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Cultivation of Chlorella sp. with livestock waste compost for lipid production
Accepted Manuscript Short Communication Cultivation of Chlorella sp. with livestock waste compost for lipid production L.-D. Zhu, Z.-H. Li, D.-B. Guo, F. Huang, Y. Nugroho, K. Xia PII: DOI: Reference:
S0960-8524(16)31361-X http://dx.doi.org/10.1016/j.biortech.2016.09.094 BITE 17117
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 July 2016 19 September 2016 22 September 2016
Please cite this article as: Zhu, L.-D., Li, Z.-H., Guo, D.-B., Huang, F., Nugroho, Y., Xia, K., Cultivation of Chlorella sp. with livestock waste compost for lipid production, Bioresource Technology (2016), doi: http:// dx.doi.org/10.1016/j.biortech.2016.09.094
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Cultivation of Chlorella sp. with livestock waste compost for lipid production
L.-D. Zhu 1,2,5,*, Z.-H. Li 1, D.-B. Guo 3, F. Huang 4, Y. Nugroho 2, K. Xia 2
1
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, and Faculty of
Resources and Environmental Science, Hubei University, Wuhan 430062, P.R. China 2
Department of Energy Technology, Faculty of Technology, University of Vaasa, Vaasa 65101,
Finland 3
School of Environmental Science & Engineering, Huazhong University of Science and Technology,
Wuhan 430074, China 4
Laboratory of Tropical Agro-Environment, Ministry of Agriculture, South China Agricultural
University, Guangzhou 510642, China 5
Renewable Energy Research Group, Vaasa Energy Institute, Vaasa 65101, Finland
* Corresponding author: E-mail: [email protected]; [email protected] Tel: +358408480438; Fax: +35863248467
ABSTRACT
Cultivation of microalgae Chlorella sp. with livestock waste compost as an alternative nutrient source was investigated in this present study. Five culture media with different nutrient concentrations were prepared. The characteristics of algal growth and lipid production were examined. The results showed that the specific growth rate together with biomass and lipid productivities was different among all the cultures. As the initial nutrient concentration decreased, the lipid content of Chlorella sp. increased. The variations in lipid 1
productivity of Chlorella sp. among all the cultures were mainly due to the deviations in biomass productivity. The livestock waste compost medium with 2000 mg L–1 COD provided an optimal nutrient concentration for Chlorella sp. cultivation, where the highest productivities of biomass (288.84 mg L–1 day–1) and lipid (104.89 mg L–1 day–1) were presented.
Keywords: microalgae, Chlorella sp., livestock waste compost, lipids, biodiesel
1. Introduction
In response to the energy shortage, global warming and climate changes, microalgae have received an increasing attention as a biodiesel feedstock. The main arguments in favor of microalgae for biodiesel production mainly include the following aspects (Zhu et al., 2015; Zhu et al., 2016). First, microalgae have high photosynthetic efficiency and growth rate. The doubling time for algal biomass during the exponential phase can be as short as 3.5 hours. Second, most algal species have high lipid contents, occupying between 20 and 50% of the dried weight. Third, microalgal cultivation will not compete with food production for farmlands, and this is because arid or semiarid earth, saline soils, infertile farms, polluted land and other land with low economic values can be applied to launch algal biorefinery facilities. Forth, flue gas that is rich in CO2 can be introduced into algal growth system as a carbon source, and thus algal biofuels are carbon neutral. Fifth, wastewaters can be used to cultivate algae, reducing the consumption of freshwater and fertilizers. Finally, algal biomass
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harvest methods are simple, though the costs are high, thus speeding up the biodiesel production process in practice. However, large-scale microalgal cultivation requires substantial amount of fertilizers. The wide application of fertilizers will cause the eutrophication of water bodies, since they are easily dissolved in rainwater or runoff. In addition, fertilizer production also requires much energy input that comes from fossil fuels, leading to an unsustainable practice. Clarens et al. (2010) suggest that during algal cultivation 50% of energy consumption and greenhouse gas emissions are related to fertilizer production. Thus, it is necessary to seek for alternative nutrient sources with low environmental impacts to grow microalgae. Using wastewaters to grow microalgae as nutrient sources appears as a promising choice to decrease the costs and environmental impacts. On one hand, wastewater contains rich nitrogen and phosphorus which favor the growth of microalgae. On the other hand, during the algal growth wastewater can be purified before discharging into water bodies, reducing environmental pollution. Plenty of on-going research on wastewater treatment using algal systems has been reported in literature (Yuan et al., 2013; Ji et al., 2014; Passero et al., 2014; Zhu, 2015; Kim et al., 2016). Apart from wastewater, using livestock waste compost to cultivate algae seems to be another alternative solution. Livestock waste has found to be rich in nitrogen and phosphorus (Ho et al., 2013). After composting, the compost is seen as a type of organic fertilizers, since an improved fertility occurs. Zhu and Hiltunen (2016) propose a feasible framework to integrate microalgal cultivation with livestock waste compost, without exhibiting any empirical data for this process. Thus, it is extremely necessary to investigate the integrated approach in practice and demonstrate the relevant data with importance. The objective of this paper was to determine algal biomass accumulation for biodiesel production when algal cultivation with livestock waste compost was combined. An optimal 3
concentration level for algal cultivation would be found, and productivities of biomass and lipids would also be specified in this study.
2. Material and methods
2.1. Microalgae pre-culture conditions
The microalgae used in this study were Chlorella sp., which was originally isolated from local fresh-water habitats by Utex and grown in a BG11 medium (Zhu et al., 2014). The seed culture was preserved and grown in 0.4-L flasks, and each flask contained 250 mL of the medium with the pH value of 6.8. The surrounding temperature was 25 ± 1 °C and cool white fluorescent lamps were placed on one side of flasks to reach the light intensity of 240 ± 10 µmol m–2 s–1.
