MACROELEMENTS AND HEAVY METALS CONTENT IN PANICUM VIRGATUM CULTIVATED ON CONTAMINATED SOIL UNDER DIFFERENT FERTILIZATION

Abstract

Heavy metal contamination of soils is a major problem worldwide. As a result, arable land polluted with heavy metals is unsuitable for food production. Utility of energy crops which allow the commercial exploitation of these soils by establishing biofuel feedstock production systems can offer a solution. Additionally, plant cultivation offers opportunities for site remediation.
Field experiments have been performed on heavy metal contaminated arable soil located in southern Poland, in the vicinity of a former smelting factory. Although heavy metal concentration exceeded standards, the area has been used for agricultural purposes. Experiments involved testing Switchgrass (Panicum virgatum) cultivated with standard NPK fertilizers and commercially available microbial inoculum. Biomass water, macronutrients (N, P, K, Mg, Ca) and heavy metal (Cd, Pb, Zn) content in aboveground plant organs were assessed at the end of two growing seasons.
Switchgrass biomass water content was higher after the second year for nearly 40%. Additionally, after the first as well as the second year fertilizers increased it. Magnesium content, essential in chlorophyll biosynthesis, was higher in the first year and additionally more evident in fertilized variants after every year. Heavy metals accumulation in aboveground organs was lower after the second year compare with the first year. Similar trend was observed for Ca and N plant accumulation. However P and K accumulations were higher after the second year of experiment.
In conclusion, due to acclimatization, switchgrass reduce heavy metal uptake, what could result in increase of two biogenic elements (P, K) essential in plant growth.

Full text

Agriculture & Forestry, Vol. 63 Issue 1: 69-76, 2017, Podgorica
69
DOI: 10.17707/AgricultForest.63.1.08
Marta POGRZEBA, Szymon RUSINOWSKI, Jacek KRZYŻAK
1
MACROELEMENTS AND HEAVY METALS CONTENT IN
PANICUM VIRGATUM CULTIVATED ON CONTAMINATED
SOIL UNDER DIFFERENT FERTILIZATION
SUMMARY
Heavy metal contamination of soils is a major problem worldwide. As a
result, arable land polluted with heavy metals is unsuitable for food production.
Utility of energy crops which allow the commercial exploitation of these soils by
establishing biofuel feedstock production systems can offer a solution.
Additionally, plant cultivation offers opportunities for site remediation.
Field experiments have been performed on heavy metal contaminated
arable soil located in southern Poland, in the vicinity of a former smelting
factory. Although heavy metal concentration exceeded standards, the area has
been used for agricultural purposes. Experiments involved testing Switchgrass
(Panicum virgatum) cultivated with standard NPK fertilizers and commercially
available microbial inoculum. Biomass water, macronutrients (N, P, K, Mg, Ca)
and heavy metal (Cd, Pb, Zn) content in aboveground plant organs were assessed
at the end of two growing seasons.
Switchgrass biomass water content was higher after the second year for
nearly 40%. Additionally, after the first as well as the second year fertilizers
increased it. Magnesium content, essential in chlorophyll biosynthesis, was
higher in the first year and additionally more evident in fertilized variants after
every year. Heavy metals accumulation in aboveground organs was lower after
the second year compare with the first year. Similar trend was observed for Ca
and N plant accumulation. However P and K accumulations were higher after the
second year of experiment.
In conclusion, due to acclimatization, switchgrass reduce heavy metal
uptake, what could result in increase of two biogenic elements (P, K) essential in
plant growth.
Keywords: Switchgrass, Heavy metals, Macronutrients, Inoculum,
Biomass composition.
INTRODUCTION
Heavy industry (i.e., smelters, coal mine) is main emitter of heavy metals
(HMs) to the environment. The most dangerous directly for human health is
spreading HMs to vicinity agricultural areas where it can be introduce to food
1
Marta Pogrzeba (corresponding author: mag@ietu.katowice.pl), Szymon Rusinowski, Jacek
Kryżak Institute for Ecology of Industrial Areas, Katowice, POLAND
Paper presented at the 7th International Scientific Agricultural Symposium "AGROSYM 2016".
Notes: The authors declare that they have no conflicts of interest. Authorship Form signed online.

