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Utilization of grasses for potential biofuel production and phytoremediation of heavy metal contaminated soils a
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Ronald A. Balsamo , William J. Kelly , Justinus A. Satrio , M. Nydia Ruiz-Felix , Marisa a
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Fetterman , Rodd Wynn & Kristen Hagel
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Department of Biology, College of Liberal Arts and Sciences, Villanova University, Villanova, PA, USA. b
Department of Chemical Engineering, College of Engineering, Villanova University, Villanova, PA, USA. Accepted author version posted online: 07 Jul 2014.
Click for updates To cite this article: Ronald A. Balsamo, William J. Kelly, Justinus A. Satrio, M. Nydia Ruiz-Felix, Marisa Fetterman, Rodd Wynn & Kristen Hagel (2014): Utilization of grasses for potential biofuel production and phytoremediation of heavy metal contaminated soils, International Journal of Phytoremediation, DOI: 10.1080/15226514.2014.922918 To link to this article: http://dx.doi.org/10.1080/15226514.2014.922918
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ACCEPTED MANUSCRIPT Utilization of grasses for potential biofuel production and phytoremediation of heavy metal contaminated soils 1
Ronald A. Balsamo, 2William J. Kelly, 2Justinus A. Satrio, 2M. Nydia Ruiz-Felix, 1Marisa
Fetterman, 2Rodd Wynn, and 2Kristen Hagel
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1
Department of Biology, College of Liberal Arts and Sciences, Villanova University, Villanova,
PA 19085 USA.
2
Department of Chemical Engineering, College of Engineering, Villanova University, Villanova,
PA 19085 USA.
Corresponding author: Ronald A. Balsamo, Department of Biology, Villanova University, Villanova, PA 19085 USA. [email protected]; 610-519-6621
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ABSTRACT
This research focuses on investigating the use of common biofuel grasses to assess their potential as agents of long-term remediation of contaminated soils using lead as a model heavy
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metal ion. We present evidence demonstrating that switch grass and Timothy grass may be potentially useful for long term phytoremediation of heavy metal contaminated soils and describe novel techniques to track and remove contaminants from inception to useful product. Enzymatic digestion and thermochemical approaches are being used to convert this lignocellulosic feedstock into useful product (sugars, ethanol, biocrude oil + biochar). Preliminary studies on enzymatic hydrolysis and fast pyrolysis of the Switchgrass materials that were grown in heavy metal contaminated soil and non-contaminated soils show that the presence of lead in the Switchgrass material feedstock does not adversely affect the outcomes of the conversion processes. These results indicate that the modest levels of contaminant uptake allow these grass species to serve as phytoremediation agents as well as feedstocks for biofuel production in areas degraded by industrial pollution.
Keywords: Phytoremediation, enzymatic hydrolysis, pyrolization, switch grass, timothy grass, lead.
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INTRODUCTION There are over a million hectares of land in the US that currently lies fallow due to past industrial and mining activities. In the case of heavy metals like lead there has been little
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research into the potential use of grasses as phytoremediation agents as they have not been shown to be hyper-accumulators of toxic materials. However, since hyper-accumulators identified thus far are for the most part dicotyledonous species, (Baker and Brooks 1989; Watanabe, 1997) the utilization of these plants in the phyto-extraction of toxic materials from areas of contaminated soils is limited as the harvest will be a singular event with limited opportunity for re-growth and re-establishment as the apical meristems are on the growing tips of shoots. While many if not most grasses are not hyper-accumulators of toxic metals (Baker and Brooks 1989), if they accumulate any metals at all they have a distinct advantage due to their architecture (basal apical meristems) of being able survive and thrive in environments where the loss of significant amounts of above ground biomass does not result in the removal of meristems and death of the organism. Hence repeatedly harvesting above ground leaf material typically does not significantly impact their survival. Thus if species of grasses can be identified that survive and accumulate toxic metals in their leaves in soil (that would preclude the planting of food crops for human or livestock consumption), then significant amounts of biomass could be harvested over several seasons and eventually reduce the amounts of toxic metals. This remediated land could then either be allowed to revert to a natural ecosystem or be productively utilized for agricultural crops. Additionally, the biomass that is harvested over several years in
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ACCEPTED MANUSCRIPT the process of cleaning up the soil could potentially be utilized as feedstock for processes generating biofuels and other potential products, provided the presence of the heavy metals is not detrimental to these processes. Some potential products includes:: sugar via enzymolysis of the grass (for use in the fermentation of ethanol), marketable chemicals produced from grass
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pyrolysis and products utilizing recovered heavy metals. Switchgrass (Panicum virgatum) is a species of grass that has garnered considerable interest in the biofuels industry. It is fast growing, can withstand a variety of adverse environmental conditions from freezing to drought, and is native to most of the continental US. Despite this, it has received little attention as a potential agent for the phytoremediation of lead contaminated soils (Salt et al. 1998, Gleeson, 2007). Traditionally Switchgrass has been used for forage production, soil conservation, and as ornamental grass. With the recent interests in utilizing biomass as an energy source, Switchgrass has gained attention as one of the most fibrous crops for energy production due its hardiness in poor soil and climate conditions, rapid growth, and low fertilization and herbicide requirements (Gupta and Demirbas 2010). The annual production of Switchgrass has been reported to range from 6 to 9 ton/acre/year (Lewandoski et al. 2003) Sources for fuel, other than gasoline, are now actively being sought to meet the world's ballooning energy requirements. Bio-fuels are one potential source of renewable energy that may supplement petroleum-based energy. Bio-fuel feed stocks are often classified into three categories: simple-sugar-derived, starch-derived and lignocellulosic-derived. Lignocellulose (found in corn stover, trees and grasses) is the most abundant renewable source of bio-mass with yearly supplies of approximately 200 billion metric tons worldwide (Watanabe, 1997; Sun and
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ACCEPTED MANUSCRIPT Cheng 2002). Switchgrass is a perennial grass with potential as a source of lignocellulosic biomass in a biofuels process. It may also have potential for assisting with soil remediation, while being grown for use as a biofuels feedstock. It is estimated that there are currently over 2 million acres of unremediated superfund fund sites in the US with lead as the primary contaminate(US EPA 1993; US EPA 1998; Watanabe, 1997)
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Timothy grass (Phleum pretense) is a perennial grass native to central Europe that was introduced to New England in the early 18th century. It is fast growing with a high fiber content and is today grown throughout much of the US as a component feedstock for cattle and horses. Similar to Switchgrass, it is moderately cold and drought tolerant, and can grow in soils with low nutrient content (Claessens et al. 2004). Little work has been conducted on the potential of Timothy grass in phytoremediation but in one study a similar grass (Ryegrass) was found to accumulate heavy metals at much higher levels in the roots versus the leaves (Jones et al. 1973). . The focus of this study primarily is to evaluate the potential of two perennial grasses (Switchgrass and Timothy grass) as agents for phytoremediation of heavy metal contaminated soils. This study used lead as the heavy metal to evaluate since it is arguably the most common and important heavy metal contaminant. The effects of the lead concentration in the soil on the growth rates of the grasses were evaluated. In addition to the plant study, grasses that were grown in lead contaminated soil were used as feedstock for two biomass conversion routes, i.e. enzymatic hydrolysis and fast pyrolysis processes.