2.2. Preparation of culture media
Livestock waste compost used in this study was from a local collection point, where windrow composting technology was applied to compost cattle wastes. The compost was immersed in water, and stirred using a magnetic stirrer. The mixture was filtered with filter paper to remove non-soluble particulate solids. The liquid was stored at 4 °C for microalgal growth studies. Characteristics of media were measured (Table 1), following the Hach DR 2700 Spectrophotometer Manual.
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2.3. Microalgal cultivation with livestock waste compost
The original livestock waste compost medium was diluted using fresh water to four different concentrations (2000, 1500, 1000 and 500 mg L–1 COD). The undiluted media and the four diluted media were applied accordingly as the cultures for microalgal cultivation. In comparison, BG11 media (control group) were also used to grow algae. Afterwards, a volume of 225 mL of livestock waste compost media with the variable COD concentrations mentioned above were introduced into 0.4-L flasks. Subsequently, a volume of 25 mL of seed microalgal suspension with an optical density (OD540) of 3.125 was introduced into each flask. The culture conditions were identical to those described in Section 2.1. All cultures including the control group were conducted in duplicates. In all experiments, microalgae were cultivated for 10 days.
2.4. Determination of microalgal growth
The optical density of Chlorella sp. at 540 nm was measured every day using spectrophotometer. A correlation between OD540 and the dried biomass weight was predetermined by the following steps. To fall within the linear testing range, algal cultures were first appropriately diluted to several levels. The actual OD was measured by multiplying by the dilution factor. Dried weight of biomass was determined by filtering 20-ml culture samples using a pre-weighed 0.45-µm filter paper, rinsing with dH2O and drying at 80 °C overnight to a constant level. The correlation was found as shown below: Dry weight (g L–1) = 0.4001 × OD540, R² = 0.9909
5
(1)
The specific growth rate µ during the exponential growth phase was measured by using Eq. (2) (Zhu et al., 2013a): µ (day–1)= ln (N2/N1)/(t2–t1)
(2)
where N1 and N2 are dry biomass weight (g L–1) at time t1 and t2, respectively. The biomass productivity (Pb) was measured using the following expression: Pb (mg L–1 day–1)= (DWi – DW0)/(ti–t0)
(3)
where DWi and DW0 are dry biomass weight (g L–1) at time ti and t0, respectively. The lipid productivity (Pl) was determined, according to the following equation: Pl (mg L–1 day–1)= Pb × Cl/100
(4)
where Cl represents lipid content (% of dw).
2.5. Harvest and drying
Microalgal cells from each flask were collected after 10-day growth, and centrifuged at 5000 rpm for 15 min. Supernatants were decanted and cell pellets were at 80 °C overnight to a constant level. The dried microalgal biomass was collected and sealed in empty containers for lipid extraction.
2.6. Lipid extraction
According to Zhu et al. (2014), the following steps were applied to determine the lipid content. 100–150 mg freeze-dried algal samples were weighed and extracted with 2 mL methanol containing 10% dimethyl sulfoxide (DMSO) in a water bath shaker at 45 °C for 45 min. The mixture was centrifuged at 3000 rpm for 10 min. Then, the supernatant was 6
collected and leftover was re-extracted twice following the same process. Afterwards, the leftover was extracted with 4 mL mixture of hexane and ether (1:1, v/v) in a water bath shaker at 45 °C for 60 min. The mixture was centrifuged at 3000 rpm for 10 min. Then, the supernatant was collected and leftover was re-extracted twice following the same process. All the supernatants were combined, after which 6 mL water was added to the incorporated extracts to form a ratio of methanol with 10% DMSO, diethyl ether, hexane and water of 1:1:1:1 (v/v/v/v). The organic phases with lipids were transferred into a pre-weighed glass tube and evaporated to dryness under nitrogen protection. Subsequently, the lipids were continuously dried at 80 °C overnight to a constant level. Afterwards, the total lipids were determined gravimetrically, and lipid content was expressed as % of dry weight.
3. Results and discussion
3.1. Microalgal growth
The growth characteristics of Chlorella sp. under five nutrient concentration levels within ten days were illustrated in Fig.1. These curves indicated all algal growth phases in batch culture except the lysis phase. The lack of a visible lysis phase was due to a short cultivation period in this study. In all cultures the lag phase lasted around 3 days. In the lag state, microalgal cells needed to adapt to the new environment with livestock waste compost media which were quite different from BG 11 medium. Meanwhile, microalgal cells were sensitive to light intensity in the beginning, since photoinhibition might occur due to a low cell concentration and thus lack of self-shading (Zhu et al., 2014; Sutherland et al., 2016). 7
However, photoadaptation to light intensity also took place (Belzile and Gosselin, 2015), which was beneficial to the growth of microalgal cells. After lag phase, exponential growth in all cultures lasted around 4 days, followed the stationary phase. During the exponential phase, microalgae experienced a fast growth and biomass accumulation. Day 7 was the last day of exponential growth for all cultures. Similarly, Passero et al. (2014) pre-treated dairy wastewater by ultraviolet radiation for the cultivation of algae C. vulgaris, and found that the exponential growth phase duration was 5 days with the tenth day as the end of this period. After the lag phase, microalgae experienced a rapid growth with specific growth rate µ ranging from 0.275 to 0.375 day–1 and doubling time ranging from 2.52 to 1.85 days (Table 2). As the culture nutrient concentration increased, specific growth rate increased, while doubling time decreased. This is in line with the study by Ji et al. (2014), who suggested that the increase of monosodium glutamate wastewater concentration stimulated the increase of the specific growth rate of algae Chlorella vulgaris. The specific growth rate µ of 1.26 day–1 was achieved by Atta et al. (2013), who grew Chlorella vulgaris by using light-emitting diode in batch culture for 8 days. The final biomass increase and productivity in the livestock waste compost medium with initial COD concentration at 2000 mg L–1 were the highest among the compost media, correspondingly reaching 2.888±0.076 g L–1 and 288.84±8.05 mg L–1 day–1, while the culture in 500 mg L–1 COD medium showed the lowest biomass increase and biomass productivity, reaching 1.842±0.051 g L–1 and 184.22±6.56 mg L–1 day–1, respectively. The biomass productivities in this study were similar to those in the study by Tan et al. (2016), who achieved 110.63–375.71 mg L–1 day–1 biomass when they cultured Chlorella pyrenoidosa with the effluent of anaerobically digested activated sludge. In contrast, applying highly efficient light-emitting diode-based photobioreactor to cultivate
8
Chlorella vulgaris, Fu et al. (2012) obtained the maximum biomass increase of 20 g L–1 and productivity of 2.11 g L–1 day–1.
