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Integrated Haematococcus pluvialis biomass production and nutrient removal using bioethanol plant waste effluent
Accepted Manuscript Title: Integrated Haematococcus pluvialis biomass production and nutrient removal using bioethanol plant waste effluent Authors: Fatima Haque, Animesh Dutta, Mahendra Thimmanagari, Yi Wai Chiang PII: DOI: Reference:
S0957-5820(17)30194-5 http://dx.doi.org/doi:10.1016/j.psep.2017.06.013 PSEP 1094
To appear in:
Process Safety and Environment Protection
Received date: Revised date: Accepted date:
18-2-2017 12-6-2017 19-6-2017
Please cite this article as: Haque, Fatima, Dutta, Animesh, Thimmanagari, Mahendra, Chiang, Yi Wai, Integrated Haematococcus pluvialis biomass production and nutrient removal using bioethanol plant waste effluent.Process Safety and Environment Protection http://dx.doi.org/10.1016/j.psep.2017.06.013 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.
Integrated Haematococcus pluvialis biomass production and nutrient removal using bioethanol plant waste effluent
Fatima Haque1, Animesh Dutta1, Mahendra Thimmanagari2, Yi Wai Chiang1*
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School of Engineering, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada,
N1G 2W1 2
Ontario Ministry of Agriculture, Food and Rural Affairs, 1 Stone Road West, Guelph,
Ontario, Canada, N1G 4Y1 *
Corresponding author; Email: [email protected]; Tel.: +1-519-824-4120 ext: 58217
Graphical abstract
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Highlights
Enhanced microalgal biomass production was achieved using air-lift photobioreactor. 5% CO2 supplementation resulted in maximum biomass density and specific growth rate. Wastewater made environmentally safe with 91.67% TN and 100% TP removal. Astaxanthin-extracted H. pluvialis biomass is a potential bioenergy feedstock. Integrated H. pluvialis cultivation-ethanol production is economically beneficial.
Abstract: The integrated system of astaxanthin and microalgal biomass production, and wastewater treatment is a promising process. Haematococcus pluvialis is a suitable microalgae for this coupled system to achieve this dual purpose in a single approach. In this study, biomass production and nutrient removal from bioethanol plant wastewater by H. pluvialis were investigated. An air-lift photobioreactor was used to utilize the wastewater as well as a CO2enriched gas supply. The maximum biomass density and maximum specific growth rate achieved were 4.37 ± 0.007 g/l and 0.317 day-1, respectively, by culturing H. pluvialis in an air-lift photobioreactor, supplemented with 5% CO2 in air. Removal of 91.7 % total nitrogen and 100 % total phosphorous from the wastewater was achieved. The residual microalgal biomass, obtained after astaxanthin extraction (1.109 ± 0.009 mg/g DW), was characterized as a potential bioenergy feedstock due to its elevated higher heating value 15.6 ± 0.01 MJ/kg. Hence, integrating H. pluvialis cultivation with bioethanol effluent serves the combined purpose of wastewater treatment, CO2 utilization, and simultaneous production of
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astaxanthin, which has a potential market value of over USD 2000 per kg, and carbohydrate rich microalgal biomass, which can be applied to bioenergy production.
Abbreviations AL
Air-lift
BBM
Bold basal media
BOD
Biological oxygen demand
COD
Chemical oxygen demand
CAPEX
Capital expenditure
DW
Dry weight
HHV
Higher heating value
MC
Microcystin
OPEX
Operational expenditure
PBR
Photobioreactor
PC
Process condensate
TGA
Thermo gravimetric analysis
TN
Total nitrogen
TOC
Total organic carbon
3
TP
Total phosphorous
TS
Thin stillage
VM
Volatile matter
µ
Specific growth rate
Keywords: Haematococcus pluvialis, bioethanol wastewater, air-lift photobioreactor, astaxanthin, bioenergy.
1. Introduction Haematococcus pluvialis is a freshwater unicellular microalgae belonging to the Chlorophyceae family (Chekanov, 2014). Recently, this microalgae has gained attention because of its potential to efficiently produce 0.5 – 4% (dry weight) of astaxanthin, an antioxidant, which is predominantly higher as compared to other natural sources like Pandalus borealis (shrimp) and Paracoccus carotinifaciens (macroalgae), which produce 0.1% and 2.2% astaxanthin, respectively (Jiao et al., 2015; Schmidt et al., 2011). Astaxanthin (C40H52O4) is a xanthophyll ketocarotenoid, which possess strong antioxidant properties. The conjugated double bonds, present in the structure, donate the electrons that react with free radicals, and hence terminates the free radical chain reaction (Higuera-Ciapara et al., 2006). As a result, it finds application as a coloring agent, nutraceutical, and pharmaceutical compounds (Giannelli et al., 2015). Astaxanthin is produced during the second stage of the
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life cycle of H. pluvialis, whereas the green vegetative cells exist during the first stage. Under environmentally stressed conditions, such as light, nitrate or salinity, astaxanthin accumulation takes place. During these cell stress conditions, there is an increase in the lipid content of H. pluvialis and astaxanthin is produced inside the TAG-rich globules (Razon and Tan, 2011; Zhekisheva et al., 2002). These astaxanthin-rich red cysts are surrounded by thick sporopollenin cell walls, which comprises of 53 - 70% carbohydrates and 6-19% proteins (Hagen et al., 2002). Hence, H. pluvialis is a good source of astaxanthin as well as lipid and carbohydrate rich microalgal biomass. Though astaxanthin constitutes a minute amount of the total biomass, it has a great potential market value of over USD 2000 per kg of astaxanthin (Li et al., 2011). For an effective production of this valuable product, high density H. pluvialis culture is a pre-requisite, and this depends on the nutrient composition of the growth media, and the choice of photobioreactor (PBR). Different types of photobioreactor has been used to cultivate algae, such as bubble column reactor (Ranjbar et al., 2008) and air-lift reactor (Kaewpintong et al., 2007). H. pluvialis cells are shear sensitive, so mechanically stirred reactor is not a desirable option, and pneumatic agitation is preferred. For example, Kaewpintong et al. (2007) studied the cultivation of H. pluvialis in basal medium using air-lift photobioreactor (AL-PBR) and supplemented with 1% CO2, and was able to achieve 0.31 day-1 of specific growth rate. B. Santhose et al. (2014) cultivated a new strain of H. pluvialis in modified BBM media in an AL-PBR and was able to achieve 3g of dried algal biomass from 2.5 L of culture. Ranjbar et al. (2008) was able to achieve 5 g/l of algal biomass by cultivating H. pluvialis in basal media and using a continuous supply of CO2 in a bubble column reactor.
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The main purpose of this study is to examine the integrated system of H. pluvialis cultivation in bioethanol waste effluent in an AL-PBR. In a previous study, Haque et al. (2016) investigated the H. pluvialis culture in bioethanol waste stream (thin stillage and process condensate), and a growth media denoted as the GroAst media, which supported astaxanthin production, was formulated. The first objective of this study is to attain high density H. pluvialis culture in GroAst media using AL-PBR supplemented with CO2 and to access the environment impact of the treated wastewater henceforth. The second objective is to analyze the bioenergy potential of the residual H. pluvialis biomass obtained after astaxanthin extraction, as microalgae is seen as a potential bioenergy feedstock in recent years (Hannon et al., 2010). 2. Materials and Methods 2.1. Microorganism, media, and cultivation condition H. pluvialis CPCC 93 strains were obtained from the Canadian Phycological Culture Centre. The stock cultures were maintained on modified M1B5, a modification of the media used by Tocquin et al. (2012): 0.5 ml/L Flora Micro and 2.5 ml/L Flora Bloom (General Hydroponics). The cultures were maintained at pH 7 and incubated at 23 ± 2°C, 12h/12h light/dark cycle (600W MH light) with gentle agitation twice a day. The culture media used for the experiment was prepared using the wastewater procured from a local bioethanol plant, where corn kernels are used as the raw material. The wastewater samples used were the thin stillage (TS) obtained from the distillation unit, and the process condensate (PC) obtained from the evaporation unit of the production plant. As reported in
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the previous study, 60 times diluted TS (denoted as modified TS) supported microalgal growth, and addition of PC coupled with continuous light resulted in astaxanthin induction. Hence this formulation of 60% modified TS and 40% PC, denoted as the GroAst media, is used for H. pluvialis cultivation (Haque et al., 2016). 2.2. Enhanced Biomass Production of H. pluvialis 2.2.1. Experimental set-up H. pluvialis culture was carried out in a 2.2 L AL-PBR. Figure 1 demonstrates the experimental set-up. The reactor column was made of glass (11.2 cm internal diameter and 22.5 cm height), with a draft tube (5.6 cm internal diameter and 11 cm height) centrally installed. The draft tube was made from silicone tubing with a rubber gasket base to hold the tube in position (Figure 1a). Prior to each experiment, the column reactor and the draft tube were sterilized by spraying with isopropyl alcohol (70%). The CO2 gas cylinder (purity 99.99%, Linde) and the air pump (Tetra, Whisper 10) were connected as shown in the Figure 1b, and the gas flow was controlled by an individual panel-mounted flow meter with control valve (Key Instruments, Cole Parmer, CA). The AL-PBR was placed inside a temperature controlled, illuminated 3’ X 3’ Expert growth tent at 23 ± 2°C and 12h/12h light/dark cycle (600W MH light). The growth tent is made of reflective 600D Mylar fabric, which helped maintain even lighting conditions and prevented the effect of any other light source on the reactor. The distance between the light source and the AL-PBR was maintained at 70 cm. The average illumination intensity incident to the outer surface of the reactor was measured using a digital lux meter (Fisher Scientific) and it was maintained at 15 klx for growth conditions (green phase) and 30 klx for astaxanthin induction (red phase).