The performance of lead-contaminated
switchgrass as conversion feedstock was studied by evaluating the production of sugar from enzymatic hydrolysis and the chemical product distribution in the bio-oil product from fast
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ACCEPTED MANUSCRIPT pyrolysis process. The biomass feedstock was also subjected to mild acid hydrolysis pretreament process prior to the conversion processes.
MATERIALS AND METHODS Plant Materials: 36 trays (3 per treatment, per species) of sufficient volume to
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accommodate 3 kg of soil media (Metromix) were set up in the Villanova Greenhouse and either saturated with distilled water or treated with lead citrate solution as follows: 50, 80,120,200, or 500 mg Pb/kg of soil. 100 seeds of Switchgrass or Timothy grass were sown in each tray and watered 3X per week with tap water for 6 weeks.
Germination and Growth Rates: Germination was determined by counting the number of successful germinations and establishment of plants and are reported here as the average germination rate for each of the 6 soil lead levels. Growth rates were determined by weekly measurements using the highest leaf and measuring from the level of the hypocotyl (root/shoot junction). The effect on germination rates of seeding density was also evaluated in normal and lead-contaminated soil, where seed spacings ranged from 2.5 to less than 1 cm.
Determination of Metals in Plant Material: Lead concentrations were determined by C oven for 48 h and then using an energy dispersive X-ray analysis attachment to a Joel S570 Scanning electron microscope. All elements detected by the device were standardized according to known concentrations of Potassium.
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ACCEPTED MANUSCRIPT Acid Hydrolysis: Switch grass and Timothy grass samples, grown either on normal soil, or grown on soil that has been treated with lead (120g Pb/kg soil), were obtained from Villanova University green house. The sample materials were dried by using an oven at 45oC overnight. A portion of the dried samples were acid hydrolyzed by using 4% phosphoric acid solutions. During hydrolysis, the grass samples were placed in a round bottom flask filled with the acid
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solution, which was then heated and left boiling for one hour. The grass materials were recovered and washed with distilled water three times to remove remaining acid and then dried overnight.
Enzyme Hydrolysis of Leaf Materials: In preparation for the grass enzymolysis, each of three three cellulase-producing fungi (Aspergillus niger, ATCC6275; Trichoderma reesei, ATCC56765; and Humicola grisea, ATCC16298) were grown separately in Potato dextrose medium until 105 spores/ml was achieved, at which point 1 ml of this solution was added to the MS medium to give the desired starting concentration of spores of at least 103 spores/ml. Experiments were then conducted in which Switchgrass and Timothy grass were both used as a cellulose source in sterile Murashige and Skoog medium (Sigma Aldrich, M5519-10L, 040M8803), buffered to pH 4.8 with 3 g/l succinic acid and 9 g/l succinate. Some of the grass, grown on normal soil or on soil that has been treated with lead (120 mg Pb/kg soil), was acid hydrolysed. The 250 ml flasks contained 100 ml media and 2 grams of grass, and were agitated at 120 RPM. Single and mixed cultivation of these organisms were explored in flasks at 25 C, which is the optimum growth temperature for all three fungi. To facilitate spore counting, Flourescein Diacetate (Sigma Aldrich, Lot # 38997A) was used to fluoresce the spores under the
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ACCEPTED MANUSCRIPT microscope. For determination of sugar concentration in each flask, test tubes were setup the same way as detailed in Colorimetric Method for Determination of Sugars and Related Substances (Dubois et al. 1956). Fast Pyrolysis: For preparing the fast pyrolysis reaction, the hydrolyzed and non-
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hydrolyzed grass materials were ground into fine particles.
The fast pyrolysis study was
performed by using a micropyrolyzer, which is CDS Pyroprobe 5200 (CDS analytical, Oxford, PA, USA). The micropyrolyzer is coupled with gas chromatograph/mass spectrometer (HP 6890/HP 5972) for fast pyrolysis vapor product analysis.
Switchgrass and Timothy grass
samples were pyrolyzed at 500oC under and inert atmosphere. The temperature was ramped at 20 C/ms and kept at 700oC for 10 s. The pyrolysis vapors were directly swept into the GC/MS using helium as the carrier gas. The chromatographic separation of pyrolysis products was performed using an alloy capillary column having high thermal resistance (30 m x 0.250 mm and 0.250 mm film thickness. An injector temperature of 300oC and a split ratio 1:100 was used. The GC oven temperature program began with a 3 min hold at 35 oC followed by heating to 130oC at 5oC/min and then to 250oC at 20oC/min RESULTS Effectiveness of Grass in Remediating soil: Results demonstrate that both switch grass and Timothy grass have the capacity to survive under conditions that far exceed the minimum levels deemed toxic by the Environmental Protection Agency. Figures 1 a and 3a show average plant height (each data point is an average of 30 results), at the end of a 6 week growing period, in soil with different lead concentrations. A two-way Anova analysis of this data indicates that plant height is significantly effected by both grass type (p-value < 0.0001) and soil lead concentration
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ACCEPTED MANUSCRIPT (p-value < 0.0001). A follow up one-way Anova analysis indicates that Switchgrass plant height is significantly lower (as compared to the control soil) when lead levels are ≥ 120 mg/kg of soil. The p-value for soil at 120 mg/kg of soil versus control soil was 0.0069, while p-values for higher soil lead levels were < 0.0001.
For Timothy grass, one-way Anova analysis indicates
that plant height is significantly lower when lead levels are ≥ 50 mg/kg of soil (p-vlaue =
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0.0088), The p-value for soil at 80 mg/kg of soil versus control soil was 0.0037, while p-values for higher soil lead levels were < 0.0001.
Figures 1 b and 3b show average germination rates (each data point is an average of 3), at the end of a 6 week growing period, in soil with different lead concentrations. A two-way Anova analysis of this data indicates that germination rate is significantly effected by both grass type (pvalue < 0.0001) and soil lead concentration (p-value < 0.0001). With only three replicates, the GLM statistical procedure was used to confirm this analysis by producing identical p-values. A follow up one-way Anova analysis indicates that Switchgrass germination rate is significantly lower than in control soil only when lead levels are ≥ 500 mg/kg of soil (p-vlaue = 0.0084). For Timothy grass, one-way Anova analysis indicates that germination rate is significantly lower when lead levels are ≥ 200 mg/kg of soil (p-vlaue = 0.047). These plant height and germination rate results indicate that Timothy grass growth is more sensitive than Switch grass growth to lead concentrations in contaminated soils. For Switchgrass, germination rates and growth rates are only impacted by lead concentrations in the soil that are triple the minimum standard set by the EPA
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ACCEPTED MANUSCRIPT Studies utilizing a scanning electron microscope equipped with energy dispersive X-ray diffraction indicate that appreciable quantities of lead are incorporated into both root (Fig. 2b and 4b) and leaf (Fig. 2a and 4a) tissues. Each data point on these graphs represents an average of 12 experimental values. A two-way Anova analysis of this data indicates that lead uptake by the roots is significantly effected by soil lead concentration (p-value < 0.0001), however there is
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no statistical difference in uptake of lead by roots between the two grass types (p-value = 0.46). A follow up one-way Anova analysis indicates that lead uptake by roots in Switchgrass is the same (p-value = 0.687) with soil lead levels of 50 and 80 mg/kg of soil, but then increases significantly at levels of ≥ 120 mg/kg of soil. In comparing lead uptake at a soil lead level of 50 versus higher levels, the p-values were all ≤ 0.0002.