3.2. Lipid content and productivity
The lipid contents of Chlorella sp. in five cultures with different nutrient concentrations were determined and exhibited in Fig. 2. The culture with the initial COD concentration at 500 mg L–1 experienced the highest algal lipid accumulation, reaching 44.30% of dry weight. The reason why algal cells in culture with low initial nutrient concentration received more lipid accumulation might be due to the fact that low biomass concentration in the culture could make more algal cells access and receive more light, which triggered and benefited lipid storage. This finding is in line with preceding studies by Seo et al. (2015) and Mandotra et al. (2016), who suggested that the increase of the amount of light available within the optimal range could favor the accumulation of lipids in algal cells. The results also showed the initial nutrient concentration affected lipid accumulation of microalgae in this study. As the initial COD concentration increased from 500 to 2680 mg L–1, the lipid content decreased from 44.30% to 33.90%. This tendency in the variation of lipid contents in microalgal cells was found to be in accordance with the study conducted by Zhu et al. (2013b), who cultivated Chlorella zofingiensis with piggery wastewater under six different nutrient concentration levels. Fig. 2 also exhibited the lipid productivities of Chlorella sp. in five cultures. The findings demonstrated that the productivities of lipids in five cultures ranged from 81.61 to 104.89 mg L–1 day–1, following the relationship 2000 mg L–1 COD culture (104.89) > 1500 mg L–1 COD culture (92.16) > 2680 mg L–1 COD culture (86.83) > 1000 mg L–1 COD 9
culture (84.76) > 500 mg L–1 COD culture (81.61). The findings were evidently higher than 49.1 ± 0.41 mg L–1 day–1 lipid productivity achieved by Arora et al. (2016), who optimized N and P concentration for attainment of maximum lipid productivity using oleaginous microalgae C. minutissima. Han et al. (2013) acquired the maximum lipid productivity of 115 mg L–1 day–1 via the culture strategy of semi-continuous cultivation of C. pyrenoidosa with nitrogen limitation and pH regulation by CO2. In this investigation the variations in lipid productivity of Chlorella sp. among different cultures mainly attributed to their biomass differences, since the lipid content deviations within different cultures were not so wide in contrast to the biomass. Although the lipid contents of Chlorella sp. in 1000 and 500 mg L–1 COD cultures were high, their biomass productivities presented as a limiting factor, thus causing relevantly low productivities of lipids. Our previous investigation showed that around one third to one fourth of lipids could be effectively converted into fatty acid methyl esters, the efficient ingredients of biodiesel (Zhu et al., 2013ab; Zhu et al., 2014). The reason is that some lipid types such as phospholipid, glycolipid and chlorophyll are not the efficient ingredients for the production of biodiesel and thus cannot be converted into biodiesel. Based on this estimation, the expected biodiesel productivity in this study will range from 20 to 35 mg L–1 day–1, following in the sequence 2000 mg L–1 COD culture (26–35) > 1500 mg L–1 COD culture (23–31) > 2680 mg L–1 COD culture (22–30) > 1000 mg L–1 COD culture (21–28) > 500 mg L–1 COD culture (20–27).
3.3. Scale-up potential and challenges
3.3.1. Source availability
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Microalgal production requires large amount of nutrients. To avoid the negative environmental impacts of chemical fertilizer application, livestock waste compost can offer a cleaner medium for the cultivation of microalgae on a large scale. In total, every year around 56 billion livestock such as pig, cattle, lamb, duck and chicken are cultivated worldwide to meet the meat consumption for human beings (Zhu and Hiltunen, 2016). Due to the increasing meat demand especially in developing countries, the number of livestock domestication is going to robustly increase, and intensive animal farming will prevail globally. Therefore, abundant livestock wastes are available, and the compost can meet the nutrient demand for microalgal cultivation upscaling. According to Choi et al. (2014), in the EU-27 countries over 1500 million fresh tons of livestock wastes are produced annually, while in USA around 35 million dry tons of livestock wastes are generated every year. Taking China as an example, more than 4.8 billion tons of livestock manures are generated annually, and the number is going to gradually increase (Schneider, 2016). To summarize, from the nutrient source point of view, there is a guarantee for the large-scale production of microalgal biomass.
3.3.2. Environmental advantages Environmental merits achieved through pollution control and CO2 mitigation should be included as advantages in practice. This study shows that Chlorella sp. can be grown with livestock waste compost, providing a potential method for waste management. In addition, no fertilizers need to be employed to a microalgae system, presenting as an added beneficial value for this approach. There is no denying the fact that CO2 is released during composting, but the amount is found to be lower in contrast to the production of chemical fertilizers. It is suggested that 4–67 and 4–82 kg of CO2 can be reduced by composting one ton of garden 11
waste and food waste, respectively (Boldrin et al., 2009). Additionally, CO2 emitted during composting can be re-absorbed by microalgae. All of these factors can add values to microalgal production with livestock waste compost.