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2.2.2. Effect of CO2 on microalgal growth CO2 not only acts as a carbon source for the photoautotrophic growth of H. pluvialis but the gas bubbles help in providing sufficient mixing inside the PBR. The concentrations of CO2, in the bubbled gas, studied were 2.5, 5.0 and 10.0 vol%. For the control, only compressed air (0.04% ambient CO2) was introduced inside the AL-PBR. 1.5 L of GroAst media was inoculated with 10% (v/v) four-day old seed culture and the initial pH of the media was maintained at 7. The flow rate of the gas was maintained at 45 ml/min, which provides sufficient mixing to keep the cells in suspension. Astaxanthin was extracted and quantified by employing the ultrasonication method as reported in Haque et al. (2016). The microalgal samples were centrifuged (Thermo Scientific Sorvall ST 24) at 5000 x g for 15 min. Cells were dried overnight at room temperature, before carrying out ultrasonication (VC 750, Sonics & Materials Inc.) in sodium hydroxide (2M) for 25 min, using a 1.3 cm diameter probe (horn) at 35% amplitude and 20 kHz operating frequency. Ultrasonicated samples were centrifuged again, followed by extraction using methanol (99.8%). All chemicals used are Fisher Scientific analytical grade. 2.3. Biomass density estimation For the analysis of the biomass density, 10 ml of the microalgal broths were centrifuged at 5,000 g for 15 minutes; the supernatant was discarded, and the dry weight was determined, using an analytical balance (Mettler Toledo), after drying the cell pellet overnight at room temperature. Specific growth rate (µ day-1) was determined using the following formula (L. Shuler and Kargi, 2002):
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µ=
ln (DW𝑖 ) – ln (DW0 ) t𝑖 − t0
Where DW0 and DWi are the dry weight (g/l) of the microalgal sample on the initial day (t0) and ith day (ti), respectively. Also, pH of the media was measured, using a pH meter (Oakton, Cole - Parmer, CA), at the end of the lag phase, the exponential phase, and the stationary phase to analyze the effect of CO2 concentration on the pH of the media. 2.4. Assessment of the treated wastewater 2.4.1. Analysis of the wastewater The wastewater samples were characterized on the basis of pH, total nitrogen (TN) (Hach method 10071), total organic carbon (TOC) (TOC analyzer, Shimadzu), total phosphorous (TP) (Hach method 8190) and acetic acid content (Siedlecka et al., 2008). These parameters were tested for: a) TS and PC initially; b) modified TS (60 times diluted), i.e., GroAst media for the green phase; c) GroAst media for the red phase, i.e., when 40% PC is added to 60% modified TS; and d) residual media at the end of the experimental run. Nutrient removal (%) was calculated as:
% Nutrient Removal =
[Nutrient]𝑓𝑖𝑛𝑎𝑙 − [Nutrient]𝑖𝑛𝑖𝑡𝑖𝑎𝑙 ∗ 100 [Nutrient]𝑖𝑛𝑖𝑡𝑖𝑎𝑙
Where, [Nutrient]final and [Nutrient]initial are the nutrient composition (g/l) of the GroAst media finally and initially, respectively. 2.4.2. Toxin detection test
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The European Food and Safety Authority (EFSA) have reported about the generation of a cyanotoxin, microcystin (MC), by H. pluvialis (EFSA, 2014). For the safe discharge of the treated wastewater into the environment, the presence of MC was tested. The EnviroLogix QuantiPlate Kit was used for the quantitative laboratory detection of MC toxin in the microalgal samples, which uses the principle of a competitive Enzyme–Linked Immuno Sorbent Assay (ELISA). The experiment was carried out as mentioned in the kit manual (Catalog number EP 022, EnviroLogix), using a negative control and standard Microcystin (0.16 ppb, 0.6 ppb, and 2.5 ppb) as calibrators. The optical density (OD) was measured at 450 nm using a UV-VIS spectrophotometer (Multiskan Go Microplate Spectrophotometer, Thermo Fisher Scientific). A standard curve was obtained by plotting % Bo and the calibrator’s concentration on a semi log graph, where: % B𝑜 =
Average OD of calibrator or sample ∗ 100 Average OD of negative control
The calibration curve was used to determine the microcystin concentration in the algal samples. The green as well as the red cells of H. pluvialis, cultured in modified M1B5 and in GroAst media were tested for microcystin presence. The detection limit of this ELISA kit is 0.16 ppb MC. 2.5. Residual microalgal biomass characterization 2.5.1. Proximate and Ultimate analyses, and Higher Heating Value ASTM standards were used to perform the proximate analysis (ash, volatile matter and fixed carbon) of the solid residual biomass. A measured amount of the sample was placed in a
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muffle furnace (Thermo Scientific F48055-60, Waltham, MA) maintained at 103 ± 2°C for at least 15 h and then cooled in a desiccator containing silica gel. The samples were then reweighed and the change in sample weight was expressed as percentage moisture (ASTME871). The dried samples were then burnt in air at 575°C for 5 h in the muffle furnace to determine percentage ash (ASTM-E1755). For determining the volatile matter (VM), the samples were heated in inert atmosphere at 950°C for 7 min (ASTM-E872). The fixed carbon content was calculated as the difference between 100% and the three other percentages (moisture, volatile and ash), according to ASTM-D7582. A Thermo Fisher Flash EA 1112 (Waltham, MA) elemental analyzer was used to perform the ultimate analysis (carbon (C), hydrogen (H), nitrogen (N), sulphur (S), and oxygen (O)) of the samples. The Higher Heating Values (HHV) of the samples were determined using an IKA-C200 bomb calorimeter (Wilmington, NC). Approximately 0.5g of the sample was placed in the steel container fitted with a ceramic crucible. Pure oxygen gas at 30 bar was used to pressurize the adiabatic vessel, and a cotton thread connected to an ignition wire in the steel container was used to ignite the sample. 2.5.2. FTIR Analysis FTIR spectrum of the dried microalgal sample was recorded on a Nicolet 4700 FTIR spectrometer (Thermo Scientific) at room temperature. The sample was pressed with potassium bromide powder and a wavelength spectrum of 4000-400 cm-1 was used for scanning.
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2.5.3. Thermal Analysis To study the combustion behavior of the residual biomass sample, thermogravimetric analysis (TGA) was performed. The dried residual biomass was ran in a TG/DT analyzer (Thermo Scientific Nicolet 700 FTIR) in nitrogen atmosphere at heating rate of 10 °C/min. Approximately 10 mg of the sample was heated from ambient temperature to 750 °C. Nitrogen gas of high purity (99.99%) was supplied at a constant flow rate of 100 ml/min as an inert purge gas. Nitrogen gas eliminates unwanted oxidation of the sample by displacing the air in the pyrolytic zone. The sample weight (W) as a function of temperature (T) is recorded and plotted; derivative TGA (DTG) curves (dW/dT) were also plotted. The volatile compounds evolved during the TGA process were analyzed using a coupled FTIR (Thermo Scientific Nicolet 700 FTIR). 2.6. Data Analysis All readings were taken in triplicates and mean results were represented along with standard deviation. The data in the graphs have been represented along with standard deviation interval bars. 3. Results 3.1. Photobioreactor performance Figure 2 shows the biomass density curve at different CO2 concentrations. It can be seen that as the CO2 concentration is increased from atmospheric to 5%, there is an increase in microalgal density. For 5% CO2 concentration, maximum growth of 4.37 ± 0.07g/l (DWmax) was obtained, and total astaxanthin content (TAC) extracted was 1.109 ± 0.009 mg/g DW.
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Table 1 presents values of TAC at other CO2 concentrations. Samples cultivated with 2.5% and 5% CO2 gas supply showed similar growth patterns. As the CO2 concentration is increased to 10%, a decline in the growth occurs, suggesting that more CO2 than required for sufficient growth is detrimental to cell survival. Samples grown under atmospheric CO2 concentration gas supply showed less growth compared to the samples supplied with 2.5% and 5% CO2, hence CO2 availability was the limiting factor for culture growth. In comparison to the cultivation method used in the previous study (Haque et al., 2016), where H. pluvialis was cultured in erlenmeyer flasks without aeration, AL-PBR provides better mode for wastewater as well as CO2 utilization, with aeration resulting in 84.3% increase in biomass density. The change in the pH of the GroAst media under the effect of different concentration of CO2 is summarized in Table 1, along with the specific growth rates (µ). When only air is supplied, the pH increased from 7.00 to 9.64 by the end of the stationary phase and a biomass density of 2.028 ± 0.09 g/l was obtained. However, when the growth media was supplied with 5% CO2, the pH changed to 6.32 at the end of the experimental run and the maximum biomass density was achieved on the 11th day (T max) with a specific growth rate of 0.317 day-1. 10% CO2 supply resulted in an acidic media (pH 3.91) and a stunted microalgal growth. 3.2. Environmental impact assessment of wastewater Table 2 shows the nutrient composition of the wastewater samples (TS and PC) and the GroAst media used for astaxanthin production, along with the amount of nutrient removal at the end of the experimental run (residual media). The modified TS sample has similar nutrient composition to that of the standard media, modified M1B5. The GroAst media contained
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0.215 g/l of acetic acid, which is absent in the standard media, and hence additional supplement of acetic acid is not required. H. pluvialis culture resulted in 91.7% total nitrogen (TN) removal and 100% total phosphorus (TP) removal. Astaxanthin synthesis also results in 42.3% acetic acid being consumed, and the final media contained 33.0% of the initial total organic carbon (TOC).