For Timothy grass, one-way Anova
analysis indicates that for all soil lead levels > 50 mg/kg of soil, lead uptake in the roots exceeds the uptake observed at 50 mg/kg of soil. The p-value was 0.0195, when comparing lead uptake by roots in soil at lead concentrations of 50 and 80 mg/Kg soil.. The p-values were < 0.0001 when comparing uptake in soil with lead level of 50 mg/kg of soil versus soil with lead level of ≥ 120 mg/kg of soil.
A two-way Anova analysis of this data indicates that lead uptake by leaves is significantly effected by both grass type (p-value < 0.0001) and soil lead concentration (p-value < 0.0001). For Switch grass, one-way Anova analysis indicates that for all soil lead levels > 50 mg/kg of soil, lead uptake in the leaves exceeds the uptake observed at 50 mg/kg of soil (p-values ≤ 0.002). One-way Anova analysis indicates that lead uptake by leaves in Timothy grass is the same (p-value = 0.12) with soil lead levels of 50 and 80 mg/kg of soil, but then increases
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ACCEPTED MANUSCRIPT significantly at levels of ≥ 120 mg/kg of soil. In comparing lead uptake at a soil lead level of 50 versus lead levels ≥12
/K
l, the p-values were all ≤ 0.0002.
Lead uptake into leaves and roots increases with lead concentration in the soil. At all lead concentrations, the lead content in the roots exceeds that in the leaves as expected. For Timothy
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grass, however, substantially more lead accumulated in the roots as compared to the leaves and leaf accumulations were substantially less than for Switchgrass (Figs. 4 a,b). Although not shown in Figures 2 or 4, a concentration of lead in the leaves or roots of the control grass, grown in soil without lead present, was not detectable. Finally, it was observed that germination rates for Switchgrass grown in normal soil and that contaminated with lead was effected in the same way by the spacing of the planted seeds. At 2.5 cm seed spacing, germination rates averaged 68% while at 1 cm spacing the germination rates averaged 31%.
Enzymatic Hydrolysis of Grass with Select Fungal Cultures: Sugar production from fungal fermentations using Switchgrass (“SG”)
(“TG”) was measured versus
Timothy
fermentation time. For both grasses, the highest sugar levels were achieved when the grass was w
lc ll
c
l
(“L”), p
l (“SGL AH ALL”) p
w (“ALL”).
c
c F
l
(“AH”),
Switchgrass (Figure 5), these
18 mg/l of sugar after 27 days of fermentation, when the
fermentation was finalized despite still increasing sugar levels. For Timothy grass, these same conditions produced 25 mg/l of sugar after 35 - 40 days of fermentation, after which sugar levels began to decline. The results from both of these experiments indicate that production of sugar by
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ACCEPTED MANUSCRIPT these three fungal species is not adversely effected by the lead taken up by the grasses. In fact, Figure 5
w
c
l
l p c
(“AN”, HG”
“TR”) c
ll p
c
slightly more sugar when digesting the grass that is higher in lead content. In addition, the results from the experiments with these two grasses supports the importance of acid hydrolysis pre-
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treatment and co-cultivation of all three cultures in achieving maximal sugar levels.
Work on Biomass Pre-treatment and Fast Pyrolysis: Fast pyrolysis of biomass carbohydrates and lignin produces valuable products in the forms of gas, oil, and solid char; these products may be used as a heat source, fuel, or feedstock for petrochemicals and other applications (Yanik, et al. 2007). Although similar in appearance to crude petroleum oil, bio-oil is chemically different. It is comprised of many oxygenated species such as carboxylic acids, hydroxyaldehydes, hydroxyl ketones, anhydro sugars, phenolic compounds and lignin fragments. Many of these compounds are used in a variety of manufacturing industries, such as chemical and pharmaceutical. The chemical composition of bio-oil depends on several factors such as – lignocellulosic composition of biomass, effect of minerals, residence time of the pyrolysis vapors pertaining to particular reactor configuration and the reaction temperature. It is known that cellulose and hemicellulose mainly result in the formation of furan/pyran ring derivatives, sugar/anhydro sugars and other low molecular weight compounds like acetic acid, acetol etc. whereas lignin mainly yields phenolic compounds and lignin oligomers. Figure 6 shows the chromatograms of the bio-oil product distribution from fast pyrolysis performed on dry switch grass (A), dry switch grass contaminated with lead (B), acid hydrolyzed switch grass with an acid solution of 4% H3PO4 (C), acid hydrolyzed switch grass contaminated
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ACCEPTED MANUSCRIPT with lead with an acid solution of 4% H3PO4 (D), enzymatic hydrolyzed switch grass (E), and enzymatic hydrolyzed switch grass contaminated with lead (F). Comparing figures A and B, figures C and D, and figures E and F, it can be seen that the chemical product distributions of the bio-oil from fast pyrolysis remains similar between the two feedstocks, showing that the lead present in the contaminated grass has only minimal effect, if any, regardless of the processes
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that the grasses were subjected to prior to fast pyrolysis. The comparison of figures A, C and E shows that, the acid hydrolysis and enzymatic hydrolysis treatments do have a larger affect effect on the chemical product distribution of the bio-oil. This effect can also be seen with the switchgrass that contains lead.
DISCUSSION There has been little research into the potential use of grasses as phytoremediation agents for heavy metals as they have not been shown to be hyper-accumulators of toxic materials. If these grasses accumulate any metals at all they have a distinct advantage due to their architecture (basal apical meristems) of being able survive and thrive in environments where the loss of significant amounts of above ground biomass does not result in the removal of meristems and death of the organism. Hence, by repeatedly harvesting above ground leaf material significant amounts of biomass could be harvested over several seasons and eventually reduce the amounts of toxic metals to the point where the affected land could either be allowed to revert to a natural ecosystem or be productively utilized for agricultural crops. The results from this research show that Timothy grass growth is more sensitive than Switch grass growth to lead concentrations in
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ACCEPTED MANUSCRIPT contaminated soils, and that Switchgrass, germination rates and growth rates are only impacted by lead concentrations in the soil that are triple the minimum standard set by the EPA . Lead uptake into leaves and roots was higher with Switchgrass as compared to Timothy grass at all soil lead concentrations. At a soil lead concentration of 120 mg Pb/kg for Switchgrass, the lead concentration in the grass leaves was 0.028 ± 10% of leaf dry weight.
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Assuming an optimal switch grass planting density gives rise to 7.5 ton/acre/year (Lewandoski et al 2003), it is estimated from the data in Figure 2a that that 0.02 ton/acre/year can be removed in the harvested leaves from soil contaminated at 80 – 120 mg Pb/kg soil. This calculation further assumes that the lead concentration in 6-8 foot tall Switchgrass (typical field plant height at harvest) is comparable to that measured in the approximately 1 foot tall Switchgrass grown during this study. This contaminated biomass that harvested over several years in the process of cleaning up the soil could potentially be utilized as feedstock in the production of biofuels. Cellulose and hemicellulose and pectins can be broken down by enzymes into glucose or other sugars that can then be fermented by yeast to produce bio-ethanol. Several organisms, including White rot fungi Phanerochaete chrysoporium (Alam et al. 2009) and Humicola grisea (ATCC product data sheet # 16298, 2011) have been reported to produce lignases. H. Grisea also produces pectinase. Trichoderma reesei (ATCC 13631) is a genetically modified fungus known for producing a slew of cellulase enzymes including: endonuclease (Kyriacou et al. 1987), cellobiohydrolase (Ehsani et al. 1996), xylase (Bailey et al. 1993), galactosidase (Kristufek et al. 1994), and mannase (Stralbrand et al. 1993). Aspergillus niger has been reported to produce low levels of l c
b
l
l
β-glucosidase (Kang et al. 2004). Trichoderma reseei,
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ACCEPTED MANUSCRIPT Aspergillus niger and Humicola grisea all grow optimally at similar conditions; temperature near 25°C, a Potato Dextrose nutrient mix at pH of 5-6 [ATCC, 2011]. This provides then the possibility for co-cultivation to produce the fungi and the cellulosic enzymes made by these fungi. Some research has focused on evaluating co-cultivation of cellulose producing organisms. In a published study, enhanced enzyme levels were achieved when T. reesei was co-cultured
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with A. niger using bagasse, corncobs and saw dust as the substrates in solid state fermentation (Maheshwari et al. 1994; Madamwar and Patel 1992). A similar result was achieved in this research, as indicated by Figures 5a and 5b. For both Timothy and Switchgrass, the highest sugar levels were achieved when pretreated with acid hydrolysis and co-cultivated with Trichoderma reseei, Aspergillus niger a