3.3.3. A scale-up roadmap Upscaling the cultivation of Chlorella sp. with livestock waste compost is possible in practice. In an effort to promote microalgal biodiesel production especially in terms of economics, a scale-up roadmap for Chlorella sp. production using livestock waste compost is proposed, as illustrated in Fig. 3. The post-extracted algal residues can be used to produce either biogas via anaerobic digestion or fertilizers. Alternatively, the leftovers or residuals appeared at any step can also be recycled into microalgal cultivation systems as the nutrient sources. In addition, apart from biodiesel, biogas and fertilizer, as exhibited in Fig. 3, valueadded bioproducts such as bio-ethanol, high-value proteins, cosmetics, food, feed and fine chemicals can be further produced through algal biorefinery. This paper can potentially serve as a starting point for authorities, stakeholders and practitioners to conduct the growth of microalgal biomass with livestock waste compost. Energy companies like the Finnish Fortum Corporation might potentially benefit if the upscaling was successful. In addition, this research might help livestock farms or enterprises (e.g., Pellon Group) solve the waste problems, putting forward an efficient solution for waste management.
3.3.4. Main uncertainties With the contribution of this investigation, the literature will no longer indicate a lack of empirical information on microalgal cultivation with livestock waste compost. According to the batch culture studies, microalgae can be successfully and robustly grown with livestock 12
waste compost. However, several main uncertainties also exist during the upscaling process. First, certain competitive microorganisms such as pathogens and bacteria from compost might be introduced into algal cultivation system, potentially hindering the growth. Thus, pretreatment methods such as sterilization should be considered, which might also affect the upscaling process. Second, chemical profiles of animal wastes vary significantly among various sources, which will largely impact the production strategy and management. This study investigates algal cultivation with cattle manure compost, but further investigation still needs to be done when other types of animal waste compost are applied. Third, different algal species exhibit distinct growth properties during the cultivation with compost. Thus, it is necessary to isolate the most suitable strains that show robust growth in compost media. Finally, algal cultivation with livestock waste compost requires complicated system integration to reduce the energy consumption and production costs. System operation is involved with many steps, such as energy input, cleaning, sterilization and maintenance, all of which need to be well planned before facility installment. To conclude, future research and development is still required in order to move the upscaling process forward.
4. Conclusions
The specific growth rate of Chlorella sp. grown in five cultures ranged from 0.275 to 0.375 day–1 with the doubling time from 2.52 to 1.85 days. Initial nutrient concentration could affect the lipid accumulation, and the lipid content ranged from 33.90% to 44.30%. The lipid productivity ranged from 81.61 to 104.89 mg L–1 day–1. The 2000 mg L–1 COD culture presented as the optimal medium for algal cultivation, since the highest biomass and 13
lipid productivities were realized. Scale-up of the process is greatly possible but requires further investigation.
Acknowledgements
This work was supported by the Kone Foundation Project in Finland and TransAlgae Project from EU’s Botnia-Atlantica programme. It was also supported by the Ministry of Housing and Urban-Rural Development (No.2016K4032), China Postdoctoral Science Foundation (No.2015M580644, 2016M592339). In addition, the authors are grateful to the support from Wuhan International Science and Technology Cooperation Project (2016030409020221). The authors would like to thank the anonymous reviewers for their helpful comments and suggestions that greatly improved the manuscript.
References
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Figure 1. Growth characteristics of Chlorella sp. cultivated under five nutrient levels within ten days (means ± sd).
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Figure 2. Lipid contents and productivities of Chlorella sp. biomass cultured in different nutrient concentrations of livestock waste compost media within ten days (means ± sd).
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Figure 3. Proposed scale-up roadmap for the cultivation of Chlorella sp. using livestock waste compost for biofuels production. This image is meant to show the linkages between each process, but the scheme eludes to the physical layout of the combined approach, as the microalgal production facilities must be located close to the livestock waste compost point.
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Table 1 Characteristics of livestock waste compost medium used in the experiments (means ± sd). Parameter Concentration pH 6.9±0.0 –1 Suspended Solid (mg L ) 286 ± 17 COD (mg L–1) 2680±51 + –1 NH4 (mg L ) 78.8±4.0 TN (mg N L–1) 222.0±3.0 TP (mg PO4–3 –P L–1) 103.0±4.0 –1 K (mg L ) 124±12 Cu (mg L–1) 8.2±0.9 –1 Zn (mg L ) 3.9±0.3
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Table 2 Growth parameters of Chlorella sp. in flasks under five concentration levels within ten days (means ± sd). Initial nutrient Biomass Doubling Biomass Specific growth concentration (mg L–1 increase time productivity rate µ (day–1) (g L–1) (days) (mg L–1 day–1) COD) * 2680 0.375±0.005 1.85±0.02 2.562±0.117 256.18±11.66 2000 0.357±0.015 1.94±0.08 2.888±0.076 288.84±8.05 1500 0.321±0.015 2.17±0.10 2.523±0.189 252.30±18.92 1000 0.306±0.015 2.27±0.11 2.066±0.065 206.64±6.95 500 0.275±0.008 2.52±0.07 1.842±0.051 184.22±6.56 BG11 medium 0.346±0.013 2.01±0.08 2.924±0.012 292.43±1.25 * COD concentration was applied to present the initial nutrient concentration in all cultures in this study.
