Methods:
Sample preparation
Prior to analysis, samples were oven dried at 70 degrees C for 48 hours, ground to 20-mesh and homogenised.
Proximate carbon fraction analysis
Carbon constituent analyses were performed using a series of extraction's modified from those used by Ryan, et. al. Two grams of material were refluxed with 75 ml of dichloromethane for five hours to remove the non-polar component of the sample (fats, waxes, and other organic soluble materials) using a block digestor and condenser apparatus modified from a TAPPI procedure. Polar extractives such as phenols, simple sugars, starch and simple amino acids were removed by boiling a subsample (1.6 g) of tissue from the non-polar analysis for three hours in deionised water and then filtering. Sample size was increased from the recommended 0.5 g to 1.6 g to ensure sufficient post polar sample quantity for the acid digestion. Lignin and cellulose content were determined by a modified wood-products chemistry procedure. A 0.5 g subsample of post polar material was digested in 72% sulphuric acid for 1 hour in a 30 degree C water bath. This was then diluted with water until a 28:1 water to acid ratio was formed to carry out secondary hydrolysis while boiling for 4 hours. To wet the sample thoroughly we used 1.5 ml of acid for each 100 mg of sample, a 0.5 ml increase in acid over the quantity Effland recommended. At the end of this process the sample was filtered. Residual tissue was considered lignin and mass lost during digestion was considered cellulose. The percentages of the different constituents were determined gravimetrically. Oven-dried samples were temporarily stored in dessicators to prevent gaining moisture from the atmosphere before weighing.
Calculations for carbon chemistry
% non polar = (Pre non polar mass - post non polar mass)/ pre non polar
mass; % polar = ((pre polar mass - post polar mass)/ pre polar mass) * (1-% non polar); % cellulose = ((pre digest mass - post digest mass)/ pre digest mass) * (1- % non polar - % polar); and % lignin = (post digest mass / pre digest mass) * (1- % non polar- % polar).
Nitrogen analyses
Nitrogen determination was performed using a Perkin-Elmer 2400 CHN Elemental Analyser. The CHN analyser combusts a sample to convert elements to gases (CO2, H2O, N2). The resulting data are weight percentage of carbon, hydrogen and nitrogen (CHN). In addition, a total Kjeldahl nitrogen (TKN) determination was performed on a subset of 100 samples. This method uses sulphuric acid and a digestion catalyst of potassium sulphate, cupric sulphate and selenium powder on ground leaf samples to generate a liquid sample for colorimetric analysis, and was included as a alternate method of nitrogen determination included for QA/QC as assessment. In addition, three replicate samples of each of the three standard materials were sent to Oregon State University for a similar Kjeldahl analysis. Finally, three National Bureau of Standards (NBS) 1.2% nitrogen pine foliage standards were included in each of four Kjeldhal runs.
Analysis Methods
Method ID: 1 Name: Near Infrared Spectroscopy. Description: Samples were dried at 70oC, and ground to pass a 1mm mesh screen. The dried and ground sample was scanned using a NIRSystems spectrophotometer. Foliar N, lignin and cellulose concentrations were predicted from the resulting spectral signatures. Citation: Bolster, K. L.; Martin, M. E., and Aber, J. A. 1996. Determination of carbon fraction and nitrogen concentration in tree foliage by near infrared reflectance: a comparison of statistical methods. Canadian Journal of Forest Research. 26(4):590-600.
Method ID: 2. Name: ICP Analysis. Description: Digestate was analyzed for Al, Ca, Fe, K, Mg, Mn, P and Sr using an inductively coupled plasma spectrometer.
Method ID: 3. Name: TKN digestion. Description: Samples were dried at 70oC, and ground to pass a 1mm mesh screen and digested using a modified TKN digestion using a selenium catalyst (Isaac and Johnson 1976). Citation: Isaac, R.A. and Johnson, W.C. 1976. Determination of total nitrogen in plant tissue using a block digestor. Journal of the AOAC 59: 98-100.
Method ID: 4. Name: Microwave digestion. Description: Samples were dried at 70oC, and ground to pass a 1mm mesh screen. Dried and ground foliage was digested using a microwave assisted acid digestion procedure. Citation: 1996. EPA method 3052: Microwave assisted acid digestion of siliceous and organically based matrices. In Test methods for evaluating solid waste update (III). U.S. Environmental protection agency, Washington, DC.
Method ID: 5. Name: TKN Analysis. Description: Analysis for the digestion of total foliar N was carried out using a Bran+Luebbe (Technicon) TRAACS 800 Autoanalyzer.
Method ID: 6. Name: CHN Analysis
Method ID: 7. Name: DCP Analysis. Description: Digestate was analyzed for Al, Ca, Fe, K, Mg, Mn, and P using a SpectraSpan III, ARL direct current plasma AES by Fisons Instruments Inc.. Citation: A.L. Page, R.H. Miller and D.R. Keeney (Eds), Methods of soil analysis chemical and microbiological properties, 2nd Edn, Vol. 2. America Society of Agronomy, Madison, WI (1982).
Method ID: 8. Name: Dry Ash. Description: Foliage samples were dry ashed at 485 degrees C; taken up in 10% HCl. Citation: Munter, R.C. 1982. Current methods used by Research Analytical Laboratory University of Minnesota, St. Paul.