The concentration of microcystin in the H. pluvialis sample has been tabulated in Table 3, along with the % Bo values. H. pluvialis samples show a % Bo value in the range of 92.01– 93.05 %, and, the samples had negative results (below detection limit) for the microcystin test. Hence, the treated wastewater is devoid of microcystin, and safe for discharge. 3.3. Assessment of the residual H. pluvialis biomass 3.3.1. Proximate and Ultimate Analyses The characterization of H. pluvialis biomass as a biofuel feedstock is itemized in Table 4. The ash content, volatile matter content, moisture content and fixed carbon content of the algal biomass is 5.43 ± 0.25%, 71.28 ± 0.27%, 7.93 ± 0.12% and 15.36 ± 0.56%, respectively. The ultimate analysis shows that the residual biomass has 43.57% carbon, 6.26% hydrogen, 48.04% oxygen, 1.97% nitrogen and 0.47% sulphur. This helps to understand the elemental composition of the sample, and the empirical formula of the residual algal biomass is found to be C3.63 H6.26 N0.14 S0.01 O3. The H/C an O/C molar ratios (on the ash free dry basis) were calculated from the elemental composition as 1.72 and 0.83, respectively. The HHV of the
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astaxanthin-rich un-extracted algal biomass is measured to be 22.78 MJ/kg, whereas the HHV of the post-extracted residual biomass is 15.60 MJ/kg. 3.3.2. FTIR Spectra FTIR spectroscopy helps in the rapid analysis of microalgal biomass and offers a viable screening approach to determine the bioenergy potential of microalgae. The FTIR spectra of the astaxanthin-rich biomass and the residual biomass are shown in Figure 3. The identification of the unknown components is derived by comparing the bands of the recorded FTIR spectrum with those available in the literature (Bi and He, 2013; Ceylan and Goldfarb, 2015; Sudhakar and Premalatha, 2015). In the spectrum of the residual biomass, the band at 3,370 cm-1 is due to the O-H stretching, primarily attributable to water and hydroxyl groups. The weak band centered at 2,920 cm-1 is due to the presence of asymmetric C-H stretching vibrations, which is likely due to the lipid’s methylene group. A sharp peak at 1,630 cm1
corresponds to C=O amide stretching from proteins present in the residual algal biomass.
The peak spanning between 1,800 – 1,500 cm-1 is the characteristic bands for proteins and 1,450 – 1,400 cm-1 is likely due to C-H stretching vibration in methyl, methylene and methyne groups. The absorbance peak at 1,150 cm-1, 1,060 cm-1, and 1,010 cm-1 are likely the C-O and C-O-H deformation in the secondary and tertiary alcohol or aliphatic ethers. The C-O-H vibrational stretching suggests the presence of polysaccharides (Sudhakar and Premalatha, 2015). The intensity of the absorbance corresponding to polysaccharides (1,010 cm-1) is much higher than the one for the lipids (2,920 cm-1). Also, the intensity of the spectrum of the residual biomass is lower than the un-extracted biomass. There is a general decrease in the lipid and
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protein content as indicated in the 3,000-2,800 cm-1 region and 1,800-1,400 cm-1 region, respectively. The FTIR spectrum of H. pluvialis residual biomass marks distinct fingerprint regions for triglycerides, lipids and polysaccharides.
3.3.3. Thermal Analysis 3.3.3.1. TGA Figure 4 shows the TGA–DTG curves of the residual algal biomass. There are three distinct decomposition zones observed during the pyrolysis process of the microalgae. The dehydration zone is the initial stage, at around 110 ºC, where the weight loss occurs due to the loss of moisture from the biomass. The second zone is the devolatilization zone, where the biomass sample loses the volatile matter content. Two overlapped weight loss regions can be distinguished, one from approximately 250 ºC to 320 ºC and another from 320 ºC to 450 ºC. Active pyrolysis zone is the region between 110 ºC – 320 ºC, and 320 ºC – 420 ºC region is the zone of passive pyrolysis, which terminates at 450 ºC (Phukan et al., 2011). The DTG peaks occur at 300 ºC and 420 ºC, in the vicinity of 350 ºC, which is believed to be lipid loss and some lignocellulosic polymer decomposition. Major volatile matter loss occurs in the range: 72%; Sudhakar and Premalatha, (2015) suggests that cellulose decomposition usually occurs between 290 ºC – 390 ºC. Solid residue decomposition zone is the last stage, where slow decomposition of the solid residue, left from the preceding heating range, occurs. As seen from Figure 4, beyond 450 ºC slow loss of weight continues until about 750 ºC, but without any marked DTG peak. This degradation profile of the microalgal biomass suggests
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that it can be a potential bioenergy feedstock and maybe suitable for thermo-chemical conversion (Phukan et al., 2011). 3.3.3.2. Analysis of the gas evolved during TGA process The volatile compounds evolved during the TGA process were analyzed using FTIR. Figure 5 presents the 3D IR spectrum with respect to wavenumbers (cm-1), time (min) and absorbance. In the region between 2,000 – 2,300 cm-1 some significant peaks for C=O stretching of CO2 were obtained.
The regions between 4,000 – 3,400 cm-1 give the
characteristic signals for water and the main gaseous products of O2, CO, H2O and some organics (a mixture of acids, aldehydes, alkanes, and ethers), which is characteristic of pyrolysis end-products (Sudhakar and Premalatha, 2015).
4. Discussion High density cultivation of H. pluvialis in an AL-PBR. A high-density H. pluvialis culture was obtained using bioethanol waste stream, in an air-lift photobioreactor supplemented with 5% CO2. The specific growth rate (0.317 day-1) reported in this study is similar to the one reported by Kaewpintong et al. (2007), where the maximum growth rate of 0.45 day -1 was achieved for a continuous culture and 0.31 day-1 for a semicontinuous culture of H pluvialis in an air-lift reactor. The highest biomass density obtained was 4.37 ± 0.07 g/l, which is slightly lower than the one achieved by Ranjbar et al. (2008) who reported 5 g/l cell concentration weight using modified basal media and 1 L bubble
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column reactor, but more than the 2.62 g/l achieved by Jun et al. (2012), who cultivated H. pluvialis in a 6 L bag type reactor supplemented with 2% CO2. Both air and 10% CO2 supply resulted in a moderate growth, however, atmospheric CO2 supply resulted in a basic media (pH of 9.64) and 10% CO2 supply led to an acidic media (pH of 3.91). The relation between CO2 supply and algal growth can be attributed to the pH change. The CO2 supply/consumption ratio plays a major role in maintaining a suitable pH of the medium. At atmospheric CO2 condition, the CO2 consumption is higher than the CO2 supply, hence the pH of the medium increases to 9.64 at the end of the stationary phase. At 5% CO2 gas supply the rate of CO2 supply is balanced by the photosynthetic CO2 consumption, thereby the pH of the medium is close to 6.32. However, at 10% CO2 gas supply, the CO2 supply exceeds the CO2 consumption rate, hence the pH drops to a greater extent (3.91), and this carbonic acid-rich acidic medium do not support algal growth. It has been reported that a pH more than 10 or less than 5.5 is detrimental to the algal growth (Kang et al., 2005). The thin stillage (TS) has a high concentration of suspended solids (turbidity greater than 1000 NTU, (Haque et al., 2016)); hence, in this study, it was diluted 60 times dilution to reduce turbidity while still providing a sufficient amount of nutrients required for H. pluvialis growth. However, in an industrial plant, thin stillage could be centrifuged to obtain the distiller’s grain, which finds application as animal feed (Mustafa et al., 2000; Van Leeuwen et al., 2015), thus reducing or eliminating the need for dilution. Alternatively, treated water could be used as dilution water, thus reducing the water demand of dilution.
Environmentally safe water from wastewater.
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H. pluvialis successfully utilized inorganic wastes, 91.67% total nitrogen and 100% total phosphorous, from the wastewater sample. These results are in accordance with Wu et al. (2013), who reported 93.8% N removal and 97.8% P removal, when H. pluvialis were cultured in the domestic secondary effluent, and Ledda et al. (2016), who reported up to 99% removal for NO3-N and NH4-N, 98% for TP, and 26% for chemical oxygen demand, when H. pluvialis were cultured in swine slurry primary filtrate. Also, Kang et al. (2006) studied the biosynthesis of astaxanthin by cultivating H. pluvialis in primary treated sewage and primary treated piggery wastewater, and the author reported that 4-8 fold dilution of primary treated piggery wastewater resulted in 100% N and P removal at the end of the process. In addition to the inorganic nutrients, H. pluvialis cultivation resulted in 67% removal of TOC, thereby reducing the biological oxygen demand (BOD) and chemical oxygen demand (COD) of the wastewater. As excess BOD can deplete the dissolved oxygen of aquatic water, its removal is one of the primary aims of wastewater treatment, in order to protect the aquatic organisms (Abdel-Raouf et al., 2012). The samples showed negative results for the presence of microcystin toxin (detection limit 0.16 ppb). According to the WHO guidelines for drinking water quality, the guiding value for the total MC is 1 ppb (WHO, 1998). Also, in 2014, EFSA published a report on the safety of their astaxanthin product due to the absence of microcystin (EFSA, 2014). Hence, it can be seen that H. pluvialis cultivation not only helps in nutrient removal but also eliminates the threat of microcystin production, and thus the treated wastewater is free of cyanotoxins. This makes the discharge of wastewater into the water bodies safer, as this wastewater is devoid of nutrients and does not support algal growth. For example, Lake Erie (Canada-USA) suffers
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from severe toxic algal bloom because of the naturally occurring cyanobacteria that are fueled by the presence of warm water, sunshine and nutrient-rich industrial wastewater and agricultural run-off.