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HIGHLIGHTS 1. Cultivation of microalgae Chlorella sp. with livestock waste compost was investigated. 2. Biomass productivity ranged from 184.22 to 288.84 mg L–1 day–1. 3. As the initial nutrient concentration decreased, the lipid content increased. 4. Lipid productivity ranged from 84.76 to 104.89 mg L–1 day–1. 5. The livestock waste compost medium with 2000 mg L–1 COD was optimal for algal growth
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S0960-8524(16)31361-X http://dx.doi.org/10.1016/j.biortech.2016.09.094 BITE 17117
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 July 2016 19 September 2016 22 September 2016
Please cite this article as: Zhu, L.-D., Li, Z.-H., Guo, D.-B., Huang, F., Nugroho, Y., Xia, K., Cultivation of Chlorella sp. with livestock waste compost for lipid production, Bioresource Technology (2016), doi: http:// dx.doi.org/10.1016/j.biortech.2016.09.094
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cultivation of Chlorella sp. with livestock waste compost for lipid production
L.-D. Zhu 1,2,5,*, Z.-H. Li 1, D.-B. Guo 3, F. Huang 4, Y. Nugroho 2, K. Xia 2
1
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, and Faculty of
Resources and Environmental Science, Hubei University, Wuhan 430062, P.R. China 2
Department of Energy Technology, Faculty of Technology, University of Vaasa, Vaasa 65101,
Finland 3
School of Environmental Science & Engineering, Huazhong University of Science and Technology,
Wuhan 430074, China 4
Laboratory of Tropical Agro-Environment, Ministry of Agriculture, South China Agricultural
University, Guangzhou 510642, China 5
Renewable Energy Research Group, Vaasa Energy Institute, Vaasa 65101, Finland
* Corresponding author: E-mail: [email protected]; [email protected] Tel: +358408480438; Fax: +35863248467
ABSTRACT
Cultivation of microalgae Chlorella sp. with livestock waste compost as an alternative nutrient source was investigated in this present study. Five culture media with different nutrient concentrations were prepared. The characteristics of algal growth and lipid production were examined. The results showed that the specific growth rate together with biomass and lipid productivities was different among all the cultures. As the initial nutrient concentration decreased, the lipid content of Chlorella sp. increased. The variations in lipid 1
productivity of Chlorella sp. among all the cultures were mainly due to the deviations in biomass productivity. The livestock waste compost medium with 2000 mg L–1 COD provided an optimal nutrient concentration for Chlorella sp. cultivation, where the highest productivities of biomass (288.84 mg L–1 day–1) and lipid (104.89 mg L–1 day–1) were presented.
Keywords: microalgae, Chlorella sp., livestock waste compost, lipids, biodiesel
1. Introduction
In response to the energy shortage, global warming and climate changes, microalgae have received an increasing attention as a biodiesel feedstock. The main arguments in favor of microalgae for biodiesel production mainly include the following aspects (Zhu et al., 2015; Zhu et al., 2016). First, microalgae have high photosynthetic efficiency and growth rate. The doubling time for algal biomass during the exponential phase can be as short as 3.5 hours. Second, most algal species have high lipid contents, occupying between 20 and 50% of the dried weight. Third, microalgal cultivation will not compete with food production for farmlands, and this is because arid or semiarid earth, saline soils, infertile farms, polluted land and other land with low economic values can be applied to launch algal biorefinery facilities. Forth, flue gas that is rich in CO2 can be introduced into algal growth system as a carbon source, and thus algal biofuels are carbon neutral. Fifth, wastewaters can be used to cultivate algae, reducing the consumption of freshwater and fertilizers. Finally, algal biomass
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harvest methods are simple, though the costs are high, thus speeding up the biodiesel production process in practice. However, large-scale microalgal cultivation requires substantial amount of fertilizers. The wide application of fertilizers will cause the eutrophication of water bodies, since they are easily dissolved in rainwater or runoff. In addition, fertilizer production also requires much energy input that comes from fossil fuels, leading to an unsustainable practice. Clarens et al. (2010) suggest that during algal cultivation 50% of energy consumption and greenhouse gas emissions are related to fertilizer production. Thus, it is necessary to seek for alternative nutrient sources with low environmental impacts to grow microalgae. Using wastewaters to grow microalgae as nutrient sources appears as a promising choice to decrease the costs and environmental impacts. On one hand, wastewater contains rich nitrogen and phosphorus which favor the growth of microalgae. On the other hand, during the algal growth wastewater can be purified before discharging into water bodies, reducing environmental pollution. Plenty of on-going research on wastewater treatment using algal systems has been reported in literature (Yuan et al., 2013; Ji et al., 2014; Passero et al., 2014; Zhu, 2015; Kim et al., 2016). Apart from wastewater, using livestock waste compost to cultivate algae seems to be another alternative solution. Livestock waste has found to be rich in nitrogen and phosphorus (Ho et al., 2013). After composting, the compost is seen as a type of organic fertilizers, since an improved fertility occurs. Zhu and Hiltunen (2016) propose a feasible framework to integrate microalgal cultivation with livestock waste compost, without exhibiting any empirical data for this process. Thus, it is extremely necessary to investigate the integrated approach in practice and demonstrate the relevant data with importance. The objective of this paper was to determine algal biomass accumulation for biodiesel production when algal cultivation with livestock waste compost was combined. An optimal 3
concentration level for algal cultivation would be found, and productivities of biomass and lipids would also be specified in this study.
2. Material and methods
2.1. Microalgae pre-culture conditions
The microalgae used in this study were Chlorella sp., which was originally isolated from local fresh-water habitats by Utex and grown in a BG11 medium (Zhu et al., 2014). The seed culture was preserved and grown in 0.4-L flasks, and each flask contained 250 mL of the medium with the pH value of 6.8. The surrounding temperature was 25 ± 1 °C and cool white fluorescent lamps were placed on one side of flasks to reach the light intensity of 240 ± 10 µmol m–2 s–1.
