Team:NAWI-Graz - 2021.igem.org

Team:NAWI-Graz

Qualitative detection of soluble phosphate of agricultural land in Austria

"Without phosphorus there would be no thoughts."
- Ludwig Buchner

Abstract

The bioavailability of ortho-phosphate as well as other phosphorus compounds is an essential aspect in agricultural economics and therefore the food industry as well as countless other industries that are connected to the further processing of plants and plant based compounds. The goal of this experiment was to give people without any scientific background an insight in the state of bioavailable phosphate. To achieve this, 18 samples different pieces of land that are agriculturally cultivated respectively were taken and the amount of soluble ortho-phosphate was determined. We decided to follow a qualitative approach to visualize the average state of bioavailable phosphate in Austria’s agricultural land. The samples were dried for five days to get rid of excess humidity and were pre-treated through CAL (Calcium-Acetate-Lactate)-extraction to free the contained phosphate in the samples from excess particles and ions. The photometric analysis was achieved through the calorimetric determination of molybdenum blue. The phosphate extracts were mixed with an acidic MoVI solution (ammonium molybdate) as well as an acidic Sb(III) solution (antimony potassium tartrate) to form a phosphoantimonylmolybdate complex. This procedure is followed by a reduction with ascorbic acid to yield the photoactive molybdenum blue complex. The ortho-phosphate concentration was determined through an Absorbance Plate Reader for easier and time effective measuring of multiple samples at the same time.

Introduction

Phosphorus (P) is one of the limiting nutrients to crop production due to its low solubility, ease of fixation and relative immobility in soils. Meanwhile, P leaching is the main factor causing eutrophication of water if over P fertilizer was applied to soil. [2] So additionally to our main project, we wanted to raise awareness of the continuous decrease of bioavailable phosphate in agricultural land all over the globe. Different models estimate the exhaustion of natural occurring phosphate between fifty years to a century, which could have devastating consequences for humanity as well as nature in general. Three differently located fields were chosen to draw samples from in the way it is depicted in the pictures below.

Every field was divided into three subfields, each of which yielded six samples for further processing. The molybdenum blue-ascorbic acid (MA) is the most commonly used method besides the malachite green (MG) method to determine the amount of phosphate in soil samples. [5]

Experimental

1 CAL-Extraction

Reagents:

Calcium Lactate
Calcium Acetate
Acetic Acid (100%)

CAL-Extraction solution:
Dissolve 15.4 g calcium lactate for soil analysis and 15.4 g calcium acetate for soil analysis separately in 300 ml of water for analysis whilst warming. Combine the two solutions in a 1-l volumetric flask and add 17.9 ml acetic acid 100 %. Allow to cool, make up to the mark with water for analysis and mix well.

Sample preparation:
Air-dry the soil sample, subsequent to removing coarse particles such as stones and plant material. Sieve the sample through 2 mm mesh.
Place 5 g of sample in a separating flask of approx. 300 ml volume and add 100 ml of extraction solution. Shake the closed flask overnight at 175 shakes per minute.
Filter the extract through a folded filter and dispose of the first 10 ml filtrate and are stored for future use in 50ml CornigTM Falcon tubes.

Analysis:
Samples containing more than 14 mg/l P2O5 should be diluted with an extraction solution. If the extract has a yellow color, it should be measured, without the addition of reagent, as a blank and the result subtracted from the sample value.

Determination of orthophosphate through molybdenum Blue method

Preparation:

Reagents:
Sulfuric acid (98%)
Ascorbic acid
Ammonium molybdate
Antimony potassium tartrate
Potassium dihydrogen phosphate
Sodium thiosulfate pentahydrate
Anhydrous Sodium carbonate

Preparation of reaction solutions:

Sulfuric Acid solution 1,, c(H₂SO₄) - 9 mol/l
Fill 250ml H₂O dest. into a 500ml beaker and under continuous stirring and cooling carefully add 250ml of conc. sulfuric acid.
Mix well and let cool down to room temperature.

Sulfuric Acid solution 2,, c(H₂SO₄) – 3,5 mol/l Fill 153ml H₂O dest. into a 500ml beaker and under continuous stirring and cooling carefully add 97ml of the sulfuric solution 1 (9mol/l). Mix well and let cool down to room temperature.

Sulfuric Acid solution 3,, c(H₂SO₄) – 2,5 mol/l
Fill 542ml H₂O dest. into a 1l beaker and under continuous stirring and cooling carefully add 208ml of the sulfuric solution 1 (9mol/l). Mix well and let cool down to room temperature.

MA solution
Dissolve 6g of ammonium molybdate [(NH₄)₆Mo₇O₂₄ * 4 H₂O] in 125ml H₂O dest. and in a separate flask 0,1454g antimony potassium tartrate [K(SbO)C₄H₄O₆ * ½ H₂O) in 50ml H₂O dest. Add the ammonium molybdate solution to 500ml of the previously prepared sulfuric acid solution 3 while stirring, followed by the addition of the antimony potassium tartrate solution and mixed well and filled up to 1000ml Dissolve 1,056g ascorbic (C₆H₈O₆) in 200ml of the acidic molybdenum antimony solution (The solution is stable for up to two weeks if stored in a Winchester bottle when cooled)

Orthophosphate stocksolution,, = 100 mg/l
Dry a few grams of potassium dihydrogen phosphate (KH₂PO₄) at 105°C to constant mass. Dissolve 0,439g KH₂PO₄ in 500ml in a 1l-volumetric flask, add 25ml of the sulfuric acid solution 2 and fill up to 1l with H2O dest.

