Abstract
Field and laboratory experiments were conducted to evaluate the feasibility of bioethanol production using the juice of sugarcane grown in heavy metal-contaminated soils. The results suggest that the sugar concentration was not adversely affected when the sugarcane was grown in the heavy metal-contaminated soil. Although the juice of sugarcane grown in contaminated soil contained elevated levels of heavy metals, sugar fermentation and ethanol production were not adversely affected when five selected yeast species were used to mediate the processes. The preliminary research findings obtained from this study have implications for developing cost-effective technologies for simultaneous bioethanol production and soil clean-up using heavy metal-contaminated soils for energy sugarcane farming.
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Bioethanol Production from Sugarcane Grown in Heavy Metal-Contaminated Soils
Jun Xie,a,b,c,* Qiang Weng,a,b,c Guangying Ye,a,b,c Sishi Luo,a,b,cRui Zhu,a,b,c Aiping Zhang,a,b,c Xiaoyang Chen,a,b,c and Chuxia Lin d,*
Field and laboratory experiments were conducted to evaluate the feasibility of bioethanol production using the juice of sugarcane grown in heavy metal-contaminated soils. The results suggest that the sugar concentration was not adversely affected when the sugarcane was grown in the heavy metal-contaminated soil. Although the juice of sugarcane grown in contaminated soil contained elevated levels of heavy metals, sugar fermentation and ethanol production were not adversely affected when five selected yeast species were used to mediate the processes. The preliminary research findings obtained from this study have implications for developing cost-effective technologies for simultaneous bioethanol production and soil clean-up using heavy metal-contaminated soils for energy sugarcane farming.
Keywords: Sugarcane; Ethanol; Fermentation; Yeast; Contamination; Heavy metals
Contact information: a: Institute of New Energy and New Materials, South China Agricultural University, Guangzhou 510642 P.R. China; b: Key Laboratory of Energy Plant Resources and Utilization under Ministry of Agriculture, South China Agricultural University, Guangzhou 510642 P.R. China; c: Key Laboratory of Guangdong Regular Higher Education Institutions, South China Agricultural University, Guangzhou 510642 P.R. China; d: School of Environment and Life Science, University of Salford, Salford, Greater Manchester, M5 4WT, UK;
* Corresponding authors: C.Lin@salford.ac.uk; xiejun@scau.edu.cn
INTRODUCTION
The importance of biofuel has been increasingly recognized due to the ever-increasing price and negative environmental and human impacts of fossil fuels (Demirbas 2008; Amin 2009; Ibeto et al. 2011). Biofuel has the advantages of being renewable and arguably more eco-friendly than fossil fuels (Goldemberg 2007). Bioethanol is the principal biofuel that has been commercially used as a petrol substitute for road transport vehicles (Anderson 2012). Many countries have implemented or are implementing programs for the addition of ethanol to petrol/gasoline (Sánchez and Cardona 2008).
Bioethanol can be produced from biomass by hydrolysis and sugar fermentation processes (Jamai et al. 2007; Bai et al. 2008). Candidate crops for ethanol production include sugarcane, sugar beet, corn, cassava, wheat, barley, and rice. Currently, the US and Brazil are the two major bioethanol-producing countries in the world. Corn and sugarcane are used as major feedstocks for bioethanol production by the former and the latter, respectively (Motta and Ferreira 1988; Luchansky and Monks 2009). Sugarcane-based ethanol production is relatively simpler, as compared to the corn-based process, because it does not require an additional treatment to convert starch to sugar prior to fermentation. Sugarcane ethanol has an energy balance seven times greater than ethanol produced from corn (International Energy Agency (IEA) 2004).
Sugarcane is a C4 plant that has a high photosynthetic efficiency (Bull 1969). Warm and humid climate conditions favor the growth of sugarcane (Verheye 2010). Therefore, there is potential to use tropical and subtropical areas to produce energy sugarcane crops to meet the increasing demand for bioethanol. However, most of the humid tropical and subtropical areas are heavily populated and there is a strong demand for food from limited available arable lands. Development of energy crop production at the cost of reducing food production in such areas is unlikely to be politically and socially feasible, economically viable, and culturally acceptable. Therefore, energy crop farming has to be confined to marginal lands such as heavy metal-contaminated lands that are not suitable for food crop production due to elevated levels of heavy metals in the edible portion of the crops (Ustyak and Petrikova 1996; Wang et al. 2001; Lin et al. 2005). While there is no human health concern with sugarcane farming for bioethanol production, it remains unclear whether the elevated levels of heavy metals in the juice of sugarcane could affect fermentation processes. It has been noted that the presence of heavy metals might or might not affect the activities of some fermenting yeasts (Perego and Howell 1997; Pearce and Sherman 1999; Azenha et al. 2000). However, direct tests for fermentation of heavy metal-contaminated sugarcane juices are lacking. In this communication, we present experimental results to address this issue.