2.2. Preparation of culture media
Livestock waste compost used in this study was from a local collection point, where windrow composting technology was applied to compost cattle wastes. The compost was immersed in water, and stirred using a magnetic stirrer. The mixture was filtered with filter paper to remove non-soluble particulate solids. The liquid was stored at 4 °C for microalgal growth studies. Characteristics of media were measured (Table 1), following the Hach DR 2700 Spectrophotometer Manual.
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2.3. Microalgal cultivation with livestock waste compost
The original livestock waste compost medium was diluted using fresh water to four different concentrations (2000, 1500, 1000 and 500 mg L–1 COD). The undiluted media and the four diluted media were applied accordingly as the cultures for microalgal cultivation. In comparison, BG11 media (control group) were also used to grow algae. Afterwards, a volume of 225 mL of livestock waste compost media with the variable COD concentrations mentioned above were introduced into 0.4-L flasks. Subsequently, a volume of 25 mL of seed microalgal suspension with an optical density (OD540) of 3.125 was introduced into each flask. The culture conditions were identical to those described in Section 2.1. All cultures including the control group were conducted in duplicates. In all experiments, microalgae were cultivated for 10 days.
2.4. Determination of microalgal growth
The optical density of Chlorella sp. at 540 nm was measured every day using spectrophotometer. A correlation between OD540 and the dried biomass weight was predetermined by the following steps. To fall within the linear testing range, algal cultures were first appropriately diluted to several levels. The actual OD was measured by multiplying by the dilution factor. Dried weight of biomass was determined by filtering 20-ml culture samples using a pre-weighed 0.45-µm filter paper, rinsing with dH2O and drying at 80 °C overnight to a constant level. The correlation was found as shown below: Dry weight (g L–1) = 0.4001 × OD540, R² = 0.9909
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(1)
The specific growth rate µ during the exponential growth phase was measured by using Eq. (2) (Zhu et al., 2013a): µ (day–1)= ln (N2/N1)/(t2–t1)
(2)
where N1 and N2 are dry biomass weight (g L–1) at time t1 and t2, respectively. The biomass productivity (Pb) was measured using the following expression: Pb (mg L–1 day–1)= (DWi – DW0)/(ti–t0)
(3)
where DWi and DW0 are dry biomass weight (g L–1) at time ti and t0, respectively. The lipid productivity (Pl) was determined, according to the following equation: Pl (mg L–1 day–1)= Pb × Cl/100
(4)
where Cl represents lipid content (% of dw).
2.5. Harvest and drying
Microalgal cells from each flask were collected after 10-day growth, and centrifuged at 5000 rpm for 15 min. Supernatants were decanted and cell pellets were at 80 °C overnight to a constant level. The dried microalgal biomass was collected and sealed in empty containers for lipid extraction.
2.6. Lipid extraction
According to Zhu et al. (2014), the following steps were applied to determine the lipid content. 100–150 mg freeze-dried algal samples were weighed and extracted with 2 mL methanol containing 10% dimethyl sulfoxide (DMSO) in a water bath shaker at 45 °C for 45 min. The mixture was centrifuged at 3000 rpm for 10 min. Then, the supernatant was 6
collected and leftover was re-extracted twice following the same process. Afterwards, the leftover was extracted with 4 mL mixture of hexane and ether (1:1, v/v) in a water bath shaker at 45 °C for 60 min. The mixture was centrifuged at 3000 rpm for 10 min. Then, the supernatant was collected and leftover was re-extracted twice following the same process. All the supernatants were combined, after which 6 mL water was added to the incorporated extracts to form a ratio of methanol with 10% DMSO, diethyl ether, hexane and water of 1:1:1:1 (v/v/v/v). The organic phases with lipids were transferred into a pre-weighed glass tube and evaporated to dryness under nitrogen protection. Subsequently, the lipids were continuously dried at 80 °C overnight to a constant level. Afterwards, the total lipids were determined gravimetrically, and lipid content was expressed as % of dry weight.
3. Results and discussion
3.1. Microalgal growth
The growth characteristics of Chlorella sp. under five nutrient concentration levels within ten days were illustrated in Fig.1. These curves indicated all algal growth phases in batch culture except the lysis phase. The lack of a visible lysis phase was due to a short cultivation period in this study. In all cultures the lag phase lasted around 3 days. In the lag state, microalgal cells needed to adapt to the new environment with livestock waste compost media which were quite different from BG 11 medium. Meanwhile, microalgal cells were sensitive to light intensity in the beginning, since photoinhibition might occur due to a low cell concentration and thus lack of self-shading (Zhu et al., 2014; Sutherland et al., 2016). 7
However, photoadaptation to light intensity also took place (Belzile and Gosselin, 2015), which was beneficial to the growth of microalgal cells. After lag phase, exponential growth in all cultures lasted around 4 days, followed the stationary phase. During the exponential phase, microalgae experienced a fast growth and biomass accumulation. Day 7 was the last day of exponential growth for all cultures. Similarly, Passero et al. (2014) pre-treated dairy wastewater by ultraviolet radiation for the cultivation of algae C. vulgaris, and found that the exponential growth phase duration was 5 days with the tenth day as the end of this period. After the lag phase, microalgae experienced a rapid growth with specific growth rate µ ranging from 0.275 to 0.375 day–1 and doubling time ranging from 2.52 to 1.85 days (Table 2). As the culture nutrient concentration increased, specific growth rate increased, while doubling time decreased. This is in line with the study by Ji et al. (2014), who suggested that the increase of monosodium glutamate wastewater concentration stimulated the increase of the specific growth rate of algae Chlorella vulgaris. The specific growth rate µ of 1.26 day–1 was achieved by Atta et al. (2013), who grew Chlorella vulgaris by using light-emitting diode in batch culture for 8 days. The final biomass increase and productivity in the livestock waste compost medium with initial COD concentration at 2000 mg L–1 were the highest among the compost media, correspondingly reaching 2.888±0.076 g L–1 and 288.84±8.05 mg L–1 day–1, while the culture in 500 mg L–1 COD medium showed the lowest biomass increase and biomass productivity, reaching 1.842±0.051 g L–1 and 184.22±6.56 mg L–1 day–1, respectively. The biomass productivities in this study were similar to those in the study by Tan et al. (2016), who achieved 110.63–375.71 mg L–1 day–1 biomass when they cultured Chlorella pyrenoidosa with the effluent of anaerobically digested activated sludge. In contrast, applying highly efficient light-emitting diode-based photobioreactor to cultivate
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Chlorella vulgaris, Fu et al. (2012) obtained the maximum biomass increase of 20 g L–1 and productivity of 2.11 g L–1 day–1.
