon Mineralization Across Northern European Peatlands. Geomicrobiology Journal. Volume: 32, Number: 10, pp 914-923. http://dx.doi.org/10.1080/01490451.2015.1025318. Access to the published version may require journal subscription. Published with permission from: Taylor & Franics. Epsilon Open Archive http://epsilon.slu.se This article was downloaded by: [SLU Library] On: 17 June 2015, At: 03:32 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Geomicrobiology Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ugmb20 Patterns of fungal and bacterial carbon mineralization across northern European peatlands Yosef Amhaab, Heike Bohnea & Beatrix Alsaniusc a Faculty of Natural Science, Leibniz University of Hannover (LUH), Hannover, Germany b United Nations Economic Commission for Africa, Africa Climate Policy Center (UNECA- ACPC), Addis Ababa, Ethiopia c Department of Biosystems and Technology, Swedish University of Agricultural Sciences (SLU), Alnarp, Sweden Accepted author version posted online: 21 May 2015. Click for updates To cite this article: Yosef Amha, Heike Bohne & Beatrix Alsanius (2015): Patterns of fungal and bacterial carbon mineralization across northern European peatlands, Geomicrobiology Journal, DOI: 10.1080/01490451.2015.1025318 To link to this article: http://dx.doi.org/10.1080/01490451.2015.1025318 Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also. 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Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions ACCEPTED MANUSCRIPT Microbial activity in peatlands Patterns of fungal and bacterial carbon mineralization across northern European peatlands Yosef Amha1,2,, Heike Bohne1, Beatrix Alsanius3 1Faculty of Natural Science, Leibniz University of Hannover (LUH), Hannover, Germany 2United Nations Economic Commission for Africa, Africa Climate Policy Center (UNECA- ACPC), Addis Ababa, Ethiopia 3Department of Biosystems and Technology, Swedish University of Agricultural Sciences 5 (SLU), Alnarp, Sweden 1 0 2 e n Ju Abstract 7 1 2 3 The fungal and bacterial activity was determined in 20 northern European peatlands ranging 3: 0 at ] from ombrotrophic bogs to eutrophic fens with key differences in degree of humification, pH, y r a r b Li dry bulk density, carbon (C) content and vegetation communities using the selective inhibition U L S [ (SI) technique. These peatlands were partly disturbed and the respective water tables lowered y b d de below the surface layer. Basal respiration ranged from 24 to 128 µg CO -C g-1 dry peat d-1. a 2 o nl w Do Bacterial contributions to CO2 production were high in most peatlands and showed the following pattern: eutrophic>>transitional≥mesotrophic>> ombrotrophic peatland types. The fungal-to- bacterial (F:B) ratios varied substantially within peatland type, and this was mainly attributed to differences in peat botanical compositions and chemistry. The computed mean Inhibitor Additivity Ratio (IAR) was quite close to 1 to suggest that the SI techniques can be used to partition eukaryotic and prokaryotic activity in wide range of peatlands. Overall, basal respiration, microbial biomass-C, fungal and bacterial activities varied across the studied 1 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT peatland types, and such differences could have consequences for C- and nutrient-cycling as well as how bogs and fens will respond to environmental changes. Keywords antibiotics, basal respiration, carbon dioxide, microbial activity, microbial biomass, peat Current mailing address: Yosef Amha, UNECA-ACPC, P.O.Box 3001, Addis Ababa, Ethiopia, Email: [email protected] 5 1 0 2 e n u J 7 1 2 3 3: 0 at ] y r a r b Li U L S [ y b d e d a o nl w o D 2 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Introduction Northern peatlands contain 30% of the global soil C that corresponds to 455 Pg C (Gorham 1991). Carbon mineralization in these peatlands is principally controlled by geochemical, environmental and microbiological factors. According to Updegraff et al. (1996), the C mineralization rates in most peatlands correlated well with the chemical compositions of peats such as water and acid soluble carbohydrates, the C/N and lignin/N ratios, or lignin contents and the peat-forming vegetation (e.g., Bryophytes, Carex, Sedge, Eriophorum or shrub/tree plants). 5 The hydrological conditions including the position of redox boundaries associated with the water 1 0 2 e table and peat moisture content are also known to influence the amount and rate of C n u J 7 1 mineralization in peatlands where short periods of flooding may stimulate decomposition, while 2 3 3: 0 at prolonged flooding slow rates (Baker et al. 2001). Severe drought, and subsequent re-wetting, ] y r ra could also destabilize peatland carbon stocks (Fenner and Freeman 2011). Similarly, the b Li U L decomposition of complex organic matter of peats to carbon dioxide (CO ), methane and S 2 [ y b d dissolved organic carbon is controlled by climatic factors. CO2 emission, for instance, tends to e d a o nl increase by a factor of 2 to 3 for every 10 0C temperature increase (Yavitt et al. 1997). w o D The importance of microorganisms as a factor of