EXPERIMENTAL
Sugarcane Plants and Juice Preparation
A total of 16 sugarcane cultivars (Table 1) were grown in soils contaminated by heavy metal-laden acidic mine water derived from the Guangdong Dabaoshan Mine (Lin et al. 2005; Chen et al. 2010). The soil was amended by adding acid neutralizing agent (red mud) to raise soil pH, which aimed to simultaneously improve the soil conditions and reduce the pollution of groundwater. In addition, a selected cultivar (YT94-128) was also grown in a small plot of un-amended contaminated soil, as well as a small plot of the soil that was not affected by mine water (i.e. the non-contaminated soil, which was about 600 m away from the main experimental site). This was to allow a comparison of sugar concentration in the juice among the three different soil conditions: (a) non-contaminated soil (control), (b) contaminated soil (T1), and (c) amended contaminated soil (T2).
At harvest, the average diameter and height of stalk for each sugarcane cultivar grown in the amended contaminated soil was obtained, and then the yield of each cultivar was estimated. For Cultivar YT94-128 grown in non-contaminated soil and un-amended contaminated soil, diameter and height of the stalks were not measured because the trial plots were too small to allow comparable estimate of the yield. In each of the trial plots, 15 randomly collected sugarcane stalks were used to obtain sugarcane juice samples. Sugarcane juice was squeezed out of sugarcane stalks by a mechanical crusher. Juice samples were then stored in a refrigerator at 4 oC before being used for the experiments.
Yeast Species and Inoculum Preparation
Five yeast species were purchased from the China Center of Industrial Culture Collection (CICC): Y1347, Y1384, Y1484, Ybeer, and Y2.82. The stocks of yeast species were maintained at 4 oC in a potato dextrose agar medium (PDA). The cells were sub-cultured from stock vials onto malt agar plates at 30 oC for 24 h. The inoculum was prepared by picking well-developed colonies from the plate and suspending the material in a test tube containing 50 mL of malt agar medium. The content in the test tube was incubated at 30 oC for 48 h and stored in a refrigerator prior to experiments.
Characterization of Sugarcane Juice and Bagasse
The sugarcane juice and bagasse of the 16 selected sugarcane cultivars plus YT94-128 grown in the non-contaminated soil (control) were used to determine the concentrations of Cd, Cu, Pb, and Zn. The sugarcane juice samples were also used for the determination of sugar concentration.
Fermentation Experiment
The juice samples of YT94-128 grown in the non-contaminated soils (C) and amended soil (T2) were used to observe the changes in residual sugar and ethanol in the fermenting juices over a period of 63.5 h. For each juice sample, 50 mL of the juice was inoculated with 10 mL of Y2.82 inoculum at 31±1 oC. Samples of the fermenting juice were taken at the 25.75th, 39th, 48th, 54.5th, and 63.5th h of the experiment for determinations of residual sugar and ethanol.
A separate experiment was also conducted to compare the effects of the five different yeast species on the fermentation rate of the control and T2. Because the above experiment showed that no marked decrease in sugar and increase in ethanol were observed after the 25.75th h, the experiment was only run for 40 h. A sample of each treatment was taken for determination of residual sugar and ethanol at the end of the experiment.
All experiments were performed in triplicate.
Analytical Methods
Heavy metals in the bagasse were extracted by dissolving the non-combustible portion in a 2 M HCl solution following combustion of a crushed plant tissue sample. The concentration of heavy metals in the extracts was then determined by atomic absorption spectrometry (AAS). Heavy metals in the sugarcane juice were also determined by AAS following HNO3-HClO4 digestion.
The concentration of sugar in the sugarcane juices was determined by a dinitrosalicylic acid (DNS) assay (Chen 2008).
The concentration of ethanol was determined by gas chromatography. Nitrogen was used as a carrier gas at 30 mL/min. The column temperature was maintained at 140 oC. The temperatures of the injector and FID detector were 200 and 300 oC, respectively. n-Butyl alcohol at a concentration of 2.0% (v/v) was used as the internal standard.