3.2. Lipid content and productivity
The lipid contents of Chlorella sp. in five cultures with different nutrient concentrations were determined and exhibited in Fig. 2. The culture with the initial COD concentration at 500 mg L–1 experienced the highest algal lipid accumulation, reaching 44.30% of dry weight. The reason why algal cells in culture with low initial nutrient concentration received more lipid accumulation might be due to the fact that low biomass concentration in the culture could make more algal cells access and receive more light, which triggered and benefited lipid storage. This finding is in line with preceding studies by Seo et al. (2015) and Mandotra et al. (2016), who suggested that the increase of the amount of light available within the optimal range could favor the accumulation of lipids in algal cells. The results also showed the initial nutrient concentration affected lipid accumulation of microalgae in this study. As the initial COD concentration increased from 500 to 2680 mg L–1, the lipid content decreased from 44.30% to 33.90%. This tendency in the variation of lipid contents in microalgal cells was found to be in accordance with the study conducted by Zhu et al. (2013b), who cultivated Chlorella zofingiensis with piggery wastewater under six different nutrient concentration levels. Fig. 2 also exhibited the lipid productivities of Chlorella sp. in five cultures. The findings demonstrated that the productivities of lipids in five cultures ranged from 81.61 to 104.89 mg L–1 day–1, following the relationship 2000 mg L–1 COD culture (104.89) > 1500 mg L–1 COD culture (92.16) > 2680 mg L–1 COD culture (86.83) > 1000 mg L–1 COD 9
culture (84.76) > 500 mg L–1 COD culture (81.61). The findings were evidently higher than 49.1 ± 0.41 mg L–1 day–1 lipid productivity achieved by Arora et al. (2016), who optimized N and P concentration for attainment of maximum lipid productivity using oleaginous microalgae C. minutissima. Han et al. (2013) acquired the maximum lipid productivity of 115 mg L–1 day–1 via the culture strategy of semi-continuous cultivation of C. pyrenoidosa with nitrogen limitation and pH regulation by CO2. In this investigation the variations in lipid productivity of Chlorella sp. among different cultures mainly attributed to their biomass differences, since the lipid content deviations within different cultures were not so wide in contrast to the biomass. Although the lipid contents of Chlorella sp. in 1000 and 500 mg L–1 COD cultures were high, their biomass productivities presented as a limiting factor, thus causing relevantly low productivities of lipids. Our previous investigation showed that around one third to one fourth of lipids could be effectively converted into fatty acid methyl esters, the efficient ingredients of biodiesel (Zhu et al., 2013ab; Zhu et al., 2014). The reason is that some lipid types such as phospholipid, glycolipid and chlorophyll are not the efficient ingredients for the production of biodiesel and thus cannot be converted into biodiesel. Based on this estimation, the expected biodiesel productivity in this study will range from 20 to 35 mg L–1 day–1, following in the sequence 2000 mg L–1 COD culture (26–35) > 1500 mg L–1 COD culture (23–31) > 2680 mg L–1 COD culture (22–30) > 1000 mg L–1 COD culture (21–28) > 500 mg L–1 COD culture (20–27).
3.3. Scale-up potential and challenges
3.3.1. Source availability
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Microalgal production requires large amount of nutrients. To avoid the negative environmental impacts of chemical fertilizer application, livestock waste compost can offer a cleaner medium for the cultivation of microalgae on a large scale. In total, every year around 56 billion livestock such as pig, cattle, lamb, duck and chicken are cultivated worldwide to meet the meat consumption for human beings (Zhu and Hiltunen, 2016). Due to the increasing meat demand especially in developing countries, the number of livestock domestication is going to robustly increase, and intensive animal farming will prevail globally. Therefore, abundant livestock wastes are available, and the compost can meet the nutrient demand for microalgal cultivation upscaling. According to Choi et al. (2014), in the EU-27 countries over 1500 million fresh tons of livestock wastes are produced annually, while in USA around 35 million dry tons of livestock wastes are generated every year. Taking China as an example, more than 4.8 billion tons of livestock manures are generated annually, and the number is going to gradually increase (Schneider, 2016). To summarize, from the nutrient source point of view, there is a guarantee for the large-scale production of microalgal biomass.
3.3.2. Environmental advantages Environmental merits achieved through pollution control and CO2 mitigation should be included as advantages in practice. This study shows that Chlorella sp. can be grown with livestock waste compost, providing a potential method for waste management. In addition, no fertilizers need to be employed to a microalgae system, presenting as an added beneficial value for this approach. There is no denying the fact that CO2 is released during composting, but the amount is found to be lower in contrast to the production of chemical fertilizers. It is suggested that 4–67 and 4–82 kg of CO2 can be reduced by composting one ton of garden 11
waste and food waste, respectively (Boldrin et al., 2009). Additionally, CO2 emitted during composting can be re-absorbed by microalgae. All of these factors can add values to microalgal production with livestock waste compost.
