They also take up P, Ca, Mg, and K from the soil and provide their host plant significant amounts of these nutrients . In return for this key role in plant nutrition the host plant transfers significant amounts of fixed carbon to their EMF. A recent review by Hobbie suggests an average of approximately 15% of total fixed carbon is allocated to ectomycorrhizae, but studies have found more than 60% of recent carbon assimilation and net primary production may be allocated to EMF.In addition to their instrumental role in tree nutrition EMF are believed to be instrumental in forest biogeochemistry. They have a strong effect on processes that govern soil carbon residence time and have been the subject of much interest over the last 15 years of biogeochemistry research due to their ability to stimulate mineral weathering. A Web of Science citation search finds that since 1995, 74 articles have been generated on the topic of ectomycorrhizal weathering. Many have found that EMF do stimulate weathering, and the proposed mechanisms include acidification , nutrient uptake , production of siderophores , and production of low molecular weigh organic acids . Numerous studies point to a significant biotic contribution to mineral weathering in forest soils from prokaryotic , fungal saprotrophic , mycorrhizal , and plant components of the biota. The relative importance of these different groups in mineral weathering as well as the effects of elevated CO2 on biotic weathering remain poorly understood. Low molecular weight organic acids are actively exuded by biota in response to nutrient demand and have been proposed to be key drivers of biotic weathering . Work by Fransson & Johansson ,arandano cultivo and Johansson et al. suggests that LMWOA production by both plants and EMF may increase in response to elevated CO2, either as a result of source sink relationships within the plant caused by increased carbon fixation or as an active response to increased nutrient demand. Assessing the relative contributions of these microbial and plant components of forest biota to LMWOA production, the effects of CO2 on LMWOA production, and the effects of these LMWOA production levels on mineral weathering rates are necessary elements for a better understanding of the importance of biotic weathering. Previous work by van Hees et al. demonstrated a slight increase in weathering rates and organic acid production in mesocosms with pine seedlings compared to those without seedlings but limited effects of ectomycorrhizae on weathering rates or organic acid production. In that study pine growth responded poorly to EMF due to low nutrient levels, EMF did not persist in one of the treatments, and overall weathering was very low because a highly weathered substrate was used which was likely well coated with resistant secondary mineral coatings. Here we focus on the role of LMWOA’s in biological weathering by using a column microcosm system to elucidate the role of Scots pine seedlings, their associated EMF and the effects of elevated CO2 on weathering rates. In contrast to earlier work that used a similar system we have increased nutrient levels, used a rooting substrate derived from primary minerals, used different fungal symbionts, and incorporated an elevated CO2 treatment.Seeds of Pinus&sylvestris were surface sterilized for 30 min in 33 % H2O2, rinsed in sterilized water and sown in sterile, autoclaved water agar for 6S8 weeks for germination . Ectomycorrhizal and non ectomycorrhizal seedlings were prepared in petri dishes of peat:vermiculite:Modified Melin Norkrans media as detailed in Fransson and Johansson . The ectomycorrhizal fungal species Suillus&variegatus O. Kuntze and Piloderma&fallaxStalpers , growing on half strength Modified Melin Norkrans media were used for EMF inoculum. After 12 weeks seedlings were removed from petridishes and planted in 10 cm X 10 cm X 10 cm pots filled with a 1:10 sterilized, autoclaved peat:quartz sand mixture. The collected soil was subjected to sequential centrifugation in winogradsky salt solution , followed by nicodenz extraction at ultra high speed . The resulting bacterial suspension was tested for the presence of culturable fungi by plating on potato dextrose agar, which yielded no observable fungal colonies. The bacterial suspension was used for inoculation within 2 days of extraction and was stored at 4S8°C in the meanwhile.A sand culture system nearly identical to that of van Hees et al. was employed with opaque plexiglas tubes serving as vertical growth columns. Each column was filled with 405 grams of a mineral mix comprised of 50% quartz sand, 28% oligioclase, 18% microcline, 1.8% hornblende, 0.9% vermiculite, and 0.9% biotite. The quartz sand was acid washed overnight and washed with DI water until the solution pH was >6 before being mixed with the other ground minerals. The complete mix was approximately 40% silt size class and 60% sand size class . The tubes were drained by applying suction at the base of the columns with a ceramic lysimeter cup and column leachate was collected in opaque 250Sml glass bottles. When tested before planting, this drainage system maintained the mineral mix at a moisture content of 5±2%. Rhizon SMSSMOM suction lysimeter samplers were inserted horizontally 10 cm below the soil surface for the purpose of extracting rhizosphere soil solution to measure LMWOA production. The columns were maintained at 20 ± 0.5°C during the “daytime” and 14S16°C during the “nighttime” by the use of plexiglass chambers placed over the column system with peltier coolers used to regulate temperature. CO2 levels were maintained at 330S380 ppm and 700S750 ppm under each chamber. Light was supplied by a high pressure sodium lamp with an intensity of 300 PPFD at the seedling tops . The columns were watered 3 times a week with 24S36 ml nutrient solution . The watering solution contained 33 µmol 2HPO4, 407 µmol NH4NO3, 27.5 µmol K2HP04, 55 µmol Ca 2, 27.5 µmol K2SO4, 5.5 µmol H3BO3, 1 µmol FeCl3, 0.1 µmol Na2MoO4, 0.1 µmol ZnSO4, 0.1 µmol CuSO4, 55 µmol Mg2. The pH was adjusted to 5.0. The molar ratio of N/K/P was 100:10:6, which is comparable to the optimal nutrient use efficiency values for conifers of 100:15:6,frambuesa maceta as determined by Ingestad , except it is slightly depauperate in K. After addition of the mineral mixture to each column the columns were allowed to equilibrate with the nutrient solution for three weeks. In April 2008, one seedling was planted in each column . At this time 2 seedlings of each colonization treatment were dried for future analysis. Black plastic beads were placed on top of the soil after planting to prevent the growth of algae. One week after planting, each column was inoculated with 5 ml of the fungus free bacterial inoculum described above.Treatments were factorial with +/seedlings, +/S EMF , and +/SCO2 . N=4 for the non mycorrhizal and non-planted treatments, and 5 for the EMF treatments; in total there were 36 columns, 28 of which were planted. The experiment was run for 9 months. Organic acid and phosphate concentrations were measured in rhizosphere soil solution, and elemental concentrations and pH were measured in column leachate collected during the experiment. Upon harvest,the cation exchange capacity of the mineral mix pre and post 9 months of growth, the weight and elemental contents of seedlings,and the chitin contents of the roots, and mineral mix were assessed. This information was used to construct whole column nutrient budgets. Rhizosphere soil solution was extracted for low molecular weight organic acid analysis by applying suction for up to 3h with a 50Sml plastic syringe to lysimeter samplers. LMWOA samples were collected five times from each column at 5, 6, 7, 8, and 9 months post planting, with sample volumes ranging from 2 to 12 ml. Sampling was performed 24–36 h after watering, and immediately frozen at S20oC for later analysis. Column leachate was sampled every 3S4 weeks. Total volume in each bottle was measured and 2 duplicate 15 ml aliquots from each bottle were sampled and frozen for future elemental analysis. Leachate was sampled a total of 11 times for each column. Upon harvest seedlings were removed from the mineral mix, and all possible adhering mineral particles were shaken/brushed off the roots under dry conditions.Each seedling was separated into roots, stem, and needles and dried at 60oC for 24 S72 hours until no more mass loss was noted, and we then measured dry weights . The plant DW is the sum of the three compartments and the root:shoot was calculated as root DW / . Seedlings sampled before planting into columns were given the same treatment. The mineral mix from the columns was separated into three fractions: bottom, top, and rhizosphere. The bottom fraction consisted of the portion of the column below the extent of lowest roots in that column , the rhizosphere fraction consisted of all of the soil which remained adhering to the roots when the roots were gently removed from the columns, and the top fraction was the remainder. Each fraction was weighed moist and a sub-sample of approximately 50 grams was collected and dried at 60°C for 24S72 h until no further mass loss was noted. Additional smaller sub-samples were freeze dried for chitin analysis. Low molecular weight organic acids were determined by capillary electrophoresis by the method of Dahlen et al. . Briefly, LMWOA’s were analyzed on an Agilent 3DCE capillary electrophoresis system . The concentrations of 12 different LMWOA’s were analyzed: acetate, butyrate, citrate, formate, fumarate, lactate, malate, malonate, oxalate, proprionate, succinate, and shikimate, as well as phosphate. To determine oxalate and citrate, EDTA was added in a separate run to eliminate interference from Al and Fe ions. LMWOA data is presented in µmol/L solution collected from rhizosphere lysimeters and as µmol/L/gram plant DW. Acid digestion of plant material was undertaken following the procedure of Zarcinas et&al. as follows: 0.1 gram of each seedling component was separately digested at room temperature overnight in 2 ml concentrated HNO3 , heated up to and refluxed at 130oC with a funnel lid for 5S7 hours and subsequently diluted with 12S15 ml deionized water. Exchangeable ions were measured for each of the three postSharvest mineral fractions for each sample as well as for 9 replicates of the pre-experimental mineral mix. Extractions were performed in a 1:10 mineral mix:1M NH4AOc suspension by shaking for 5 hours at 100 rpm at room temperature. The supernatant was separated by centrifugation and filtered through a pre-washed 0.45 µm NaAcetate filter syringe. Before cation exchange, the pre-experimental mineral mix was equilibrated with the experimental nutrient solution 3 separate times for 12 hours each to mimic the period that the columns were allowed to equilibrate for three weeks before planting. Plant digests, CEC extracts, and column leachate were all analyzed for elemental contents of Al, Ca, Fe, K, Mg, Mn, Na, P, S, and Si on a Perkin Elmer atomic optical emission inductively coupled plasma emission spectrometer . A set of four standards was established based on preliminary analysis for each sample type. In addition to hourly rerunning of standards, duplicates and an internal scandium standard were run to ensure an accuracy of elemental contents to +/S 1%. Elemental loss though column leachate was calculated from the leachate concentration and the total volume of leachate. Plant roots and growth substrate were assayed for chitin content post harvest to assess fungal biomass. Chitin was extracted and analyzed by HPLC at the Department of Forest Ecology & Management, SLU , according to the method in Ekblad and Näsholm . Chitin concentration of each column fraction was multiplied by the mass of that fraction and these sums were added to obtain total chitin content per column. Significant quantities of chitin were not found in any of the bottom fraction samples. To relate fungal biomass to plant biomass the total chitin content was divided by the total plant biomass in each column.Except where explicitly stated all data are presented as the mean per column. LMWOA and chitin were also presented as mean per column per unit seedling mass. In this experiment we investigate 2 different independent variables: CO2 and seedling treatment . If significant interaction effects were found they were indicated. Otherwise, when looking at the effects of seedling treatment the two CO2 treatments were combined .