Pogrzeba et al.
70
chain by accumulation in consumed plant organs (Järup, 2003). Such
contaminated arable lands should be excluded from agricultural production. The
alternative for such production can be establishing biofuel feedstock production
systems on those areas. Except biomass production for energy purposes there is
additional benefit in those land management what protect soil from erosion
(Meerset al., 2010). Mineral macro- and micronutrients are essential in plant
development and proper growth of plants is undeniable depends on its content in
soil. Mineral macronutrients are need for plants in higher amount in comparison
to micronutrients. Mineral macronutrients can be divided in to two groups:
primary mineral macronutrients (N, P, K) and secondary mineral macronutrients
(Ca, Mg, Fe, S) (Tripathi et al., 2014). There are reports indicates negative
impact of HM on macronutrients accumulation in above ground organs in
agricultural plant as well as in plant cultivated for medical purposes (Bello et al.,
2004, Siedlecka, 2014). There is a dearth of papers concerning on macronutrients
status in energy crop cultivated HMs contaminated soils. Panicum virgatum
(Switchgrass) is C4 photosynthesis perennial grass belongs to second generation
energy crops, however it can be also cultivated for feed purposes. It is originated
from prairie of Northern America where it is dominant species. Switchgrass due
to those features is plant very resistant to drought conditions (Parrish and Fike,
2005).
The aim of the study was to describe relationships in HMs and mineral
macronutrients accumulation in fertilized Switchgrass during two growing season
(2014 and 2015) after plantation establishment. Plants were treated with standard
NPK fertilizer and commercial available microbial inoculum (EmFarma,
Probiotics Magdalena Górska, Poland).
MATERIALS AND METHODS
Site description
The trial was established in Bytom (Upper Silesia Region, 50°20'43.0"N
18°57'19.6"E, Poland) on arable land contaminated with HMs. Soil was
contaminated over the last century due to dust fall with HMs deposition (Zn, Cd
and Pb) emitted by already non-existent Zn/Pb smelter. The climate conditions at
the site are moderate, with monthly average temperature and precipitation
presented in Figure 1 (based on Institute of Meteorology and Water Management
data). Experiment design
Switchgrass plots were established at the beginning of May 2014 from the
seedlings obtained from seeds, pre-cultivated in growing room. On each of the
three plots 49 plants were planted with density of 3plants per 1 m2. Due to high
soil homogeneity (Tab. 1) and apprehension of uncontrolled fertilizer application
pseudo-replication were performed. On each plot four section were distinguish
(Fig. 2): edge plants excluded from further analysis(i) and three section (pseudo-
replication) from which samples were taken(ii). Each plot was treated in a
different way and 4 meter buffer zone between each experimental plot was set:

Macroelements and heavy metals content in Panicum virgatum cultivated on …
71
•P - C - control (no treatment);
•P - NPK - NPK standard fertilization, applied directly to the soil once before
planting (nitrogen 70 kg ha-1, phosphorus 30 kg ha-1 as P2O5 and potassium 45 kg
ha-1 as K2O);
•P - INC - commercial microbial inoculum (EmFarma, ProBioticsPolska
Magdalena Górska, Poland) applied on seedlings roots before plantation and on
the leaves as aerosol in the middle of every month during the growing seasons
(from May to September 2014 and 2015).
Initial soil samples were collected before planting, one from each plot sections.
The plant samples for HMs and mineral macronutrients analysis were collected
in the middle of October each year: one sample were collected from the middle
plot section and two samples were collected from upper and down plot section.
Figure 1. Meteorological data for growing seasons 2014-2015
Soil and plant samples analysis
Soil pH was measured in H2O (1:2.5 m/v) with a combination
glass/calomel electrode (OSH 10-10, METRON, Poland) and a pH-meter (CPC-
551, Elmetron, Poland) at 20°C.
The conductivity was determined by an ESP 2ZM electrode (EUROSENSOR,
Poland) according to the Polish norm PN-ISO 11265:1997.
Soil texture was evaluated by the hydrometric method according to the
Polish norm PN-R-04032:1998.
The total content of metals in the soil and plant tissues was obtained using
hot plate digestion (HNO3 and HClO3, ratio 4:1) and flame atomic absorption
spectrometry (SpektrAA 300, Varian INC., USA).
Total nitrogen content (N) in plant (PB.06 edition 1 from 2011.09.01) and soil
(ISO 13878:1998), available phosphorus (P) in plant (PB.03 edition 1 from
2011.09.01) and soil (PN-R-04023:1996) as well as potassium (K) content in