RESULTS AND DISCUSSION
The yield of the sugarcane cultivars grown in the amended contaminated soils ranged from 14440 to 30156 kg/ha (Table 1). This suggests that selection of appropriate cultivars is important to maximize the capacity of the contaminated soils for sugarcane production.
Figure 1 gives a comparison of sugar concentration in the juice among the same sugarcane cultivar (YT94-128) grown in the three different soils. The juice from the sugarcane grown in the non-contaminated soil contained significantly lower sugar contents than that from either the contaminated soil or amended contaminated soil.
Table 1. Growth Performance and Yield of the Various Sugarcane Cultivars Grown in the Contaminated Soil
Fig. 1. Comparison of sugar concentration in the juice among the same sugarcane cultivar (YT94-128) grown in non-contaminated soil (control), contaminated soil (T1), and amended contaminated soil (T2). Different letters above bars indicate significant difference at P < 0.05.
There was also a significant difference in juice sugar concentration between the sugarcane grown in the un-amended soil and that grown in the amended soil; the former contained more sugar than the latter. However, it must be realized that the soil used for the control was in another location, which had different fertility status. Therefore, this only allows a rough comparison being made. It is not intended to conclude that there was a higher juice sugar concentration in sugarcane grown in the contaminated soil than in the non-contaminated soil.
Table 2. Concentration of Heavy Metals in the Juice and Bagasse of Various Sugarcane Cultivars Grown in the Contaminated Soil
YT94-128C, grown in non-contaminated soil, served as the control for YT94-128, grown in the amended contaminated soil
The heavy metal concentration in the juice and bagasse of the same sugarcane cultivar differed markedly between the non-contaminated soil (YT94-128C) and the contaminated soil (YT94-128). Sugarcane plants grown in contaminated soils tended to have higher heavy metal concentrations than did those grown in non-contaminated soil, particularly for Zn.
Most of the other 15 sugarcane cultivars showed even higher concentrations of Zn, Cu, Cd, and Pb in the juice, as compared to YT94-128 (Table 2).
There was no clear relationship between the yield of sugarcane and any of the heavy metals tested in this study (Fig. 2). This suggests that the increased uptake of heavy metals by the sugarcane plants did not affect the growth of the plants.
Fig. 2. Relationship between the yield of sugarcane and (a) Zn, (b) Cu, (c) Cd and (d) Pb contained in the juice and bagasse of the sugarcane cultivars
Both the control and T2 showed a similar pattern of temporal variation in sugar in the fermenting juice; the concentration of sugar dropped rapidly to a very low level within the initial 25 h (Fig. 3). This was accompanied by a sharp increase in ethanol content during the same period of time. After the 25.75th h, the sugar concentration underwent little change during the rest of the experiment. However, different variation trends of ethanol content were observed for the control and T2; the ethanol content in the control decreased from the 25.75th h to the 48th h and then increased to the 63.5th h while the ethanol content in T2 kept increasing until the 48th h and then decreased to the 54.5th h, followed by re-increase to the 63.5th h.
Fig. 3. Change in (a) residual sugar and (b) ethanol in the fermenting sugarcane juice (using Y2.82 as the fermenting yeast) during the experimental period
The content of ethanol in the fermented juices varied among the 9 selected cultivars grown in the contaminated soil (Table 3). However, there was no clear relationship between the ethanol content and the concentration of heavy metals present in the juices (Fig. 4). This further suggests that the heavy metals contained in the juices did not adversely affect the production of ethanol under the experimental conditions set for this study.
There was no statistically significant difference (P <0.05) in sugar concentration of the fermented sugarcane juice among the fermentation experiments using the five different yeast species for either the control or T2. However, the concentration of residual sugar was consistently lower (P <0.05) in the control than in T2.
Table 3. Content of Ethanol in the Fermented Juice of Various Sugarcane Cultivars Grown in the Contaminated Soil
Fig. 4. Scatter plots showing the relationship between the ethanol produced during fermentation experiment and (a) Zn, (b) Cu, (c) Cd and (d) Pb in the sugarcane juice samples extracted from the 9 selected sugarcane cultivars
Unlike residual sugar, the ethanol content in the fermented juice varied significantly (P <0.05) among the experiments using different yeast species. For the control, the use of Y1484 and Y1347 resulted in the highest and lowest ethanol contents, respectively. The other three experiments produced similar amounts of ethanol. For T2, the effect of yeast species on the production of ethanol showed the following decreasing order: Y2.82 > Y1347 > Ybeer > Y1484 > Y1384 (Table 4).