H
c l
pH
2 C.
Previous work has clearly demonstrated that the inorganic compounds found indigenously within biomass promote the formation of gaseous species and char at the expense of bio-oil yield. Raveendran et al. (1995) studied the pyrolysis of twelve different biomass feedstock and observed that the yield of pyrolysis vapors, devolatilization rate and the initial decomposition temperature increased whereas the char yield decreased on demineralization of the biomass samples except for the case of rice husks, groundnut shell and coir pith. The exceptional behavior of these biomass sources was attributed collectively to their high contents of lignin, potassium and zinc. Potassium and zinc are known to catalyze the char gasification reactions which are prevented when they are removed by demineralization (Yuki and Shafizedeh 1984; Raveendram et al. 1995; Patwardhan et al. 2010). In this study, the product distribution of the fast pyrolysis was found to be not affected by the lead that was present in the contaminated
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ACCEPTED MANUSCRIPT Switchgrass; however, the acid hydrolysis and enzymatic hydrolysis treatments do affect the product distribution of the fast pyrolysis. For example, fast pyrolysis of acid hydrolyzed switch grass results in the formation of new compounds, including furfural, levoglucosenone, tetradecanal and stearic acid. On the other hand, when the switch grass had been treated with enzymatic hydrolysis, 1% of 2,6-dimethoxyphenol is produced and 1,4:3,6-Dianhydro-alpha-D-
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Glucopyranose is no longer formed. In conclusion, our studies demonstrate that Switchgrass and to a lesser extent Timothy grass may have great potential as a phytoremediation agents for heavy metal contaminated soils. Germination and growth rates were only inhibited at the highest concentration used in this study. Further, SEM-EDAX results suggest that appreciable quantities of lead accumulate in both root and leaf tissues. Based on the germination rates and lead uptake observed in this study, Switchgrass seems to have much more potential than Timothy grass as a phytoremediation agent for lead contaminated soils. The results for enzymatic hydrolysis suggest that production of sugar by the three fungal species evaluated in this study was not adversely effected by the lead taken up by the grasses. The product distribution of the fast pyrolysis was not affected by the lead that is present in the contaminated Switchgrass. Future efforts will focus on optimizing lead uptake by considering the effect of plant height on lead uptake rates and on optimizing the biofuels processes to maximize productivity with respect to the biofuels and chemicals produced from the contaminated Switchgrass and to maximize lead removal and collection. Our studies may potentially result in methods that would allow for the productive use of millions of hectares worldwide that are currently unusable for production of feedstocks for biofuels processes while at the same restoring those lands to their native state.
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ACCEPTED MANUSCRIPT Figure Captions
Figure 1. Switchgrass (a). Average height for plants grown for 6 weeks under various Pb concentrations (25 plants per tray X 12 replicates). (b) Percent germination of seeds planted in
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media mix pre-treated with various concentrations of Pb (CO = Controls, T50= 50 mg Pb/kg media mix, T80 = 80 mg Pb/kg media mix, T120 = 120 mg Pb/kg mediamix, T200 = 200 mg Pb/kg media mix, T500 = 500 mg Pb/kg media mix).
Figure 2. Switchgrass a) Average Pb concentrations in leaf tissues (% dry weight) after 6 weeks exposure to Pb standardized to potassium. (b) Average Pb concentrations in root tissues (% dry weight) after 6 weeks exposure to Pb standardized to potassium.
Figure 3. Timothy grass (a). Average height for plants grown for 6 weeks under various Pb concentrations (25 plants per tray X 12 replicates). (b) Percent germination of seeds planted in media mix pre-treated with various concentrations of Pb CO = Controls, T50= 50 mg Pb/kg media mix, T80 = 80 mg Pb/kg media mix, T120 = 120 mg Pb/kg media mix, T200 = 200 mg Pb/kg media mix, T500 = 500 mg Pb/kg media mix.
Figure 4. Timothy grass a) Average Pb concentrations in leaf tissues (% dry weight) after 6 weeks exposure to Pb standardized to potassium. (b) Average Pb concentrations in root tissues (% dry weight) after 6 weeks exposure to Pb standardized to potassium.
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Figure 5. Sugar concentration versus time using Switchgrass .
Figure 6. Effect of acid hydrolysis and enzymatic hydrolysis on pyrolyzed switchgrass and
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swithgrass that contains lead. The pyrolysis were performed at 500°C. A: Product distribution from the pyrolysis of dry switch grass, B: Product distribution from the pyrolysis of dry switch grass contaminated with lead, C: Product distribution from the pyrolysis of acid hydrolyzed switch grass with an acid solution of 4% H3PO4, D: Product distribution from the pyrolysis of acid hydrolyzed switch grass contaminated with lead with an acid solution of 4% H3PO4, E: Product distribution from the pyrolysis of enzymatic hydrolyzed switch grass, and F: Product distribution from the pyrolysis of enzymatic hydrolyzed switch grass contaminated with lead. (1) Toluene, (2) Furfural, (3) Furan, (4) Phenol, (5) p-Cresol, (6) 2-Methoxyphenol, (7) Levoglucosenone, (8) 2Methoxy-4-methylphenol, (9) 1,4:3,6-Dianhydro-alpha-D-Glucopyranose, (10) 4-Ethyl2-methoxyphenol, (11) 2-Methoxy-4-vinylphenol, (12) 2,6-Dimethoxyphenol, (13)2,6,6trimethyl-Bicyclo [3.1.1]heptane, (14) 3,7,11,15-Tetramethyl hexadec-2-en-1-ol, (15) Tetradecanal, (16) 2-Propylcyclohexanol, (17) Palmitic acid, (18) 9,12,15-octadecatrien1-ol, and (19) Stearic acid,
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Average Grass Height (cm)
35 30 25 20 15 10 5 0 C0
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Lead level in Soil (mg/kg of soil)
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100 % Germination
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% (DW) Lead uptake in Leaves
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Lead Level in Soil (mg/kg of soil) Figure 2a
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International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Utilization of grasses for potential biofuel production and phytoremediation of heavy metal contaminated soils a
b
b
b
Ronald A. Balsamo , William J. Kelly , Justinus A. Satrio , M. Nydia Ruiz-Felix , Marisa a
b
Fetterman , Rodd Wynn & Kristen Hagel
b
a
Department of Biology, College of Liberal Arts and Sciences, Villanova University, Villanova, PA, USA. b
Department of Chemical Engineering, College of Engineering, Villanova University, Villanova, PA, USA. Accepted author version posted online: 07 Jul 2014.
