# Spectrophotometric Determination of Biopolymers in Alabama Benchmark Soils
## 2 Materials and Methods
### Soil Analysis
#### 2.2.1 Spectroscopic Quantification of Biopolymers in Soils
The microplate was pre-incubated at 60 \({}^{\circ}\)C for one hour, allowed to cool, and then transferred to an 800 TS microplate reader (BioTek instruments, Agilent Technologies Inc. Winooski, VT, USA), set to shake for thirty seconds at 37 \({}^{\circ}\)C before reading at 562 nm. The proteins in the extracts were quantified against a bovine serum albumin (BSA) standard curve using BSA standards 0, 125, 250, 500, 750, 1000, 1500, and 2000 \(\upmu\)g/L. To calculate the autoclaved citrate extractable soil protein content of each sample, the protein contents in the extract were multiplied by the volume of the extractant and then divided by the weight of the soil used. The results were expressed as mg protein/g soil (g protein kg\({}^{-1}\) soil). Extraction and spectrophotometric quantification of cellulose in soils:  
Cellulose in the soils was determined by first extracting with the acetic acid/nitric acid reagent [29]. Air-dried soil samples (100 mg) were put into 15 mL polypropylene tubes, to which was added 3 mL of 8:2:1 (\(\
u/v/v\)) glacial acetic acid, distilled water, and nitric acid (69%) solution. The tubes were then placed in a thermostatic water bath at 90 \({}^{\circ}\)C for 30 min. This hot oxidizing solution will dissolve most cell-wall components, including hemicellulose and lignin, and leave cellulose untouched. The samples were allowed to cool to room temperature and then centrifuged at 2500\(\times\)\(g\) for 3 min, after which the supernatant was discarded. Ten (10) mL of deionized water was added to the residue and centrifuged at 2500\(\times\)\(g\) for 3 min to wash away the oxidizing acid solution. The acid-wash procedure was repeated twice to give a total of three washes. The residue was then oven-dried at 75 \({}^{\circ}\)C for 24 h. Two and a half (2.5) mL of 72% sulfuric acid was carefully added to the dry residue and allowed to react at room temperature for 1 h in a horizontal shaker. The tubes were placed in an ice bath and the volume of the sample was made up to 10 mL by carefully adding deionized water. The solution was centrifuged at 2500\(\times\)\(g\) for 3 min to remove any debris. Four hundred (400) \(\upmu\)L of the supernatant was transferred to new tubes, to which was added 5 mL of anthrone reagent (2 g in 1 L72% H\({}_{2}\)SO\({}_{4}\)). The mixture was incubated for 15 min at 95 \({}^{\circ}\)C in a water bath and the absorbance read at 690 nm against blank samples using a GENESYSTM 150 UV-Visible spectrophotometer (Thermo ScientificTM Madison, WI USA). A calibration curve was prepared using a stock solution of cellulose (0.1 g L\({}^{-1}\) in 72% sulfuric acid). Dilutions were made to cover the range of cellulose concentrations between 0.010 and 0.100 g L\({}^{-1}\). The results were expressed as mg g\({}^{-1}\) (g/kg) dry weight of soil. Extraction and spectrophotometric quantification of acetyl bromide lignin in soils:  
Lignin was quantified using the acetyl bromide method [25]. One hundred (100) mg of air-dried soil samples in triplicates was put into a 15 mL polypropylene tube, to which was added 5 mL of 70% acetone solution in distilled water (\(v/v\)). The mixture was vortexed and the tube was placed in a sonic bath (40 kHz) for 40 min. The sample was centrifuged at 200\(\times\)\(g\) for 2 min and the supernatant was carefully discarded, with special attention  
Figure 1: Soils used in the study. The Sumter clay (white) and Houston clay (dark black) were collected from contiguous fields. to avoid removing clay particles; the procedure was repeated once. However, with soils rich in soil organic matter (SOM), the procedure needs to be repeated until the acetone extraction appears colorless rather than pale yellow. The residue was oven-dried at 75 \({}^{\circ}\)C for 24 h to remove the residual acetone. The sample was transferred to a fume cupboard, to which was added 5 mL of 25% acetyl bromide solution (in glacial acetic acid, \(v/v\)).