Pogrzeba et al.
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plant (PB.01 edition 1 from 2011.09.01) and soil (PN-R-04022:1996) samples
was assessed in Institute for Ecology of Industrial Areas laboratory.
Soil organic matter (OM) was measured by loss on ignition as follows: air
dry soil was sifted to pass a 2 mm sieve, dried at 105°C for 24 h and then (5 g)
treated with 550°C for 4 h. Soil organic carbon (C-org) was assessed according to
PN-ISO 14235:2003.
Figure 2. Section on plot distinguished for sample collection. Red - section
excluded from sampling, green – section where two samples were collected,
yellow – section where one sample was collected
Statistical analysis
Data were analyzed using a one-way ANOVA, followed by a post hoc
comparison using the Fisher LSD test (P < 0.05). Statistical analyses were
performed using Statistica 10 (Statsoft, USA). Spider charts were constructed
using Excel MS Office (Microsoft, USA) and standardization of data for charts
construction were performed using Statistica 10 Software (Statsoft, USA).
RESULTS AND DISCUSSION
Results of soil analyses indicate that among experimental plots there were
no significant differences between measured parameter, except Zn (Tab. 1). Soil
Zn content was slightly higher on P – INC plot when compare to control. There
were no significant differences between P – NPK plot and P – INC plot. Soil
texture on the experimental field was classified as silty loam. According to
obtained results and regulation of Polish law (D.2002. r.165 poz.1369) soil HMs
content exceed the limits defined by government regulation. In this case
experimental area should be classified as marginal, moreover whole agriculture
production should be abandoned (Gopalakrishnan et al., 2011). Spider charts
(Fig. 3) were used as a tool for assess values pattern changes of measured
parameters in above ground organs of Switchgrass among different treatments, as
well as annual changes for two growing seasons (2014 and 2015). Spider charts
can be divided in to three representative sections: water with secondary

Macroelements and heavy metals content in Panicum virgatum cultivated on …
73
macronutrient content (Ca, Mg) (1), primary macronutrients content (N, P, K) (2)
and HMs content (Zn, Cd, Pb) (3). Overall, the highest HMs accumulation in
above ground organs were found for control plants and it was conditioned by Cd
accumulation in the first growing season. The lowest values of Mg and Ca for
control plants could be associated with that highest HMs uptake. Hermans et al.
(2011) reported that in Arabidopsis thaliana old leaves the content of Cd was
lower forplants treated with Cd and Mg, when compare to plant treated only with
Cd. It can be assumed with agreement to presented results that higher Mg content
can reduce Cd accumulation in elder plants. It is well known that magnesium
plays significant role in chlorophyll biosynthesis due to this is essential for
photosynthesis process (Cakmak, 2014). Other measured parameters were equal
among each treatment in the first growing season. In the second growing season
it was found, that there is similar trend among treatments for Mg and Ca
accumulation, however it was found that those parameter values are lower when
compare to the second growing season.
Table 1. Soil physico-chemical characteristic, heavy metal content and
macronutrients content in initial soil samples
Soil physico-chemical characteristic
pH (H2O)
pH (KCl)
EC (µS cm-3)
OM (%)
C-org (%)
P - C
6.49 ± 0.03a
5.97 ± 0.05a
89.92 ± 1.41a
5.39 ± 0.02a
2.18 ± 0.03a
P - NPK
6.57 ± 0.04a
6.06 ± 0.07a
82.28 ± 2.80a
5.52 ± 0.06a
2.11 ± 0.03a
P - INC
6.57 ± 0.03a
6.12 ± 0.02a
89.33 ± 2.89a
5.44 ± 0.06a
2.10 ± 0.01a
Soil heavy metals content
Pb (mg kg-1)
Cd (mg kg-1)
Zn (mg kg-1)
P - C
514.77 ± 14.26a
17.94 ± 0.05a
1659.50 ± 7.22b
P - NPK
487.30 ± 3.18a
18.06 ± 0.05a
1700.00 ± 6.35ab
P - INC
496.50 ± 4.33a
18.02 ± 0.11a
1750.33 ± 34.04a
Soil primary mineral macronutrients content
Ntotal (%)
P (mg kg-1)
K (mg kg-1)
P - C
0.15 ± 0.00a
834.30 ± 6.64a
950.30 ± 17.32a
P - NPK
0.15 ± 0.00a
833.20 ± 8.66a
970.60 ± 1.15a
P - INC
0.15 ± 0.00a
835.87 ± 15.90a
1014.27 ± 24.62a
Soil secondary mineral macronutrients content
Fe (mg kg-1)
Mg (mg kg-1)
Ca (mg kg-1)
P - C
9854.50 ± 44.74a
1557.00 ± 30.60a
3015.00 ± 49.07a
P - NPK
10135.00 ± 13.28a
1592.00 ± 4.04a
2998.50 ± 3.17a
P - INC
10219.00 ± 164.93a
1669.33 ± 51.49a
3185.17 ± 98.48a
P – Panicum virgatum, C – control plot; NPK – NPK fertilized plot, INC –
microbial inoculated plot. Values are means ± SE (n=3). A lower case letters (a,
b, c, d) denotes significant differences among soils samples taken from different
plots at P ≤ 0.05 according to Fisher LSD test.