Click for updates To cite this article: Ronald A. Balsamo, William J. Kelly, Justinus A. Satrio, M. Nydia Ruiz-Felix, Marisa Fetterman, Rodd Wynn & Kristen Hagel (2014): Utilization of grasses for potential biofuel production and phytoremediation of heavy metal contaminated soils, International Journal of Phytoremediation, DOI: 10.1080/15226514.2014.922918 To link to this article: http://dx.doi.org/10.1080/15226514.2014.922918
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ACCEPTED MANUSCRIPT Utilization of grasses for potential biofuel production and phytoremediation of heavy metal contaminated soils 1
Ronald A. Balsamo, 2William J. Kelly, 2Justinus A. Satrio, 2M. Nydia Ruiz-Felix, 1Marisa
Fetterman, 2Rodd Wynn, and 2Kristen Hagel
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1
Department of Biology, College of Liberal Arts and Sciences, Villanova University, Villanova,
PA 19085 USA.
2
Department of Chemical Engineering, College of Engineering, Villanova University, Villanova,
PA 19085 USA.
Corresponding author: Ronald A. Balsamo, Department of Biology, Villanova University, Villanova, PA 19085 USA. [email protected]; 610-519-6621
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ABSTRACT
This research focuses on investigating the use of common biofuel grasses to assess their potential as agents of long-term remediation of contaminated soils using lead as a model heavy
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metal ion. We present evidence demonstrating that switch grass and Timothy grass may be potentially useful for long term phytoremediation of heavy metal contaminated soils and describe novel techniques to track and remove contaminants from inception to useful product. Enzymatic digestion and thermochemical approaches are being used to convert this lignocellulosic feedstock into useful product (sugars, ethanol, biocrude oil + biochar). Preliminary studies on enzymatic hydrolysis and fast pyrolysis of the Switchgrass materials that were grown in heavy metal contaminated soil and non-contaminated soils show that the presence of lead in the Switchgrass material feedstock does not adversely affect the outcomes of the conversion processes. These results indicate that the modest levels of contaminant uptake allow these grass species to serve as phytoremediation agents as well as feedstocks for biofuel production in areas degraded by industrial pollution.
Keywords: Phytoremediation, enzymatic hydrolysis, pyrolization, switch grass, timothy grass, lead.
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INTRODUCTION There are over a million hectares of land in the US that currently lies fallow due to past industrial and mining activities. In the case of heavy metals like lead there has been little
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research into the potential use of grasses as phytoremediation agents as they have not been shown to be hyper-accumulators of toxic materials. However, since hyper-accumulators identified thus far are for the most part dicotyledonous species, (Baker and Brooks 1989; Watanabe, 1997) the utilization of these plants in the phyto-extraction of toxic materials from areas of contaminated soils is limited as the harvest will be a singular event with limited opportunity for re-growth and re-establishment as the apical meristems are on the growing tips of shoots. While many if not most grasses are not hyper-accumulators of toxic metals (Baker and Brooks 1989), if they accumulate any metals at all they have a distinct advantage due to their architecture (basal apical meristems) of being able survive and thrive in environments where the loss of significant amounts of above ground biomass does not result in the removal of meristems and death of the organism. Hence repeatedly harvesting above ground leaf material typically does not significantly impact their survival. Thus if species of grasses can be identified that survive and accumulate toxic metals in their leaves in soil (that would preclude the planting of food crops for human or livestock consumption), then significant amounts of biomass could be harvested over several seasons and eventually reduce the amounts of toxic metals. This remediated land could then either be allowed to revert to a natural ecosystem or be productively utilized for agricultural crops. Additionally, the biomass that is harvested over several years in
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ACCEPTED MANUSCRIPT the process of cleaning up the soil could potentially be utilized as feedstock for processes generating biofuels and other potential products, provided the presence of the heavy metals is not detrimental to these processes. Some potential products includes:: sugar via enzymolysis of the grass (for use in the fermentation of ethanol), marketable chemicals produced from grass
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pyrolysis and products utilizing recovered heavy metals. Switchgrass (Panicum virgatum) is a species of grass that has garnered considerable interest in the biofuels industry. It is fast growing, can withstand a variety of adverse environmental conditions from freezing to drought, and is native to most of the continental US. Despite this, it has received little attention as a potential agent for the phytoremediation of lead contaminated soils (Salt et al. 1998, Gleeson, 2007). Traditionally Switchgrass has been used for forage production, soil conservation, and as ornamental grass. With the recent interests in utilizing biomass as an energy source, Switchgrass has gained attention as one of the most fibrous crops for energy production due its hardiness in poor soil and climate conditions, rapid growth, and low fertilization and herbicide requirements (Gupta and Demirbas 2010). The annual production of Switchgrass has been reported to range from 6 to 9 ton/acre/year (Lewandoski et al. 2003) Sources for fuel, other than gasoline, are now actively being sought to meet the world's ballooning energy requirements. Bio-fuels are one potential source of renewable energy that may supplement petroleum-based energy. Bio-fuel feed stocks are often classified into three categories: simple-sugar-derived, starch-derived and lignocellulosic-derived. Lignocellulose (found in corn stover, trees and grasses) is the most abundant renewable source of bio-mass with yearly supplies of approximately 200 billion metric tons worldwide (Watanabe, 1997; Sun and
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ACCEPTED MANUSCRIPT Cheng 2002). Switchgrass is a perennial grass with potential as a source of lignocellulosic biomass in a biofuels process. It may also have potential for assisting with soil remediation, while being grown for use as a biofuels feedstock. It is estimated that there are currently over 2 million acres of unremediated superfund fund sites in the US with lead as the primary contaminate(US EPA 1993; US EPA 1998; Watanabe, 1997)
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Timothy grass (Phleum pretense) is a perennial grass native to central Europe that was introduced to New England in the early 18th century. It is fast growing with a high fiber content and is today grown throughout much of the US as a component feedstock for cattle and horses. Similar to Switchgrass, it is moderately cold and drought tolerant, and can grow in soils with low nutrient content (Claessens et al. 2004). Little work has been conducted on the potential of Timothy grass in phytoremediation but in one study a similar grass (Ryegrass) was found to accumulate heavy metals at much higher levels in the roots versus the leaves (Jones et al. 1973). . The focus of this study primarily is to evaluate the potential of two perennial grasses (Switchgrass and Timothy grass) as agents for phytoremediation of heavy metal contaminated soils. This study used lead as the heavy metal to evaluate since it is arguably the most common and important heavy metal contaminant. The effects of the lead concentration in the soil on the growth rates of the grasses were evaluated. In addition to the plant study, grasses that were grown in lead contaminated soil were used as feedstock for two biomass conversion routes, i.e. enzymatic hydrolysis and fast pyrolysis processes.
The performance of lead-contaminated
switchgrass as conversion feedstock was studied by evaluating the production of sugar from enzymatic hydrolysis and the chemical product distribution in the bio-oil product from fast
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ACCEPTED MANUSCRIPT pyrolysis process. The biomass feedstock was also subjected to mild acid hydrolysis pretreament process prior to the conversion processes.