H. pluvialis as a potential bioenergy feedstock. The characterization of the microalgal biomass helps to understand its physio - chemical properties and to assess its bioenergy potential. The residual biomass has a satisfactory HHV of 15.6 MJ/kg, which is comparable to that of Chlorella sp. (15.88 MJ/kg) (Phukan et al., 2011). The high volatile matter content (71.28 ± 0.27%) and the TGA thermogram suggest that the residual biomass can be a potential feedstock for thermochemical conversions (Phukan et al., 2011). The elevated moisture content (7.9 ± 0.12 %) is not desirable as it reduces the heating value of the biomass, but the low ash content (5.43 ± 0.25 %) of the residual biomass is desirable as it has no heating value and it also affects both the handling and processing cost of the biomass energy conversion (Ceylan and Goldfarb, 2015; Sudhakar and Premalatha, 2015). The ultimate analysis showed that the residual biomass possesses low N and S values (C3.63H6.26N0.14S0.01O3), which is favorable as this limits the formation of NOx and SOx during the combustion process, and hence proves to be environmental friendly, similar to other biomass sources like rice husk (0.63% N, 0.041% S) and Laminaria saccharina (2.4% N and 0.7% S) (Chow et al., 2013; Olupot et al., 2016). Residual Haematococcus biomass has an H/C ratio (ash free basis of 1.72, which is lower than the H/C ratio of rice husk (1.95), higher
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than that of Laminaria saccharina, a brown microalgae (1.42), and similar to the H/C ratio of Chlorella, Nannochloropsis and Spirulina species (Chow et al., 2013; Phukan et al., 2011; Zhu et al., 2013). Microalgal biomass can be converted to bioenergy by using thermochemical conversion, which produces oil and gas as the end-products, or by using biochemical processes, where ethanol and biodiesel can be produced (Amin, 2009; Patil et al., 2008). Table 5 shows the comparison between these different conversion technologies in terms of the end-product formed, operating conditions and drying requirement, as moisture content plays a key role in deciding which conversion technology can be used. Thermochemical conversion is best suited for biomass with low moisture content whereas biochemical conversion is preferred for biomass with high moisture content. Along with the moisture content, it is important to understand the main component of the residual biomass; if it is rich in carbohydrate then it can be used in bioethanol production, and if it is rich in fatty acids then biodiesel can be produced. (Bi and He, 2013). FTIR spectrum (Figure 3) of the residual Haematococcus biomass showed high peaks for carbohydrates and lipids. Therefore, based on these characteristics, a selection matrix (Table 5) is created, to find the most suitable conversion technology. According to the selection matrix, the fermentation process is the most suitable option to convert the residual biomass to biofuel. The biomass has a high moisture content, however drying is not a necessary step for fermentation. Also, the FTIR spectrum showed a higher peak for carbohydrate compared to lipids, thus indicating that the biomass is a good source of carbohydrates. As the fermentation process requires a carbohydrate-rich raw material, the
21
Haematococcus biomass fits in this requirement. In this study, H. pluvialis is cultured in wastewater obtained from the bioethanol production plant, which uses fermentation technology to produce bioethanol. So, if the residual Haematococcus biomass is fed into the saccharification unit as an additional feedstock, then this biomass could be upcycled back to the process. This integrated approach of H. pluvialis cultivation in wastewater and residual microalgal biomass utilization is illustrated in Figure 6. Economic benefits of the integrated H. pluvialis cultivation. The major manufacturers of synthetic astaxanthin are Royal DSM N.V. in the Netherlands, BASF SE in France, and Zhejiang NHU Co. Ltd. in China. The estimated cost of production of synthetic astaxanthin is about USD 1000∙kg-1 and the total market value being more that USD 200 million per year (Olaizola, 2003). However, due to the food safety concerns related to the petrochemical-derived synthetic astaxanthin, the high production cost of synthetic astaxanthin, and a high market demand for natural astaxanthin, the biological sources of astaxanthin are seen as preferable ‘green solutions’ (Li et al., 2011). Li et al. (2011) performed a comprehensive cost analysis of astaxanthin production from H. pluvialis in Shenzhen, China, for a hypothetically scaled-up plant with a production capacity of about 900 kg astaxanthin per year. They estimated about USD 163∙kg-1 CAPEX and USD 555∙kg1
OPEX respectively, and a total production cost of USD 718∙kg-1 astaxanthin.
For the present study, the proposed process could be integrated into an established bioethanol production plant, with an existing infrastructure, thereby reducing the capital investment on the infrastructure required, land procurement and wastewater (medium) supply station, as well as operational expenditures such as procurement of media, carbon dioxide, and
22
sterilization of media. Hence, using the cost analysis data from the work of Li et al. (2011), an integrated process would save a minimum of 24% in CAPEX (savings on land acquisition and improvement, medium supply station for bioreactors, and CO2 storage tank, while maintaining building area and construction costs) and 22% in OPEX (savings on potassium nitrate, disodium hydrogen phosphate, sodium bicarbonate, carbon dioxide, water and wastewater treatment). Regarding wastewater treatment, the ethanol plant utilizes ozonation to presently treat its wastewater, so no additional cost is expected by the integration. The total production cost could thus be reduced by USD 159∙kg-1 astaxanthin. 5. Conclusions A high density (4.37 ± 0.07 g/l) H. pluvialis culture was obtained using the bioethanol production waste stream as the growth media, in an air-lift bioreactor supplemented with 5% CO2. The residual Haematococcus biomass showed potential to be used as a future bioenergy feedstock. This microalga can make use of a simple and inexpensive nutrient medium, wastewater, to grow. Also, H. pluvialis resulted in 91.67 % total nitrogen and 100 % total phosphorous removal. The FTIR analysis confirmed the presence of carbohydrates and lipids, and the thermal analysis presented a useful decomposition profile of the biomass. Hence, the assessment of the biomass with respect to bioenergy production proves it to be a potential feedstock. H. pluvialis cultivation can be coupled with CO2 capture from bioethanol production, wastewater reutilization, and production of high-value astaxanthin and microalgal biomass. This proposed process should be more environmentally friendly and economical, compared to conventional astaxanthin production processes, due to integrating microalgal culture in an exisiting bioethanol plant and utilizing the waste product generated
23
in the plant. The processing costs may be further reduced with advances of separation technologies and optimization of culturing processes. Acknowledgements This research was financially supported by the Ontario Ministry of Agriculture, Food and Rural Affairs (LAAIR2014-5089). The author would like to thank Greenfield Ethanol Plant, Chatham, Ontario, for providing the wastewaters, and Joanne Ryks, Ryan Smith, and Michael Speagle, from University of Guelph, for their help.
24
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Schmidt, I., Schewe, H., Jin, C., Buckingham, J., Sandmann, G., Schrader, J., 2011. Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces dendrorhous. Appl. Microbiol. Biotechnol. 89, 555– 571. doi:10.1007/s00253-010-2976-6 Shuler, M.L., Kargi, F., 2002. How cells grow, in: Bioprocess Engineering Basic Concepts. Prentice Hall, Upper Saddle River, NJ, pp. 162–164. Siedlecka, E.M., Kumirska, J., Ossowski, T., Glamowski, P., Gołębiowski, M., Gajdus, J., Kaczyński, Z., Stepnowski, P., 2008. Determination of Volatile Fatty Acids in Environmental Aqueous Samples. Polish J. Environ. Stud. 17, 351–356. Solovchenko, A., 2014. Accumulation of Astaxanthin by a New Haematococcus pluvialis Strain BM1 from the White Sea Coastal Rocks (Russia). Mar. Drugs 12, 4504–4520. doi:10.3390/md12084504 Sudhakar, K., Premalatha, M., 2015. Characterization of Micro Algal Biomass Through FTIR / TGA / CHN Analysis : Application to Scenedesmus sp . Energy Sources, Part A 37, 2330–2337. doi:10.1080/15567036.2013.825661 Tocquin, P., Fratamico, A., Franck, F., 2012. Screening for a low-cost Haematococcus pluvialis medium reveals an unexpected impact of a low N / P ratio on vegetative growth. J. Appl. Phycol. 24, 365–373. doi:10.1007/s10811-011-9771-3 Van Leeuwen, J., Khanal Kumar, S., Pometto, A.L., 2015. Purification of thin stillage from dry-grind corn milling with fungi. US Pat. Appl. Publ. US 2008/0153149 AI. WHO, 1998. Cyanobacterial toxins : Microcystin-LR in Drinking-water. (Background
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document for development of WHO Guidelines for Drinking-water Quality). Environ. Heal. 2, 1–14. doi:10.1016/j.kjms.2011.05.002 Wu, Y.H., Yang, J., Hu, H.Y., Yu, Y., 2013. Lipid-rich microalgal biomass production and nutrient removal by Haematococcus pluvialis in domestic secondary effluent. Ecol. Eng. 60, 155–159. doi:10.1016/j.ecoleng.2013.07.066 Zhekisheva, M., Boussiba, S., Khozin-Goldberg, I., Zarka, A., Cohen, Z., 2002. Accumulation of oleic acid in Haematococcus pluvialis (Chlorophyceae) under nitrogen starvation or high light is correlated with that of astaxanthin esters. J. Phycol. 38, 325–331. doi:10.1046/j.1529-8817.2002.01107.x Zhu, Y., Albrecht, K.O., Elliott, D.C., Hallen, R.T., Jones, S.B., 2013. Development of hydrothermal liquefaction and upgrading technologies for lipid-extracted algae conversion to liquid fuels. Algal Res. 2, 455–464. doi:10.1016/j.algal.2013.07.003
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Figure 1. a) Draft tube design, b) Experimental set-up.