Orthophosphate standardsolution,, = 4 mg/l
Transfer 10ml of the orthophosphate stock solution into a 250ml volumetric flask and fill up to the mark. The solution should be prepared on the same day as it is used.

All flask and glassware were cleaned using phosphate-free detergent and optionally can be cleaned through a HCl-bath (10%) for several hours.

Measurement with Absorbance Plate Reader (96) Every sample was measured as a triplet using a Accuris™ SmartReader™ 96 microplate absorbancy reader at 700nm to determine the ortho-phosphate concentration in the soil samples. For P determination in soil samples with the MA colorimetric method 40 μL of soil CAL extract was mixed with 20 μL MA reagent in each well of 96-well microplate and shaken for 1 min, then 140 μL aliquot of deionized water added into every well. Samples' P determinations were assigned to rows 2–8 per microplate with 3 -well repeats each soil extract. The first row was for the standard solutions containing 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg L−1 P with 40 μL standard added per well. [4]

Table 1: Values of ortho-P conc. in [mg/kg] are presented as mean with standard deviation ± SD (n=3)

Results and Discussion

Soil samples	96-Well MA			96-Well MA			96-Well MA
Field 1		SD	Field 2	Conc. [mg/kg]	SD	Field 3	Conc. [mg/kg]	SD
1		1,73	19	16,01	0,35	37	14,04	0,83
2	14,53	0,15	20	19,91	0,16	38	11,63	0,15
3	17,83	1,28	21	25,74	2,28	39	20,35	0,38
4	16,74	0,36	22	22,81	0,96	40	16,22	0,47
5		0,18	23	18,08	1,32	41	18,26	0,79
6	22,79	0,47	24	16,14	0,80	42	9,97	0,80
7	20,00	0,23	25	22,44	1,17	43	20,56	1,30
8		0,99	26	22,22	0,72	44	20,56	0,33
9		0,16	27	15,72	0,30	45	17,31	0,22
10	21,48	1,19	28	16,55	0,74	46	13,60	0,27
11	27,07	2,53	29	20,66	0,07	47	12,59	2,95
12	19,69	1,06	30	22,10	0,25	48	17,23	0,30
13	17,81	1,56	31	18,94	0,35	49	16,27	0,06
14	16,84	1,87	32	20,92	1,16	50	14,61	0,04
15	15,76	0,19	33	25,26	0,30	51	12,38	1,76
16	19,03	0,94	34	24,85	1,49	52	14,91	0,22
17	22,93	0,77	35	22,66	0,53	53	20,77	1,31
18	19,43	0,45	36	20,46	0,53	54	16,07	0,59

The amount of ortho-phosphate in the soil samples is ranged between approximately 10 and 27 mg/kg, which coincides well with a study done by Vienna University of Technology in collaboration with the Ministry of Life in Austria, which would put our examined fields in category B considering the P-concentration. [6][7]
In field 1, the samples 1 to 6 were taken in close proximity to a nearby river, samples 7 to 12 were collected in an area of the field rich in vegetation and samples 13 to 18 from a part of the field where plant growth was visibly inhibited. A trend, depicting a correlation between the amount of vegetation present and the concentration of ortho-P in the soil, is recognizable but to make a clear statement about causality, more samples need to be obtained and measured accordingly. In addition, the theory, that a nearby body of water could be able to leech of soluble phosphate out of the soil, is a possibility, but as stated previously, to make any kind of verification, measuring more samples is necessary.
In field 2, the degree of vegetation was pretty homogenous and therefore no noticeable trends in difference of soil P concentration are evident, but still the values are in the same range as in field 1.
Field 3 on the other hand is the only field not commercially used for agricultural purposes, with ortho-phosphate concentrations in the lower range of the spectrum, suggesting an obvious correlation between the concentration of soluble phosphate in the soil and the rate of plant growth.
With sufficient time and funds, the scope of this project could be expanded to monitoring the seasonal development of P concentration in agricultural land over different time periods and how different factors like climate, precipitation and the level of cultivation impact the amount that is quantifiable.

References

[1] doi: 10.3390/agronomy9010029
[2] https://doi.org/10.1007/BF00748590
[3] doi: 10.1042/bj0370256
[4] https://doi.org/10.1016/j.microc.2019.01.002
[5] https://doi.org/10.1080/00103629709369813
[6] http://www.vdlufa.de/Dokumente/Veroeffentlichungen/Standpunkte/2018_Standpunkt_P-Duengung.pdf
[7] https://info.bmlrt.gv.at/dam/jcr:2eb21ff4-2f2c-4a5b-9353-6ed56fcd1118/Die%20%C3%B6sterreichische%20Phosphorbilanz_Endbericht_27%202%2014.pdf

Team:NAWI-Graz/excellence