3.3.3. A scale-up roadmap Upscaling the cultivation of Chlorella sp. with livestock waste compost is possible in practice. In an effort to promote microalgal biodiesel production especially in terms of economics, a scale-up roadmap for Chlorella sp. production using livestock waste compost is proposed, as illustrated in Fig. 3. The post-extracted algal residues can be used to produce either biogas via anaerobic digestion or fertilizers. Alternatively, the leftovers or residuals appeared at any step can also be recycled into microalgal cultivation systems as the nutrient sources. In addition, apart from biodiesel, biogas and fertilizer, as exhibited in Fig. 3, valueadded bioproducts such as bio-ethanol, high-value proteins, cosmetics, food, feed and fine chemicals can be further produced through algal biorefinery. This paper can potentially serve as a starting point for authorities, stakeholders and practitioners to conduct the growth of microalgal biomass with livestock waste compost. Energy companies like the Finnish Fortum Corporation might potentially benefit if the upscaling was successful. In addition, this research might help livestock farms or enterprises (e.g., Pellon Group) solve the waste problems, putting forward an efficient solution for waste management.
3.3.4. Main uncertainties With the contribution of this investigation, the literature will no longer indicate a lack of empirical information on microalgal cultivation with livestock waste compost. According to the batch culture studies, microalgae can be successfully and robustly grown with livestock 12
waste compost. However, several main uncertainties also exist during the upscaling process. First, certain competitive microorganisms such as pathogens and bacteria from compost might be introduced into algal cultivation system, potentially hindering the growth. Thus, pretreatment methods such as sterilization should be considered, which might also affect the upscaling process. Second, chemical profiles of animal wastes vary significantly among various sources, which will largely impact the production strategy and management. This study investigates algal cultivation with cattle manure compost, but further investigation still needs to be done when other types of animal waste compost are applied. Third, different algal species exhibit distinct growth properties during the cultivation with compost. Thus, it is necessary to isolate the most suitable strains that show robust growth in compost media. Finally, algal cultivation with livestock waste compost requires complicated system integration to reduce the energy consumption and production costs. System operation is involved with many steps, such as energy input, cleaning, sterilization and maintenance, all of which need to be well planned before facility installment. To conclude, future research and development is still required in order to move the upscaling process forward.
4. Conclusions
The specific growth rate of Chlorella sp. grown in five cultures ranged from 0.275 to 0.375 day–1 with the doubling time from 2.52 to 1.85 days. Initial nutrient concentration could affect the lipid accumulation, and the lipid content ranged from 33.90% to 44.30%. The lipid productivity ranged from 81.61 to 104.89 mg L–1 day–1. The 2000 mg L–1 COD culture presented as the optimal medium for algal cultivation, since the highest biomass and 13
lipid productivities were realized. Scale-up of the process is greatly possible but requires further investigation.
Acknowledgements
This work was supported by the Kone Foundation Project in Finland and TransAlgae Project from EU’s Botnia-Atlantica programme. It was also supported by the Ministry of Housing and Urban-Rural Development (No.2016K4032), China Postdoctoral Science Foundation (No.2015M580644, 2016M592339). In addition, the authors are grateful to the support from Wuhan International Science and Technology Cooperation Project (2016030409020221). The authors would like to thank the anonymous reviewers for their helpful comments and suggestions that greatly improved the manuscript.
References
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Figure 1. Growth characteristics of Chlorella sp. cultivated under five nutrient levels within ten days (means ± sd).
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Figure 2. Lipid contents and productivities of Chlorella sp. biomass cultured in different nutrient concentrations of livestock waste compost media within ten days (means ± sd).
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Figure 3. Proposed scale-up roadmap for the cultivation of Chlorella sp. using livestock waste compost for biofuels production. This image is meant to show the linkages between each process, but the scheme eludes to the physical layout of the combined approach, as the microalgal production facilities must be located close to the livestock waste compost point.
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Table 1 Characteristics of livestock waste compost medium used in the experiments (means ± sd). Parameter Concentration pH 6.9±0.0 –1 Suspended Solid (mg L ) 286 ± 17 COD (mg L–1) 2680±51 + –1 NH4 (mg L ) 78.8±4.0 TN (mg N L–1) 222.0±3.0 TP (mg PO4–3 –P L–1) 103.0±4.0 –1 K (mg L ) 124±12 Cu (mg L–1) 8.2±0.9 –1 Zn (mg L ) 3.9±0.3
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Table 2 Growth parameters of Chlorella sp. in flasks under five concentration levels within ten days (means ± sd). Initial nutrient Biomass Doubling Biomass Specific growth concentration (mg L–1 increase time productivity rate µ (day–1) (g L–1) (days) (mg L–1 day–1) COD) * 2680 0.375±0.005 1.85±0.02 2.562±0.117 256.18±11.66 2000 0.357±0.015 1.94±0.08 2.888±0.076 288.84±8.05 1500 0.321±0.015 2.17±0.10 2.523±0.189 252.30±18.92 1000 0.306±0.015 2.27±0.11 2.066±0.065 206.64±6.95 500 0.275±0.008 2.52±0.07 1.842±0.051 184.22±6.56 BG11 medium 0.346±0.013 2.01±0.08 2.924±0.012 292.43±1.25 * COD concentration was applied to present the initial nutrient concentration in all cultures in this study.
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HIGHLIGHTS 1. Cultivation of microalgae Chlorella sp. with livestock waste compost was investigated. 2. Biomass productivity ranged from 184.22 to 288.84 mg L–1 day–1. 3. As the initial nutrient concentration decreased, the lipid content increased. 4. Lipid productivity ranged from 84.76 to 104.89 mg L–1 day–1. 5. The livestock waste compost medium with 2000 mg L–1 COD was optimal for algal growth
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