31
5.0 4.5
Biomass Density (g/l)
4.0 3.5 3.0
2.5 2.0 1.5 1.0 0.5 0.0 1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time (days)
Air
2.5% CO2
5% CO2
10% CO2
No aeration
Figure 2: Biomass density at different CO2 gas supply.
32
15
16
Figure 3: FTIR Spectrum of astaxanthin-rich algae and residual biomass.
33
Figure 4: The TGA-DTG curve of residual biomass at 10 °C/min heating flow rate.
34
Figure 5: FTIR Spectrum of the gasses evolved during the TGA process.
35
Figure 6: Integrated H. pluvialis cultivation in bioethanol waste effluent.
36
Table 1: pH change, specific growth rate and TAC at different CO2 concentrations. CO2 Initial pH Tmax DWmax Concentration pH (day) (g/l) Lag Exponential Stationary phase phase Phase
µ (day -1) TAC (mg/g DW)
Air
7.00
7.91
8.42
9.64
8
0.236
0.523 ± 0.02
2.5%
7.00
7.33
7.93
8.22
13
0.280
0.721 ± 0.019
5%
7.00
7.84
6.61
6.32
11
0.317
1.109 ± 0.009
10%
7.00
4.13
4.09
3.91
3
0.013
-
37
2.028 ± 0.09 2.836 ± 0.03 4.37 ± 0.07 0.259 ± 0.02
Table 2: Wastewater analysis and nutrient removal. Analysis (g/l)
TS
PC
Modified TS
Modified M1B5
pH
3.82
3.11
7
6.74
6.61
6.32
Nutrient Removal (%) NA
TN
1.52
0.017
0.025
0.025
0.012
0.001
91.7
TOC
23.1
1.28
0.385
0.03
0.882
0.291
67.0
TP
1.79
0.0015
0.031
0.026
0.004
0
100
Acetic Acid
0.861
0.336
0.0144
NA
0.215
0.124
42.3
38
GroAst Residual media media
Table 3: Microcystin concentration in the H. pluvialis sample. Compound
% Bo
Microcystin Concentration ppb H. pluvialis cultured in modified M1B5 media Green Cells 93.05 Negative Red Cells 90.79 Negative H. pluvialis cultured in GroAst media Green Cells 92.01 Negative Red cells 92.08 Negative
39
Table 4: Analysis of residual Haematococcus Biomass. Properties
Haematococcus pluvialis 22.78 ± 0.24
HHV (MJ/kg) of astaxanthin-rich algal biomass HHV (MJ/kg) of residual biomass Empirical formula (on ash free basis) H/C molar ratio (on ash free basis) O/C molar ratio (on ash free basis) Elemental analysis (wt. %)
Carbon Hydrogen Nitrogen Sulphur Oxygen Moisture Volatile matter Fixed Carbon Ash
Proximate analysis (wt. %)
40
15.60 ± 0.01 C3.63 H6.26 N0.14 S0.01 O3 1.72 0.83 43.57 ± 0.61 6.26 ± 0.54 1.98 ± 0.16 0.47 ± 0.03 48.04 ± 1.5 7.93 ± 0.12 71.28 ± 0.27 15.36 ± 0.56 5.43 ± 0.25
Table 5: Selection matrix of suitable biofuel conversion technology (“-”= Not favorable, “+” = Favorable) Process
Operating conditions
Liquefaction
250-330 °C, 5-20 MPa (Amaro et al., 2011) 300-600 °C, 0.1-0.5 MPa (Patil et al., 2008) 700-1400°C, > 0.1 MPa (Patil et al., 2008) 500-1300°C, > 0.1 MPa (Boer et al., 2012) 25-30 °C (Amin, 2009)
Pyrolysis Combustion Gasification Fermentation
Transesterification 60-120 °C (Amin, 2009) Hydrothermal 280 – 370 °C, 10 – 25 MPa liquefaction (López Barreiro et al., 2013)
41
Drying Remarks Required? No High Pressure is undesirable Yes High VM
Decision Preference -
Not essential Yes
High Temperature High VM
+
No
Integration with Bioethanol plant possible. Carbohydrates present Lipids Present Lipids Present
+++
No No
+
+
++ ++
S0957-5820(17)30194-5 http://dx.doi.org/doi:10.1016/j.psep.2017.06.013 PSEP 1094
To appear in:
Process Safety and Environment Protection
Received date: Revised date: Accepted date:
18-2-2017 12-6-2017 19-6-2017
Please cite this article as: Haque, Fatima, Dutta, Animesh, Thimmanagari, Mahendra, Chiang, Yi Wai, Integrated Haematococcus pluvialis biomass production and nutrient removal using bioethanol plant waste effluent.Process Safety and Environment Protection http://dx.doi.org/10.1016/j.psep.2017.06.013 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.
Integrated Haematococcus pluvialis biomass production and nutrient removal using bioethanol plant waste effluent
Fatima Haque1, Animesh Dutta1, Mahendra Thimmanagari2, Yi Wai Chiang1*
1
School of Engineering, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada,
N1G 2W1 2
Ontario Ministry of Agriculture, Food and Rural Affairs, 1 Stone Road West, Guelph,
Ontario, Canada, N1G 4Y1 *
Corresponding author; Email: [email protected]; Tel.: +1-519-824-4120 ext: 58217
Graphical abstract
1
Highlights
Enhanced microalgal biomass production was achieved using air-lift photobioreactor. 5% CO2 supplementation resulted in maximum biomass density and specific growth rate. Wastewater made environmentally safe with 91.67% TN and 100% TP removal. Astaxanthin-extracted H. pluvialis biomass is a potential bioenergy feedstock. Integrated H. pluvialis cultivation-ethanol production is economically beneficial.
Abstract: The integrated system of astaxanthin and microalgal biomass production, and wastewater treatment is a promising process. Haematococcus pluvialis is a suitable microalgae for this coupled system to achieve this dual purpose in a single approach. In this study, biomass production and nutrient removal from bioethanol plant wastewater by H. pluvialis were investigated. An air-lift photobioreactor was used to utilize the wastewater as well as a CO2enriched gas supply. The maximum biomass density and maximum specific growth rate achieved were 4.37 ± 0.007 g/l and 0.317 day-1, respectively, by culturing H. pluvialis in an air-lift photobioreactor, supplemented with 5% CO2 in air. Removal of 91.7 % total nitrogen and 100 % total phosphorous from the wastewater was achieved. The residual microalgal biomass, obtained after astaxanthin extraction (1.109 ± 0.009 mg/g DW), was characterized as a potential bioenergy feedstock due to its elevated higher heating value 15.6 ± 0.01 MJ/kg. Hence, integrating H. pluvialis cultivation with bioethanol effluent serves the combined purpose of wastewater treatment, CO2 utilization, and simultaneous production of
2
astaxanthin, which has a potential market value of over USD 2000 per kg, and carbohydrate rich microalgal biomass, which can be applied to bioenergy production.
Abbreviations AL
Air-lift
BBM
Bold basal media
BOD
Biological oxygen demand
COD
Chemical oxygen demand
CAPEX
Capital expenditure
DW
Dry weight
HHV
Higher heating value
MC
Microcystin
OPEX
Operational expenditure
PBR
Photobioreactor
PC
Process condensate
TGA
Thermo gravimetric analysis
TN
Total nitrogen
TOC
Total organic carbon
3
TP
Total phosphorous
TS
Thin stillage
VM
Volatile matter
µ
Specific growth rate
Keywords: Haematococcus pluvialis, bioethanol wastewater, air-lift photobioreactor, astaxanthin, bioenergy.