controlling the rate and amount of C mineralization is also widely recognized across a range of peatlands (Andersen et al. 2006; Jaatinen et al. 2007; Martani 2005; Thormann 2006) although there are still gaps in our understanding of the types of and controls on microbes responsible for C mineralization. Broadly, fungi are believed to play fundamental roles in the decomposition processes of organic matter in many acidic terrestrial ecosystems (Andersen et al. 2006; Martani 2005; Thormann et al. 2002; Williams and Crawford 1983), as they have extensive hyphal growth habit and ability 3 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT to translocate nutrients through their hyphal networks. They are also tolerant to nutrient limited environments because of their lower biomass N and other nutrient requirements. Fungi principally occupy oxic layers with the exception of certain yeasts and moulds that can carry out fermentation. In contrast, bacteria might have a competitive advantage over fungi in deeper and/or more anoxic soil layers as they can also utilize alternative electron acceptors (Killham and Prosser 2007). However, bacterial diversity has been shown decrease with increasing acidity across a large range of soil types (Fierer and Jackson 2006). 5 In peatland ecosystems, a range of diversity of fungi, bacteria, archaea, protists, animals and 1 0 2 e plants have been described (Nuyim 2000). In some studies, fungi have been described as being n u J 7 1 an important group of decomposers (Andersen et al. 2006; Martani 2005; Jaatinen et al. 2007). In 2 3 3: 0 at a microscopy-based study, Golovchenko et al. (2007) have reported fungal biomass dominance ] y r ra in ombrotrophic sites while bacterial biomass dominates the minerotrophic sites. However, b Li U L recent studies by Myers et al. (2012) and Winsborough and Basiliko (2010) have indicated that S [ y b d metabolic activity in peatlands is dominated by bacteria. These authors observed bacterial e d a o nl dominance over fungi in acidic Sphagnum derived Canadian bogs and poor fens as well as in a w o D near neutral wetter rich fen with sedge peat. Lin et al. (2012) also reported strong bacterial dominance over fungi with Acidobacteria and Firmicutes as the dominant microbial groups in bogs and fens of northwestern Minnesota peatlands, respectively. The above findings warrant research to more fully understand the contribution of fungi and bacteria in peatland C cycling; as these microorganisms differ significantly in their physiology and metabolic activity (Poll et al. 2006), and perhaps thus in their responses to environmental changes . 4 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT The fungal and bacterial activities in soils can be measured using various techniques; and the selective inhibition (SI) techniques have been used to describe important characteristics of soil functioning by partitioning glucose-induced bacterial and fungal respiration in a range of agricultural and forest soils (Alphei et al. 1995; Ananyeva et al. 2006, Anderson and Domsch 1975; Bailey et al. 2003). Recent studies by Myers et al. (2012) and Winsborough and Basiliko (2010) used SI techniques to effectively inhibit and therefore quantify fungal and bacterial activity in Canadian peatlands, but only after optimizing the antibiotic and glucose additions. 5 Here, we measured bacterial and fungal activity in northern European eutrophic, ombrotrophic, 1 0 2 e mesotrophic and transitional peatlands using the SI approach. We hypothesized that n u J 7 1 ombrotrophic (bog) sites would be more dominated by fungi, while the mesotrophic and 2 3 3: 0 at eutrophic sites would favor bacteria. We also characterized bulk microbial C mineralization and ] y r ra performed correlation analyses between physical, chemical, and microbiological parameters to b Li U L evaluate potential controls on C loss from our large range of peats. S [ y b d Materials and Methods e d a o nl Characteristics of sampled peatlands w o D Samples from 20 peatlands of 7 European countries were included in this study (Table 1). These peatlands were partly disturbed (i.e., particularly through the construction of drainage ditches) and their respective water tables have been lowered below the surface layer. The living vegetation has also been scraped to facilitate peat mining for horticultural and other uses. In this study, surface peats (up to 20 cm) were considered as the drainage structures expected to affect the moisture content of the upper peat layers. From each peatland, peats were taken in 3 plastic bags (each has a volume of ≈30L). Each bag contained peats taken from six random places. They 5 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT were sampled in the month of September, packed in coolers, and transported to the Leibniz University of Hannover for analysis. All wood, bark and roots were removed and sieved to pass 5 mm sieve. Each bag was then considered as a replicate. Based on the source of water and nutrients in sites (Stewart and Kantrud 1971), the studied peatlands were classified into ombrotrophic (n = 9), transitional (n = 6), mesotrophic (n = 3) and eutrophic (n = 2). The ombrotrophic peatlands, or bogs, are entirely dependent on precipitation for hydrologic inputs and are generally nutrient poor environments (Shotyk 1988; Steinmann and Shotyk 1997). The 5 mesotrophic and eutrophic sites, or fens, received more mineral rich ground and/or surface 1 0 2 e waters. Transitional sites receive some ground/surface water, but are still influenced substantially n u J 7 1 by mineral-poor precipitation inputs. 