MATERIALS AND METHODS Plant Materials: 36 trays (3 per treatment, per species) of sufficient volume to
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accommodate 3 kg of soil media (Metromix) were set up in the Villanova Greenhouse and either saturated with distilled water or treated with lead citrate solution as follows: 50, 80,120,200, or 500 mg Pb/kg of soil. 100 seeds of Switchgrass or Timothy grass were sown in each tray and watered 3X per week with tap water for 6 weeks.
Germination and Growth Rates: Germination was determined by counting the number of successful germinations and establishment of plants and are reported here as the average germination rate for each of the 6 soil lead levels. Growth rates were determined by weekly measurements using the highest leaf and measuring from the level of the hypocotyl (root/shoot junction). The effect on germination rates of seeding density was also evaluated in normal and lead-contaminated soil, where seed spacings ranged from 2.5 to less than 1 cm.
Determination of Metals in Plant Material: Lead concentrations were determined by C oven for 48 h and then using an energy dispersive X-ray analysis attachment to a Joel S570 Scanning electron microscope. All elements detected by the device were standardized according to known concentrations of Potassium.
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ACCEPTED MANUSCRIPT Acid Hydrolysis: Switch grass and Timothy grass samples, grown either on normal soil, or grown on soil that has been treated with lead (120g Pb/kg soil), were obtained from Villanova University green house. The sample materials were dried by using an oven at 45oC overnight. A portion of the dried samples were acid hydrolyzed by using 4% phosphoric acid solutions. During hydrolysis, the grass samples were placed in a round bottom flask filled with the acid
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solution, which was then heated and left boiling for one hour. The grass materials were recovered and washed with distilled water three times to remove remaining acid and then dried overnight.
Enzyme Hydrolysis of Leaf Materials: In preparation for the grass enzymolysis, each of three three cellulase-producing fungi (Aspergillus niger, ATCC6275; Trichoderma reesei, ATCC56765; and Humicola grisea, ATCC16298) were grown separately in Potato dextrose medium until 105 spores/ml was achieved, at which point 1 ml of this solution was added to the MS medium to give the desired starting concentration of spores of at least 103 spores/ml. Experiments were then conducted in which Switchgrass and Timothy grass were both used as a cellulose source in sterile Murashige and Skoog medium (Sigma Aldrich, M5519-10L, 040M8803), buffered to pH 4.8 with 3 g/l succinic acid and 9 g/l succinate. Some of the grass, grown on normal soil or on soil that has been treated with lead (120 mg Pb/kg soil), was acid hydrolysed. The 250 ml flasks contained 100 ml media and 2 grams of grass, and were agitated at 120 RPM. Single and mixed cultivation of these organisms were explored in flasks at 25 C, which is the optimum growth temperature for all three fungi. To facilitate spore counting, Flourescein Diacetate (Sigma Aldrich, Lot # 38997A) was used to fluoresce the spores under the
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ACCEPTED MANUSCRIPT microscope. For determination of sugar concentration in each flask, test tubes were setup the same way as detailed in Colorimetric Method for Determination of Sugars and Related Substances (Dubois et al. 1956). Fast Pyrolysis: For preparing the fast pyrolysis reaction, the hydrolyzed and non-
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hydrolyzed grass materials were ground into fine particles.
The fast pyrolysis study was
performed by using a micropyrolyzer, which is CDS Pyroprobe 5200 (CDS analytical, Oxford, PA, USA). The micropyrolyzer is coupled with gas chromatograph/mass spectrometer (HP 6890/HP 5972) for fast pyrolysis vapor product analysis.
Switchgrass and Timothy grass
samples were pyrolyzed at 500oC under and inert atmosphere. The temperature was ramped at 20 C/ms and kept at 700oC for 10 s. The pyrolysis vapors were directly swept into the GC/MS using helium as the carrier gas. The chromatographic separation of pyrolysis products was performed using an alloy capillary column having high thermal resistance (30 m x 0.250 mm and 0.250 mm film thickness. An injector temperature of 300oC and a split ratio 1:100 was used. The GC oven temperature program began with a 3 min hold at 35 oC followed by heating to 130oC at 5oC/min and then to 250oC at 20oC/min RESULTS Effectiveness of Grass in Remediating soil: Results demonstrate that both switch grass and Timothy grass have the capacity to survive under conditions that far exceed the minimum levels deemed toxic by the Environmental Protection Agency. Figures 1 a and 3a show average plant height (each data point is an average of 30 results), at the end of a 6 week growing period, in soil with different lead concentrations. A two-way Anova analysis of this data indicates that plant height is significantly effected by both grass type (p-value < 0.0001) and soil lead concentration
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ACCEPTED MANUSCRIPT (p-value < 0.0001). A follow up one-way Anova analysis indicates that Switchgrass plant height is significantly lower (as compared to the control soil) when lead levels are ≥ 120 mg/kg of soil. The p-value for soil at 120 mg/kg of soil versus control soil was 0.0069, while p-values for higher soil lead levels were < 0.0001.
For Timothy grass, one-way Anova analysis indicates
that plant height is significantly lower when lead levels are ≥ 50 mg/kg of soil (p-vlaue =
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0.0088), The p-value for soil at 80 mg/kg of soil versus control soil was 0.0037, while p-values for higher soil lead levels were < 0.0001.
Figures 1 b and 3b show average germination rates (each data point is an average of 3), at the end of a 6 week growing period, in soil with different lead concentrations. A two-way Anova analysis of this data indicates that germination rate is significantly effected by both grass type (pvalue < 0.0001) and soil lead concentration (p-value < 0.0001). With only three replicates, the GLM statistical procedure was used to confirm this analysis by producing identical p-values. A follow up one-way Anova analysis indicates that Switchgrass germination rate is significantly lower than in control soil only when lead levels are ≥ 500 mg/kg of soil (p-vlaue = 0.0084). For Timothy grass, one-way Anova analysis indicates that germination rate is significantly lower when lead levels are ≥ 200 mg/kg of soil (p-vlaue = 0.047). These plant height and germination rate results indicate that Timothy grass growth is more sensitive than Switch grass growth to lead concentrations in contaminated soils. For Switchgrass, germination rates and growth rates are only impacted by lead concentrations in the soil that are triple the minimum standard set by the EPA
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ACCEPTED MANUSCRIPT Studies utilizing a scanning electron microscope equipped with energy dispersive X-ray diffraction indicate that appreciable quantities of lead are incorporated into both root (Fig. 2b and 4b) and leaf (Fig. 2a and 4a) tissues. Each data point on these graphs represents an average of 12 experimental values. A two-way Anova analysis of this data indicates that lead uptake by the roots is significantly effected by soil lead concentration (p-value < 0.0001), however there is
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no statistical difference in uptake of lead by roots between the two grass types (p-value = 0.46). A follow up one-way Anova analysis indicates that lead uptake by roots in Switchgrass is the same (p-value = 0.687) with soil lead levels of 50 and 80 mg/kg of soil, but then increases significantly at levels of ≥ 120 mg/kg of soil. In comparing lead uptake at a soil lead level of 50 versus higher levels, the p-values were all ≤ 0.0002.
For Timothy grass, one-way Anova
analysis indicates that for all soil lead levels > 50 mg/kg of soil, lead uptake in the roots exceeds the uptake observed at 50 mg/kg of soil. The p-value was 0.0195, when comparing lead uptake by roots in soil at lead concentrations of 50 and 80 mg/Kg soil.. The p-values were < 0.0001 when comparing uptake in soil with lead level of 50 mg/kg of soil versus soil with lead level of ≥ 120 mg/kg of soil.