1. Introduction Haematococcus pluvialis is a freshwater unicellular microalgae belonging to the Chlorophyceae family (Chekanov, 2014). Recently, this microalgae has gained attention because of its potential to efficiently produce 0.5 – 4% (dry weight) of astaxanthin, an antioxidant, which is predominantly higher as compared to other natural sources like Pandalus borealis (shrimp) and Paracoccus carotinifaciens (macroalgae), which produce 0.1% and 2.2% astaxanthin, respectively (Jiao et al., 2015; Schmidt et al., 2011). Astaxanthin (C40H52O4) is a xanthophyll ketocarotenoid, which possess strong antioxidant properties. The conjugated double bonds, present in the structure, donate the electrons that react with free radicals, and hence terminates the free radical chain reaction (Higuera-Ciapara et al., 2006). As a result, it finds application as a coloring agent, nutraceutical, and pharmaceutical compounds (Giannelli et al., 2015). Astaxanthin is produced during the second stage of the
4
life cycle of H. pluvialis, whereas the green vegetative cells exist during the first stage. Under environmentally stressed conditions, such as light, nitrate or salinity, astaxanthin accumulation takes place. During these cell stress conditions, there is an increase in the lipid content of H. pluvialis and astaxanthin is produced inside the TAG-rich globules (Razon and Tan, 2011; Zhekisheva et al., 2002). These astaxanthin-rich red cysts are surrounded by thick sporopollenin cell walls, which comprises of 53 - 70% carbohydrates and 6-19% proteins (Hagen et al., 2002). Hence, H. pluvialis is a good source of astaxanthin as well as lipid and carbohydrate rich microalgal biomass. Though astaxanthin constitutes a minute amount of the total biomass, it has a great potential market value of over USD 2000 per kg of astaxanthin (Li et al., 2011). For an effective production of this valuable product, high density H. pluvialis culture is a pre-requisite, and this depends on the nutrient composition of the growth media, and the choice of photobioreactor (PBR). Different types of photobioreactor has been used to cultivate algae, such as bubble column reactor (Ranjbar et al., 2008) and air-lift reactor (Kaewpintong et al., 2007). H. pluvialis cells are shear sensitive, so mechanically stirred reactor is not a desirable option, and pneumatic agitation is preferred. For example, Kaewpintong et al. (2007) studied the cultivation of H. pluvialis in basal medium using air-lift photobioreactor (AL-PBR) and supplemented with 1% CO2, and was able to achieve 0.31 day-1 of specific growth rate. B. Santhose et al. (2014) cultivated a new strain of H. pluvialis in modified BBM media in an AL-PBR and was able to achieve 3g of dried algal biomass from 2.5 L of culture. Ranjbar et al. (2008) was able to achieve 5 g/l of algal biomass by cultivating H. pluvialis in basal media and using a continuous supply of CO2 in a bubble column reactor.
5
The main purpose of this study is to examine the integrated system of H. pluvialis cultivation in bioethanol waste effluent in an AL-PBR. In a previous study, Haque et al. (2016) investigated the H. pluvialis culture in bioethanol waste stream (thin stillage and process condensate), and a growth media denoted as the GroAst media, which supported astaxanthin production, was formulated. The first objective of this study is to attain high density H. pluvialis culture in GroAst media using AL-PBR supplemented with CO2 and to access the environment impact of the treated wastewater henceforth. The second objective is to analyze the bioenergy potential of the residual H. pluvialis biomass obtained after astaxanthin extraction, as microalgae is seen as a potential bioenergy feedstock in recent years (Hannon et al., 2010). 2. Materials and Methods 2.1. Microorganism, media, and cultivation condition H. pluvialis CPCC 93 strains were obtained from the Canadian Phycological Culture Centre. The stock cultures were maintained on modified M1B5, a modification of the media used by Tocquin et al. (2012): 0.5 ml/L Flora Micro and 2.5 ml/L Flora Bloom (General Hydroponics). The cultures were maintained at pH 7 and incubated at 23 ± 2°C, 12h/12h light/dark cycle (600W MH light) with gentle agitation twice a day. The culture media used for the experiment was prepared using the wastewater procured from a local bioethanol plant, where corn kernels are used as the raw material. The wastewater samples used were the thin stillage (TS) obtained from the distillation unit, and the process condensate (PC) obtained from the evaporation unit of the production plant. As reported in
6
the previous study, 60 times diluted TS (denoted as modified TS) supported microalgal growth, and addition of PC coupled with continuous light resulted in astaxanthin induction. Hence this formulation of 60% modified TS and 40% PC, denoted as the GroAst media, is used for H. pluvialis cultivation (Haque et al., 2016). 2.2. Enhanced Biomass Production of H. pluvialis 2.2.1. Experimental set-up H. pluvialis culture was carried out in a 2.2 L AL-PBR. Figure 1 demonstrates the experimental set-up. The reactor column was made of glass (11.2 cm internal diameter and 22.5 cm height), with a draft tube (5.6 cm internal diameter and 11 cm height) centrally installed. The draft tube was made from silicone tubing with a rubber gasket base to hold the tube in position (Figure 1a). Prior to each experiment, the column reactor and the draft tube were sterilized by spraying with isopropyl alcohol (70%). The CO2 gas cylinder (purity 99.99%, Linde) and the air pump (Tetra, Whisper 10) were connected as shown in the Figure 1b, and the gas flow was controlled by an individual panel-mounted flow meter with control valve (Key Instruments, Cole Parmer, CA). The AL-PBR was placed inside a temperature controlled, illuminated 3’ X 3’ Expert growth tent at 23 ± 2°C and 12h/12h light/dark cycle (600W MH light). The growth tent is made of reflective 600D Mylar fabric, which helped maintain even lighting conditions and prevented the effect of any other light source on the reactor. The distance between the light source and the AL-PBR was maintained at 70 cm. The average illumination intensity incident to the outer surface of the reactor was measured using a digital lux meter (Fisher Scientific) and it was maintained at 15 klx for growth conditions (green phase) and 30 klx for astaxanthin induction (red phase).
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2.2.2. Effect of CO2 on microalgal growth CO2 not only acts as a carbon source for the photoautotrophic growth of H. pluvialis but the gas bubbles help in providing sufficient mixing inside the PBR. The concentrations of CO2, in the bubbled gas, studied were 2.5, 5.0 and 10.0 vol%. For the control, only compressed air (0.04% ambient CO2) was introduced inside the AL-PBR. 1.5 L of GroAst media was inoculated with 10% (v/v) four-day old seed culture and the initial pH of the media was maintained at 7. The flow rate of the gas was maintained at 45 ml/min, which provides sufficient mixing to keep the cells in suspension. Astaxanthin was extracted and quantified by employing the ultrasonication method as reported in Haque et al. (2016). The microalgal samples were centrifuged (Thermo Scientific Sorvall ST 24) at 5000 x g for 15 min. Cells were dried overnight at room temperature, before carrying out ultrasonication (VC 750, Sonics & Materials Inc.) in sodium hydroxide (2M) for 25 min, using a 1.3 cm diameter probe (horn) at 35% amplitude and 20 kHz operating frequency. Ultrasonicated samples were centrifuged again, followed by extraction using methanol (99.8%). All chemicals used are Fisher Scientific analytical grade. 2.3. Biomass density estimation For the analysis of the biomass density, 10 ml of the microalgal broths were centrifuged at 5,000 g for 15 minutes; the supernatant was discarded, and the dry weight was determined, using an analytical balance (Mettler Toledo), after drying the cell pellet overnight at room temperature. Specific growth rate (µ day-1) was determined using the following formula (L. Shuler and Kargi, 2002):
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µ=
ln (DW𝑖 ) – ln (DW0 ) t𝑖 − t0
Where DW0 and DWi are the dry weight (g/l) of the microalgal sample on the initial day (t0) and ith day (ti), respectively. Also, pH of the media was measured, using a pH meter (Oakton, Cole - Parmer, CA), at the end of the lag phase, the exponential phase, and the stationary phase to analyze the effect of CO2 concentration on the pH of the media. 2.4. Assessment of the treated wastewater 2.4.1. Analysis of the wastewater The wastewater samples were characterized on the basis of pH, total nitrogen (TN) (Hach method 10071), total organic carbon (TOC) (TOC analyzer, Shimadzu), total phosphorous (TP) (Hach method 8190) and acetic acid content (Siedlecka et al., 2008). These parameters were tested for: a) TS and PC initially; b) modified TS (60 times diluted), i.e., GroAst media for the green phase; c) GroAst media for the red phase, i.e., when 40% PC is added to 60% modified TS; and d) residual media at the end of the experimental run. Nutrient removal (%) was calculated as:
% Nutrient Removal =
[Nutrient]𝑓𝑖𝑛𝑎𝑙 − [Nutrient]𝑖𝑛𝑖𝑡𝑖𝑎𝑙 ∗ 100 [Nutrient]𝑖𝑛𝑖𝑡𝑖𝑎𝑙
Where, [Nutrient]final and [Nutrient]initial are the nutrient composition (g/l) of the GroAst media finally and initially, respectively. 2.4.2. Toxin detection test
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The European Food and Safety Authority (EFSA) have reported about the generation of a cyanotoxin, microcystin (MC), by H. pluvialis (EFSA, 2014). For the safe discharge of the treated wastewater into the environment, the presence of MC was tested. The EnviroLogix QuantiPlate Kit was used for the quantitative laboratory detection of MC toxin in the microalgal samples, which uses the principle of a competitive Enzyme–Linked Immuno Sorbent Assay (ELISA). The experiment was carried out as mentioned in the kit manual (Catalog number EP 022, EnviroLogix), using a negative control and standard Microcystin (0.16 ppb, 0.6 ppb, and 2.5 ppb) as calibrators. The optical density (OD) was measured at 450 nm using a UV-VIS spectrophotometer (Multiskan Go Microplate Spectrophotometer, Thermo Fisher Scientific). A standard curve was obtained by plotting % Bo and the calibrator’s concentration on a semi log graph, where: % B𝑜 =
Average OD of calibrator or sample ∗ 100 Average OD of negative control
The calibration curve was used to determine the microcystin concentration in the algal samples. The green as well as the red cells of H. pluvialis, cultured in modified M1B5 and in GroAst media were tested for microcystin presence. The detection limit of this ELISA kit is 0.16 ppb MC. 2.5. Residual microalgal biomass characterization 2.5.1. Proximate and Ultimate analyses, and Higher Heating Value ASTM standards were used to perform the proximate analysis (ash, volatile matter and fixed carbon) of the solid residual biomass. A measured amount of the sample was placed in a
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muffle furnace (Thermo Scientific F48055-60, Waltham, MA) maintained at 103 ± 2°C for at least 15 h and then cooled in a desiccator containing silica gel. The samples were then reweighed and the change in sample weight was expressed as percentage moisture (ASTME871). The dried samples were then burnt in air at 575°C for 5 h in the muffle furnace to determine percentage ash (ASTM-E1755). For determining the volatile matter (VM), the samples were heated in inert atmosphere at 950°C for 7 min (ASTM-E872). The fixed carbon content was calculated as the difference between 100% and the three other percentages (moisture, volatile and ash), according to ASTM-D7582. A Thermo Fisher Flash EA 1112 (Waltham, MA) elemental analyzer was used to perform the ultimate analysis (carbon (C), hydrogen (H), nitrogen (N), sulphur (S), and oxygen (O)) of the samples. The Higher Heating Values (HHV) of the samples were determined using an IKA-C200 bomb calorimeter (Wilmington, NC). Approximately 0.5g of the sample was placed in the steel container fitted with a ceramic crucible. Pure oxygen gas at 30 bar was used to pressurize the adiabatic vessel, and a cotton thread connected to an ignition wire in the steel container was used to ignite the sample. 2.5.2. FTIR Analysis FTIR spectrum of the dried microalgal sample was recorded on a Nicolet 4700 FTIR spectrometer (Thermo Scientific) at room temperature. The sample was pressed with potassium bromide powder and a wavelength spectrum of 4000-400 cm-1 was used for scanning.