Table 4. Residual Sugar and Ethanol in the Fermented Sugarcane Juice of Cultivar Yuetan 94-128 using the Five Selected Yeast Species
Means with different letters in the same columns indicate statistically significant differences at P <0.05.
The higher level of heavy metals in the juice and bagasse of the sugarcane grown in the contaminated soil, relative to that grown in the non-contaminated soil, was consistent with what has been found for many other crops (Liu et al. 2005; Jia et al. 2010). However, the sugar concentration in the sugarcane juice was not adversely affected by the elevated level of heavy metals in the soil. As a matter of fact, the contaminated soil enhanced the accumulation of sugar in the tested sugarcane cultivar (YT94-128), and amendment of the contaminated soil reduced the sugar content in the juice. These results suggest that soil amendment is not necessary when the contaminated soil is used for the production of energy sugarcane.
An additional beneficial effect of growing energy sugarcane in heavy metal-contaminated soil is the cleanup of soil through phytoextraction of soil-borne heavy metals. Sugarcane has the ability to produce a large amount of biomass per unit area of land within a given period of time. This may make it more efficient and effective than the use of heavy metal hyperaccumulating plants in terms of soil cleanup purposes. Hyperaccumulating plants are usually of low biomass output per unit of land area per unit of time (Robinson et al. 2009; Conesa et al. 2012).
In spite of elevated levels of heavy metals in the juice of sugarcane grown in the contaminated soil, the fermentation rate of sugarcane juice was not adversely affected. The higher ethanol content in T2 than in the control can be partly attributed to the higher initial sugar concentration in the former than in the latter. However, the residual sugar at and after the 25.75th h was also higher in T2 than in the control. If we assume that the residual sugar represents the non-fermentable sugar fraction in the sugarcane juice, then the proportion of non-fermentable sugar in the total sugar can be calculated using the following formula,
Non-fermentable sugar (%) = (St – Sr)/St x 100 (1)
where St denotes the initial sugar concentration (mg/L) in the sugarcane juice and Sr stands for sugar concentration (mg/L) in the fermented juice.
Figure 5 gives a comparison of non-fermentable sugar (%) in the fermented sugarcane juice between the control (the non-contaminated soil) and T2 (the amended contaminated soil) for the same sugarcane cultivar (YT94-128). It is clear that T2 had a higher percentage of non-fermentable sugar than the control did. Therefore, the higher ethanol content in T2 is also attributable to the higher fermentation rate in the former than in the latter. Calculation showed that the fermentation rate of T2 was 82% at the 25.75th h, as compared to 70% for the control at the same time. The decrease in ethanol content in the fermented juice after the 25.75th h indicated volatilization of ethanol from the fermented juice.
Fig. 5. Comparison of non-fermentable sugar (%) in the fermenting sugarcane juice between the control (the non-contaminated soil) and T2 (the amended contaminated soil) for the same sugarcane cultivar (YT94-128). Y2.82 was used as the fermenting yeast.
The similar fermentation effects of the five yeasts used in this study (Table 4) suggests that fermenting yeasts suitable for producing bioethanol using heavy metal-containing sugarcane juice are readily available, although the fermentation rate may vary slightly from species to species.
The findings obtained from this preliminary study have implications for developing cost-effective technologies for simultaneous bioethanol production and soil clean-up using heavy metal-contaminated soils for energy sugarcane farming. Further work is currently underway to obtain further insights into the mechanisms and kinetics related to the uptake of heavy metals by sugarcane plants, tolerance of sugar fermenting yeasts, etc., which can be used to optimize the technologies for using heavy metal-contaminated soils to produce sugar ethanol.
CONCLUSIONS
- The sugar content was not adversely affected when the sugarcane was grown in heavy metal-contaminated soil.
- The elevated levels of heavy metals in the sugarcane juice did not adversely affect the fermentation and the resulting ethanol production when appropriate yeast species were used to mediate the processes.
- The uptake of heavy metals by energy sugarcane may allow the cleanup of contaminated soils in a more economical way, which makes it superior to the use of low-biomass heavy metal hyperaccumulating plants.
ACKNOWLEDGMENTS
The work was supported by the “948” project of the State Forestry Administration (2011-4-75), the Key Project of the Guangdong Science and Technology Program (2011B090400437), and the “Second Five-Year” National Science and Technology Support Project (2011BAD22B01).
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Article submitted: Oct. 28, 2013; Peer review completed: Jan. 9, 2014; Revised version received and accepted: March 15, 2014; Published: March 21, 2014.