A two-way Anova analysis of this data indicates that lead uptake by leaves is significantly effected by both grass type (p-value < 0.0001) and soil lead concentration (p-value < 0.0001). For Switch grass, one-way Anova analysis indicates that for all soil lead levels > 50 mg/kg of soil, lead uptake in the leaves exceeds the uptake observed at 50 mg/kg of soil (p-values ≤ 0.002). One-way Anova analysis indicates that lead uptake by leaves in Timothy grass is the same (p-value = 0.12) with soil lead levels of 50 and 80 mg/kg of soil, but then increases
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/K
l, the p-values were all ≤ 0.0002.
Lead uptake into leaves and roots increases with lead concentration in the soil. At all lead concentrations, the lead content in the roots exceeds that in the leaves as expected. For Timothy
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grass, however, substantially more lead accumulated in the roots as compared to the leaves and leaf accumulations were substantially less than for Switchgrass (Figs. 4 a,b). Although not shown in Figures 2 or 4, a concentration of lead in the leaves or roots of the control grass, grown in soil without lead present, was not detectable. Finally, it was observed that germination rates for Switchgrass grown in normal soil and that contaminated with lead was effected in the same way by the spacing of the planted seeds. At 2.5 cm seed spacing, germination rates averaged 68% while at 1 cm spacing the germination rates averaged 31%.
Enzymatic Hydrolysis of Grass with Select Fungal Cultures: Sugar production from fungal fermentations using Switchgrass (“SG”)
(“TG”) was measured versus
Timothy
fermentation time. For both grasses, the highest sugar levels were achieved when the grass was w
lc ll
c
l
(“L”), p
l (“SGL AH ALL”) p
w (“ALL”).
c
c F
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(“AH”),
Switchgrass (Figure 5), these
18 mg/l of sugar after 27 days of fermentation, when the
fermentation was finalized despite still increasing sugar levels. For Timothy grass, these same conditions produced 25 mg/l of sugar after 35 - 40 days of fermentation, after which sugar levels began to decline. The results from both of these experiments indicate that production of sugar by
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ACCEPTED MANUSCRIPT these three fungal species is not adversely effected by the lead taken up by the grasses. In fact, Figure 5
w
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slightly more sugar when digesting the grass that is higher in lead content. In addition, the results from the experiments with these two grasses supports the importance of acid hydrolysis pre-
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treatment and co-cultivation of all three cultures in achieving maximal sugar levels.
Work on Biomass Pre-treatment and Fast Pyrolysis: Fast pyrolysis of biomass carbohydrates and lignin produces valuable products in the forms of gas, oil, and solid char; these products may be used as a heat source, fuel, or feedstock for petrochemicals and other applications (Yanik, et al. 2007). Although similar in appearance to crude petroleum oil, bio-oil is chemically different. It is comprised of many oxygenated species such as carboxylic acids, hydroxyaldehydes, hydroxyl ketones, anhydro sugars, phenolic compounds and lignin fragments. Many of these compounds are used in a variety of manufacturing industries, such as chemical and pharmaceutical. The chemical composition of bio-oil depends on several factors such as – lignocellulosic composition of biomass, effect of minerals, residence time of the pyrolysis vapors pertaining to particular reactor configuration and the reaction temperature. It is known that cellulose and hemicellulose mainly result in the formation of furan/pyran ring derivatives, sugar/anhydro sugars and other low molecular weight compounds like acetic acid, acetol etc. whereas lignin mainly yields phenolic compounds and lignin oligomers. Figure 6 shows the chromatograms of the bio-oil product distribution from fast pyrolysis performed on dry switch grass (A), dry switch grass contaminated with lead (B), acid hydrolyzed switch grass with an acid solution of 4% H3PO4 (C), acid hydrolyzed switch grass contaminated
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ACCEPTED MANUSCRIPT with lead with an acid solution of 4% H3PO4 (D), enzymatic hydrolyzed switch grass (E), and enzymatic hydrolyzed switch grass contaminated with lead (F). Comparing figures A and B, figures C and D, and figures E and F, it can be seen that the chemical product distributions of the bio-oil from fast pyrolysis remains similar between the two feedstocks, showing that the lead present in the contaminated grass has only minimal effect, if any, regardless of the processes
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that the grasses were subjected to prior to fast pyrolysis. The comparison of figures A, C and E shows that, the acid hydrolysis and enzymatic hydrolysis treatments do have a larger affect effect on the chemical product distribution of the bio-oil. This effect can also be seen with the switchgrass that contains lead.
DISCUSSION There has been little research into the potential use of grasses as phytoremediation agents for heavy metals as they have not been shown to be hyper-accumulators of toxic materials. If these grasses accumulate any metals at all they have a distinct advantage due to their architecture (basal apical meristems) of being able survive and thrive in environments where the loss of significant amounts of above ground biomass does not result in the removal of meristems and death of the organism. Hence, by repeatedly harvesting above ground leaf material significant amounts of biomass could be harvested over several seasons and eventually reduce the amounts of toxic metals to the point where the affected land could either be allowed to revert to a natural ecosystem or be productively utilized for agricultural crops. The results from this research show that Timothy grass growth is more sensitive than Switch grass growth to lead concentrations in
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ACCEPTED MANUSCRIPT contaminated soils, and that Switchgrass, germination rates and growth rates are only impacted by lead concentrations in the soil that are triple the minimum standard set by the EPA . Lead uptake into leaves and roots was higher with Switchgrass as compared to Timothy grass at all soil lead concentrations. At a soil lead concentration of 120 mg Pb/kg for Switchgrass, the lead concentration in the grass leaves was 0.028 ± 10% of leaf dry weight.
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Assuming an optimal switch grass planting density gives rise to 7.5 ton/acre/year (Lewandoski et al 2003), it is estimated from the data in Figure 2a that that 0.02 ton/acre/year can be removed in the harvested leaves from soil contaminated at 80 – 120 mg Pb/kg soil. This calculation further assumes that the lead concentration in 6-8 foot tall Switchgrass (typical field plant height at harvest) is comparable to that measured in the approximately 1 foot tall Switchgrass grown during this study. This contaminated biomass that harvested over several years in the process of cleaning up the soil could potentially be utilized as feedstock in the production of biofuels. Cellulose and hemicellulose and pectins can be broken down by enzymes into glucose or other sugars that can then be fermented by yeast to produce bio-ethanol. Several organisms, including White rot fungi Phanerochaete chrysoporium (Alam et al. 2009) and Humicola grisea (ATCC product data sheet # 16298, 2011) have been reported to produce lignases. H. Grisea also produces pectinase. Trichoderma reesei (ATCC 13631) is a genetically modified fungus known for producing a slew of cellulase enzymes including: endonuclease (Kyriacou et al. 1987), cellobiohydrolase (Ehsani et al. 1996), xylase (Bailey et al. 1993), galactosidase (Kristufek et al. 1994), and mannase (Stralbrand et al. 1993). Aspergillus niger has been reported to produce low levels of l c
b
l
l
β-glucosidase (Kang et al. 2004). Trichoderma reseei,
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ACCEPTED MANUSCRIPT Aspergillus niger and Humicola grisea all grow optimally at similar conditions; temperature near 25°C, a Potato Dextrose nutrient mix at pH of 5-6 [ATCC, 2011]. This provides then the possibility for co-cultivation to produce the fungi and the cellulosic enzymes made by these fungi. Some research has focused on evaluating co-cultivation of cellulose producing organisms. In a published study, enhanced enzyme levels were achieved when T. reesei was co-cultured
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with A. niger using bagasse, corncobs and saw dust as the substrates in solid state fermentation (Maheshwari et al. 1994; Madamwar and Patel 1992). A similar result was achieved in this research, as indicated by Figures 5a and 5b. For both Timothy and Switchgrass, the highest sugar levels were achieved when pretreated with acid hydrolysis and co-cultivated with Trichoderma reseei, Aspergillus niger a