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2.5.3. Thermal Analysis To study the combustion behavior of the residual biomass sample, thermogravimetric analysis (TGA) was performed. The dried residual biomass was ran in a TG/DT analyzer (Thermo Scientific Nicolet 700 FTIR) in nitrogen atmosphere at heating rate of 10 °C/min. Approximately 10 mg of the sample was heated from ambient temperature to 750 °C. Nitrogen gas of high purity (99.99%) was supplied at a constant flow rate of 100 ml/min as an inert purge gas. Nitrogen gas eliminates unwanted oxidation of the sample by displacing the air in the pyrolytic zone. The sample weight (W) as a function of temperature (T) is recorded and plotted; derivative TGA (DTG) curves (dW/dT) were also plotted. The volatile compounds evolved during the TGA process were analyzed using a coupled FTIR (Thermo Scientific Nicolet 700 FTIR). 2.6. Data Analysis All readings were taken in triplicates and mean results were represented along with standard deviation. The data in the graphs have been represented along with standard deviation interval bars. 3. Results 3.1. Photobioreactor performance Figure 2 shows the biomass density curve at different CO2 concentrations. It can be seen that as the CO2 concentration is increased from atmospheric to 5%, there is an increase in microalgal density. For 5% CO2 concentration, maximum growth of 4.37 ± 0.07g/l (DWmax) was obtained, and total astaxanthin content (TAC) extracted was 1.109 ± 0.009 mg/g DW.
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Table 1 presents values of TAC at other CO2 concentrations. Samples cultivated with 2.5% and 5% CO2 gas supply showed similar growth patterns. As the CO2 concentration is increased to 10%, a decline in the growth occurs, suggesting that more CO2 than required for sufficient growth is detrimental to cell survival. Samples grown under atmospheric CO2 concentration gas supply showed less growth compared to the samples supplied with 2.5% and 5% CO2, hence CO2 availability was the limiting factor for culture growth. In comparison to the cultivation method used in the previous study (Haque et al., 2016), where H. pluvialis was cultured in erlenmeyer flasks without aeration, AL-PBR provides better mode for wastewater as well as CO2 utilization, with aeration resulting in 84.3% increase in biomass density. The change in the pH of the GroAst media under the effect of different concentration of CO2 is summarized in Table 1, along with the specific growth rates (µ). When only air is supplied, the pH increased from 7.00 to 9.64 by the end of the stationary phase and a biomass density of 2.028 ± 0.09 g/l was obtained. However, when the growth media was supplied with 5% CO2, the pH changed to 6.32 at the end of the experimental run and the maximum biomass density was achieved on the 11th day (T max) with a specific growth rate of 0.317 day-1. 10% CO2 supply resulted in an acidic media (pH 3.91) and a stunted microalgal growth. 3.2. Environmental impact assessment of wastewater Table 2 shows the nutrient composition of the wastewater samples (TS and PC) and the GroAst media used for astaxanthin production, along with the amount of nutrient removal at the end of the experimental run (residual media). The modified TS sample has similar nutrient composition to that of the standard media, modified M1B5. The GroAst media contained
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0.215 g/l of acetic acid, which is absent in the standard media, and hence additional supplement of acetic acid is not required. H. pluvialis culture resulted in 91.7% total nitrogen (TN) removal and 100% total phosphorus (TP) removal. Astaxanthin synthesis also results in 42.3% acetic acid being consumed, and the final media contained 33.0% of the initial total organic carbon (TOC).
The concentration of microcystin in the H. pluvialis sample has been tabulated in Table 3, along with the % Bo values. H. pluvialis samples show a % Bo value in the range of 92.01– 93.05 %, and, the samples had negative results (below detection limit) for the microcystin test. Hence, the treated wastewater is devoid of microcystin, and safe for discharge. 3.3. Assessment of the residual H. pluvialis biomass 3.3.1. Proximate and Ultimate Analyses The characterization of H. pluvialis biomass as a biofuel feedstock is itemized in Table 4. The ash content, volatile matter content, moisture content and fixed carbon content of the algal biomass is 5.43 ± 0.25%, 71.28 ± 0.27%, 7.93 ± 0.12% and 15.36 ± 0.56%, respectively. The ultimate analysis shows that the residual biomass has 43.57% carbon, 6.26% hydrogen, 48.04% oxygen, 1.97% nitrogen and 0.47% sulphur. This helps to understand the elemental composition of the sample, and the empirical formula of the residual algal biomass is found to be C3.63 H6.26 N0.14 S0.01 O3. The H/C an O/C molar ratios (on the ash free dry basis) were calculated from the elemental composition as 1.72 and 0.83, respectively. The HHV of the
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astaxanthin-rich un-extracted algal biomass is measured to be 22.78 MJ/kg, whereas the HHV of the post-extracted residual biomass is 15.60 MJ/kg. 3.3.2. FTIR Spectra FTIR spectroscopy helps in the rapid analysis of microalgal biomass and offers a viable screening approach to determine the bioenergy potential of microalgae. The FTIR spectra of the astaxanthin-rich biomass and the residual biomass are shown in Figure 3. The identification of the unknown components is derived by comparing the bands of the recorded FTIR spectrum with those available in the literature (Bi and He, 2013; Ceylan and Goldfarb, 2015; Sudhakar and Premalatha, 2015). In the spectrum of the residual biomass, the band at 3,370 cm-1 is due to the O-H stretching, primarily attributable to water and hydroxyl groups. The weak band centered at 2,920 cm-1 is due to the presence of asymmetric C-H stretching vibrations, which is likely due to the lipid’s methylene group. A sharp peak at 1,630 cm1
corresponds to C=O amide stretching from proteins present in the residual algal biomass.
The peak spanning between 1,800 – 1,500 cm-1 is the characteristic bands for proteins and 1,450 – 1,400 cm-1 is likely due to C-H stretching vibration in methyl, methylene and methyne groups. The absorbance peak at 1,150 cm-1, 1,060 cm-1, and 1,010 cm-1 are likely the C-O and C-O-H deformation in the secondary and tertiary alcohol or aliphatic ethers. The C-O-H vibrational stretching suggests the presence of polysaccharides (Sudhakar and Premalatha, 2015). The intensity of the absorbance corresponding to polysaccharides (1,010 cm-1) is much higher than the one for the lipids (2,920 cm-1). Also, the intensity of the spectrum of the residual biomass is lower than the un-extracted biomass. There is a general decrease in the lipid and
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protein content as indicated in the 3,000-2,800 cm-1 region and 1,800-1,400 cm-1 region, respectively. The FTIR spectrum of H. pluvialis residual biomass marks distinct fingerprint regions for triglycerides, lipids and polysaccharides.
3.3.3. Thermal Analysis 3.3.3.1. TGA Figure 4 shows the TGA–DTG curves of the residual algal biomass. There are three distinct decomposition zones observed during the pyrolysis process of the microalgae. The dehydration zone is the initial stage, at around 110 ºC, where the weight loss occurs due to the loss of moisture from the biomass. The second zone is the devolatilization zone, where the biomass sample loses the volatile matter content. Two overlapped weight loss regions can be distinguished, one from approximately 250 ºC to 320 ºC and another from 320 ºC to 450 ºC. Active pyrolysis zone is the region between 110 ºC – 320 ºC, and 320 ºC – 420 ºC region is the zone of passive pyrolysis, which terminates at 450 ºC (Phukan et al., 2011). The DTG peaks occur at 300 ºC and 420 ºC, in the vicinity of 350 ºC, which is believed to be lipid loss and some lignocellulosic polymer decomposition. Major volatile matter loss occurs in the range: 72%; Sudhakar and Premalatha, (2015) suggests that cellulose decomposition usually occurs between 290 ºC – 390 ºC. Solid residue decomposition zone is the last stage, where slow decomposition of the solid residue, left from the preceding heating range, occurs. As seen from Figure 4, beyond 450 ºC slow loss of weight continues until about 750 ºC, but without any marked DTG peak. This degradation profile of the microalgal biomass suggests
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that it can be a potential bioenergy feedstock and maybe suitable for thermo-chemical conversion (Phukan et al., 2011). 3.3.3.2. Analysis of the gas evolved during TGA process The volatile compounds evolved during the TGA process were analyzed using FTIR. Figure 5 presents the 3D IR spectrum with respect to wavenumbers (cm-1), time (min) and absorbance. In the region between 2,000 – 2,300 cm-1 some significant peaks for C=O stretching of CO2 were obtained.