Pogrzeba et al.
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Figure 3. Spider charts constructed on macronutrients, heavy metals and water
content show patterns indicated changes of those parameters; among treatment
(C – control, NPK – NPK fertilizer treated plant, INC – microbial inoculum
treated plant) and growing season (a-2014 and b-2015) for Panicum virgatum(P).
Mg, Ca, P, K, N, Pb, Cd, Zn – elements accumulation in above ground plants
organs. For better data visualization all presented values were standardized. Each
measurement was performed in 5 replicate (n = 5)
It also has been found that there is overall decrease in accumulation of
HMs and N for plants from each plot, except Cd accumulation in NPK treated
plants when compare to the first growing season. Increased K concentration in
plant samples was found for each variant and P was found to be increased on
each plot treated with fertilizer, when compare to the first growing season.
Gonçalves et al. (2009) reported, that higher Cd concentration in hydroponic
solution can decrease K uptake to above ground organs. This paper is with
agreement with obtained results, however the tendency is only contrary to NPK
treated plants. Water content in plant stems was 40% higher for plant in the

Macroelements and heavy metals content in Panicum virgatum cultivated on …
75
growing season 2015 when compare to the growing season 2014 and treatments
slightly increased it. This phenomena can be associated with ability to water
capture, which is correlated to root distribution. It is known that perennial grasses
root system become fully developed after 4th growing season (Ferchaud et al.,
2015). In this case water content could be increased due to annual increase of
roots density. Spider charts with description corresponds to overall annual and
treatment changes in biomass HMs, water and mineral macronutrient content. Its
allows to track changes in tendencies while detailed statistical analysis for
measured parameters is presented in Table 2.
Table 2. Matrix of statistical significant differences among analyzed parameters
presented on spider charts (Fig. 2)
P-C
(2014)
P-NPK
(2014)
P-INC
(2014)
P-C
(2015)
P-NPK
(2015)
P-INC
(2015)
Water content
b
b
b
a
a
a
Mg
bc
ab
a
e
d
cd
Ca
ab
ab
a
b
ab
ab
P
b
b
b
b
a
a
K
b
b
b
a
a
a
N
a
a
a
b
b
b
Pb
a
a
b
c
c
c
Cd
a
bc
ab
c
bc
bc
Zn
a
a
a
ab
ab
b
P – Panicum virgatum, C – control; NPK – NPK fertilized plants, INC –
microbial inoculated plants.
A lower case letters (a, b, c, d, e, f – where “a” corresponds to the highest value
and “f” to the lowest) denotes significant differences among plants elements and
water content taken from different plots at P ≤ 0.05 according to Fisher LSD test.
Each measurement was performed in 5 replicate (n = 5)
CONCLUSION
It could be concluded that HMs in soil affect mineral macronutrients status
in Panicum virgatum, especially Mg and K. Additionally there are differences
mostly in each measured parameters when compare growing seasons. It can be
associated with acclimatization of plant to contaminated site. There is a dearth of
papers concerning mineral nutrients as well as HMs status in the first two years
after establishment, because most of them are focused on fully establishment
trials, where perennial energy crops achieves its maturity after 3rd -4th year after
establishment.
ACKNOWLEDGEMENT
Research financed by the PHYTO2ENERGY project – 7FP EU, Grant
Agreement No. 610797 and IETU statutory funds by Polish Ministry of Science
and Higher Education.

Pogrzeba et al.
76
The authors wish to extend their appreciation to dr Włodzimierz
Majtkowski from Plant Breeding and Acclimatization Institute, National
Research Institute (Poland), for providing seeds of Panicum virgatum.
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