2 3 3: 0 at The botanical compositions in the sampled peats were identified following Heikurainen and ] y r ra Huikari (1952). Briefly, air dried peat was crushed carefully with a mortar and a representative b Li U L subsample (100-200 ml) soaked in deionized water for a minimum of 2 h. Botanical S [ y b d identification was then made from the peat remains using a high resolution microscope (Meiji e d a o nl Company, Japan). Critical identification markers such as smallest pore holes, cell structure, and w o D mid and/or tip parts of the leaf were used to ascertain different plant remains. The original plant remains within a given peat sample grouped into Sphagnum, Carex, Bryales, Eriophorum, ericoid shrubs and soft/hard wood, where Sphagnum and Carex were found to be the two major peat-forming groups (Amha et al. 2010; Amha 2011). The degree of humification was determined according to the von Post humification scale (von Post 1924). In this qualitative method, a small amount of moist peat was squeezed by hand and the value determined from the 6 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT color of the running water as well as from the nature of the residue. The physical and chemical properties of these peatlands were analyzed and previously reported by Amha et al. (2010). Sample preparation for incubation The water content in each bag was adjusted to 40% of the corresponding water filled pore space (WFPS; eq. 1) with distilled and deionized water (if necessary), and stored in 4 °C room. The WFPS, as oppose to moisture content, measures the influence of moisture on microbial activity and gives direct information on the availability of water in the samples (Robertson and Groffman 5 2007). Subsamples were then taken and conditioned for a week at 20°C (Rumed®, Rubarth 1 0 2 e Apparate GmbH, Germany). These homogenized subsamples were used in the basal respiration n u J 7 1 and microbial biomass measurements, in glucose and antibiotics optimization experiments as 2 3 3: 0 at well as in a SI experiment. ] y r a r W * D Lib WFPS  m BD *100 (1) U H O *P 2 S L S [ y Where, WFPS, W , D , ρH O and P represent water filled pore space (%), gravimetric water b m BD 2 S d e d oa content (Mg/Mg), peat dry bulk density (Mg m-3), density of water (Mg m-3) and total pore space nl w o D (%), respectively. Basal respiration measurement Basal respiration in the conditioned peat samples was measured without any amendment. Briefly, triplicate conditioned subsamples (each corresponding to 10 g oven dry weight, odw) were incubated in 1.5 l glass jars for 10 d at 25 °C. The final water content was adjusted to 60% of the corresponding WFPS. Microbial respiration in these peatlands was found to increase considerably with the additions of water up to 60% of their respective WFPS (Amha and Bohne 7 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2011). Incubating KNO treated peat samples at 60% WFPS also did not induce a sizable amount 3 of anaerobic respiration, as evidenced by small emission of (N O+N )–N over 24 hrs. Headspace 2 2 gas was taken at the beginning and end of incubation period and analysed for CO on a Perkin 2 Elmer Autosystem XL gas chromatograph equipped with a thermal conductivity detector. CO 2 production (μg CO –C g-1 dry peat d-1) was calculated from a calibration with four commercial 2 standards. Microbial biomass 5 Microbial biomass was estimated by a Substrate Induced Respiration (SIR) method (Anderson 1 0 2 e and Domsch 1975) as modified by West (1986). Briefly, glucose solution was added to 10 g n u J 7 1 moist sample (n = 3) to achieve the peat-to-solution ratio of 1:2 (w/v). The final glucose 2 3 3: at 0 concentration in the sample solution was adjusted to 30 mg glucose ml-1. Jars containing samples ] y r ra (each 250 ml capacity) were closed ≈15 min after glucose addition and incubated at 22 °C. b Li U L Headspace gas was sampled at 0, 1, 2, 3, 4, 5, and 6 h after the jars were sealed. Following each S [ y b d headspace sampling, an equivalent volume of ambient air was injected back into the closed glass e d a o wnl jar to avoid a drop in pressure. Microbial biomass-C (MB-CSIR) was calculated according to the o D revised equation of Sparling et al. (1990) where MB-C (µg g-1 dry peat) = 50 x (µl CO g-1 dry SIR 2 peat h-1). Glucose and antibiotics optimization experiments One peat sample from each peat-forming environment (#9, #12, #17 and #19; Table 1) was used to determine the amount of glucose and antibiotics to be added in a SI experiment. The selected peat samples had similar humification degree (H4-H5). Moreover, the dominant peat-forming plant species in three of the four peat samples was S. fuscum (i.e., it accounted for >60% of the 8 ACCEPTED MANUSCRIPT