H
c l
pH
2 C.
Previous work has clearly demonstrated that the inorganic compounds found indigenously within biomass promote the formation of gaseous species and char at the expense of bio-oil yield. Raveendran et al. (1995) studied the pyrolysis of twelve different biomass feedstock and observed that the yield of pyrolysis vapors, devolatilization rate and the initial decomposition temperature increased whereas the char yield decreased on demineralization of the biomass samples except for the case of rice husks, groundnut shell and coir pith. The exceptional behavior of these biomass sources was attributed collectively to their high contents of lignin, potassium and zinc. Potassium and zinc are known to catalyze the char gasification reactions which are prevented when they are removed by demineralization (Yuki and Shafizedeh 1984; Raveendram et al. 1995; Patwardhan et al. 2010). In this study, the product distribution of the fast pyrolysis was found to be not affected by the lead that was present in the contaminated
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ACCEPTED MANUSCRIPT Switchgrass; however, the acid hydrolysis and enzymatic hydrolysis treatments do affect the product distribution of the fast pyrolysis. For example, fast pyrolysis of acid hydrolyzed switch grass results in the formation of new compounds, including furfural, levoglucosenone, tetradecanal and stearic acid. On the other hand, when the switch grass had been treated with enzymatic hydrolysis, 1% of 2,6-dimethoxyphenol is produced and 1,4:3,6-Dianhydro-alpha-D-
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Glucopyranose is no longer formed. In conclusion, our studies demonstrate that Switchgrass and to a lesser extent Timothy grass may have great potential as a phytoremediation agents for heavy metal contaminated soils. Germination and growth rates were only inhibited at the highest concentration used in this study. Further, SEM-EDAX results suggest that appreciable quantities of lead accumulate in both root and leaf tissues. Based on the germination rates and lead uptake observed in this study, Switchgrass seems to have much more potential than Timothy grass as a phytoremediation agent for lead contaminated soils. The results for enzymatic hydrolysis suggest that production of sugar by the three fungal species evaluated in this study was not adversely effected by the lead taken up by the grasses. The product distribution of the fast pyrolysis was not affected by the lead that is present in the contaminated Switchgrass. Future efforts will focus on optimizing lead uptake by considering the effect of plant height on lead uptake rates and on optimizing the biofuels processes to maximize productivity with respect to the biofuels and chemicals produced from the contaminated Switchgrass and to maximize lead removal and collection. Our studies may potentially result in methods that would allow for the productive use of millions of hectares worldwide that are currently unusable for production of feedstocks for biofuels processes while at the same restoring those lands to their native state.
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ACCEPTED MANUSCRIPT Figure Captions
Figure 1. Switchgrass (a). Average height for plants grown for 6 weeks under various Pb concentrations (25 plants per tray X 12 replicates). (b) Percent germination of seeds planted in
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media mix pre-treated with various concentrations of Pb (CO = Controls, T50= 50 mg Pb/kg media mix, T80 = 80 mg Pb/kg media mix, T120 = 120 mg Pb/kg mediamix, T200 = 200 mg Pb/kg media mix, T500 = 500 mg Pb/kg media mix).
Figure 2. Switchgrass a) Average Pb concentrations in leaf tissues (% dry weight) after 6 weeks exposure to Pb standardized to potassium. (b) Average Pb concentrations in root tissues (% dry weight) after 6 weeks exposure to Pb standardized to potassium.
Figure 3. Timothy grass (a). Average height for plants grown for 6 weeks under various Pb concentrations (25 plants per tray X 12 replicates). (b) Percent germination of seeds planted in media mix pre-treated with various concentrations of Pb CO = Controls, T50= 50 mg Pb/kg media mix, T80 = 80 mg Pb/kg media mix, T120 = 120 mg Pb/kg media mix, T200 = 200 mg Pb/kg media mix, T500 = 500 mg Pb/kg media mix.
Figure 4. Timothy grass a) Average Pb concentrations in leaf tissues (% dry weight) after 6 weeks exposure to Pb standardized to potassium. (b) Average Pb concentrations in root tissues (% dry weight) after 6 weeks exposure to Pb standardized to potassium.
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Figure 5. Sugar concentration versus time using Switchgrass .
Figure 6. Effect of acid hydrolysis and enzymatic hydrolysis on pyrolyzed switchgrass and
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swithgrass that contains lead. The pyrolysis were performed at 500°C. A: Product distribution from the pyrolysis of dry switch grass, B: Product distribution from the pyrolysis of dry switch grass contaminated with lead, C: Product distribution from the pyrolysis of acid hydrolyzed switch grass with an acid solution of 4% H3PO4, D: Product distribution from the pyrolysis of acid hydrolyzed switch grass contaminated with lead with an acid solution of 4% H3PO4, E: Product distribution from the pyrolysis of enzymatic hydrolyzed switch grass, and F: Product distribution from the pyrolysis of enzymatic hydrolyzed switch grass contaminated with lead. (1) Toluene, (2) Furfural, (3) Furan, (4) Phenol, (5) p-Cresol, (6) 2-Methoxyphenol, (7) Levoglucosenone, (8) 2Methoxy-4-methylphenol, (9) 1,4:3,6-Dianhydro-alpha-D-Glucopyranose, (10) 4-Ethyl2-methoxyphenol, (11) 2-Methoxy-4-vinylphenol, (12) 2,6-Dimethoxyphenol, (13)2,6,6trimethyl-Bicyclo [3.1.1]heptane, (14) 3,7,11,15-Tetramethyl hexadec-2-en-1-ol, (15) Tetradecanal, (16) 2-Propylcyclohexanol, (17) Palmitic acid, (18) 9,12,15-octadecatrien1-ol, and (19) Stearic acid,
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Average Grass Height (cm)
35 30 25 20 15 10 5 0 C0
T50
T80
T120
T200
T500
Lead level in Soil (mg/kg of soil)
Figure 1a
100 % Germination
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80 60 40 20 0 CO
T50
T80
T120
T200
T500
Lead level in Soil (mg/kg of soil) Figure 1b
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% (DW) Lead uptake in Leaves
0.05 0.04 0.03 0.02 0.01 0 T50
T80
T120
T200
T500
Lead Level in Soil (mg/kg of soil) Figure 2a
% (DW) Lead uptake in Roots
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0.08 0.06 0.04 0.02 0 T50
T80
T120
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Lead Level in Soil (mg/kg of Soil) Figure 2b
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Average Grass Height (cm)
20 15
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Lead level in Soil (mg/kg of soil) Figure 3a
80 % Germination
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60 40 20 0
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Lead level in Soil (mg/kg of soil)
Figure 3b
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% (DW) Lead uptake in Leaves
0.030 0.025 0.020 0.015 0.010 0.005 0.000 T50
T80
T120
T200
T500
Lead Level in Soil (mg/kg of soil) Figure 4a
% Lead uptake in Roots
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Lead Level in Soil (mg/kg of soil) Figure 4b
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Sugar Concentration (mg/l)
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Figure 5
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A B
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Figure 6
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