The regions between 4,000 – 3,400 cm-1 give the
characteristic signals for water and the main gaseous products of O2, CO, H2O and some organics (a mixture of acids, aldehydes, alkanes, and ethers), which is characteristic of pyrolysis end-products (Sudhakar and Premalatha, 2015).
4. Discussion High density cultivation of H. pluvialis in an AL-PBR. A high-density H. pluvialis culture was obtained using bioethanol waste stream, in an air-lift photobioreactor supplemented with 5% CO2. The specific growth rate (0.317 day-1) reported in this study is similar to the one reported by Kaewpintong et al. (2007), where the maximum growth rate of 0.45 day -1 was achieved for a continuous culture and 0.31 day-1 for a semicontinuous culture of H pluvialis in an air-lift reactor. The highest biomass density obtained was 4.37 ± 0.07 g/l, which is slightly lower than the one achieved by Ranjbar et al. (2008) who reported 5 g/l cell concentration weight using modified basal media and 1 L bubble
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column reactor, but more than the 2.62 g/l achieved by Jun et al. (2012), who cultivated H. pluvialis in a 6 L bag type reactor supplemented with 2% CO2. Both air and 10% CO2 supply resulted in a moderate growth, however, atmospheric CO2 supply resulted in a basic media (pH of 9.64) and 10% CO2 supply led to an acidic media (pH of 3.91). The relation between CO2 supply and algal growth can be attributed to the pH change. The CO2 supply/consumption ratio plays a major role in maintaining a suitable pH of the medium. At atmospheric CO2 condition, the CO2 consumption is higher than the CO2 supply, hence the pH of the medium increases to 9.64 at the end of the stationary phase. At 5% CO2 gas supply the rate of CO2 supply is balanced by the photosynthetic CO2 consumption, thereby the pH of the medium is close to 6.32. However, at 10% CO2 gas supply, the CO2 supply exceeds the CO2 consumption rate, hence the pH drops to a greater extent (3.91), and this carbonic acid-rich acidic medium do not support algal growth. It has been reported that a pH more than 10 or less than 5.5 is detrimental to the algal growth (Kang et al., 2005). The thin stillage (TS) has a high concentration of suspended solids (turbidity greater than 1000 NTU, (Haque et al., 2016)); hence, in this study, it was diluted 60 times dilution to reduce turbidity while still providing a sufficient amount of nutrients required for H. pluvialis growth. However, in an industrial plant, thin stillage could be centrifuged to obtain the distiller’s grain, which finds application as animal feed (Mustafa et al., 2000; Van Leeuwen et al., 2015), thus reducing or eliminating the need for dilution. Alternatively, treated water could be used as dilution water, thus reducing the water demand of dilution.
Environmentally safe water from wastewater.
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H. pluvialis successfully utilized inorganic wastes, 91.67% total nitrogen and 100% total phosphorous, from the wastewater sample. These results are in accordance with Wu et al. (2013), who reported 93.8% N removal and 97.8% P removal, when H. pluvialis were cultured in the domestic secondary effluent, and Ledda et al. (2016), who reported up to 99% removal for NO3-N and NH4-N, 98% for TP, and 26% for chemical oxygen demand, when H. pluvialis were cultured in swine slurry primary filtrate. Also, Kang et al. (2006) studied the biosynthesis of astaxanthin by cultivating H. pluvialis in primary treated sewage and primary treated piggery wastewater, and the author reported that 4-8 fold dilution of primary treated piggery wastewater resulted in 100% N and P removal at the end of the process. In addition to the inorganic nutrients, H. pluvialis cultivation resulted in 67% removal of TOC, thereby reducing the biological oxygen demand (BOD) and chemical oxygen demand (COD) of the wastewater. As excess BOD can deplete the dissolved oxygen of aquatic water, its removal is one of the primary aims of wastewater treatment, in order to protect the aquatic organisms (Abdel-Raouf et al., 2012). The samples showed negative results for the presence of microcystin toxin (detection limit 0.16 ppb). According to the WHO guidelines for drinking water quality, the guiding value for the total MC is 1 ppb (WHO, 1998). Also, in 2014, EFSA published a report on the safety of their astaxanthin product due to the absence of microcystin (EFSA, 2014). Hence, it can be seen that H. pluvialis cultivation not only helps in nutrient removal but also eliminates the threat of microcystin production, and thus the treated wastewater is free of cyanotoxins. This makes the discharge of wastewater into the water bodies safer, as this wastewater is devoid of nutrients and does not support algal growth. For example, Lake Erie (Canada-USA) suffers
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from severe toxic algal bloom because of the naturally occurring cyanobacteria that are fueled by the presence of warm water, sunshine and nutrient-rich industrial wastewater and agricultural run-off.
H. pluvialis as a potential bioenergy feedstock. The characterization of the microalgal biomass helps to understand its physio - chemical properties and to assess its bioenergy potential. The residual biomass has a satisfactory HHV of 15.6 MJ/kg, which is comparable to that of Chlorella sp. (15.88 MJ/kg) (Phukan et al., 2011). The high volatile matter content (71.28 ± 0.27%) and the TGA thermogram suggest that the residual biomass can be a potential feedstock for thermochemical conversions (Phukan et al., 2011). The elevated moisture content (7.9 ± 0.12 %) is not desirable as it reduces the heating value of the biomass, but the low ash content (5.43 ± 0.25 %) of the residual biomass is desirable as it has no heating value and it also affects both the handling and processing cost of the biomass energy conversion (Ceylan and Goldfarb, 2015; Sudhakar and Premalatha, 2015). The ultimate analysis showed that the residual biomass possesses low N and S values (C3.63H6.26N0.14S0.01O3), which is favorable as this limits the formation of NOx and SOx during the combustion process, and hence proves to be environmental friendly, similar to other biomass sources like rice husk (0.63% N, 0.041% S) and Laminaria saccharina (2.4% N and 0.7% S) (Chow et al., 2013; Olupot et al., 2016). Residual Haematococcus biomass has an H/C ratio (ash free basis of 1.72, which is lower than the H/C ratio of rice husk (1.95), higher
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than that of Laminaria saccharina, a brown microalgae (1.42), and similar to the H/C ratio of Chlorella, Nannochloropsis and Spirulina species (Chow et al., 2013; Phukan et al., 2011; Zhu et al., 2013). Microalgal biomass can be converted to bioenergy by using thermochemical conversion, which produces oil and gas as the end-products, or by using biochemical processes, where ethanol and biodiesel can be produced (Amin, 2009; Patil et al., 2008). Table 5 shows the comparison between these different conversion technologies in terms of the end-product formed, operating conditions and drying requirement, as moisture content plays a key role in deciding which conversion technology can be used. Thermochemical conversion is best suited for biomass with low moisture content whereas biochemical conversion is preferred for biomass with high moisture content. Along with the moisture content, it is important to understand the main component of the residual biomass; if it is rich in carbohydrate then it can be used in bioethanol production, and if it is rich in fatty acids then biodiesel can be produced. (Bi and He, 2013). FTIR spectrum (Figure 3) of the residual Haematococcus biomass showed high peaks for carbohydrates and lipids. Therefore, based on these characteristics, a selection matrix (Table 5) is created, to find the most suitable conversion technology. According to the selection matrix, the fermentation process is the most suitable option to convert the residual biomass to biofuel. The biomass has a high moisture content, however drying is not a necessary step for fermentation. Also, the FTIR spectrum showed a higher peak for carbohydrate compared to lipids, thus indicating that the biomass is a good source of carbohydrates. As the fermentation process requires a carbohydrate-rich raw material, the
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Haematococcus biomass fits in this requirement. In this study, H. pluvialis is cultured in wastewater obtained from the bioethanol production plant, which uses fermentation technology to produce bioethanol. So, if the residual Haematococcus biomass is fed into the saccharification unit as an additional feedstock, then this biomass could be upcycled back to the process. This integrated approach of H. pluvialis cultivation in wastewater and residual microalgal biomass utilization is illustrated in Figure 6. Economic benefits of the integrated H. pluvialis cultivation. The major manufacturers of synthetic astaxanthin are Royal DSM N.V. in the Netherlands, BASF SE in France, and Zhejiang NHU Co. Ltd. in China. The estimated cost of production of synthetic astaxanthin is about USD 1000∙kg-1 and the total market value being more that USD 200 million per year (Olaizola, 2003). However, due to the food safety concerns related to the petrochemical-derived synthetic astaxanthin, the high production cost of synthetic astaxanthin, and a high market demand for natural astaxanthin, the biological sources of astaxanthin are seen as preferable ‘green solutions’ (Li et al., 2011). Li et al. (2011) performed a comprehensive cost analysis of astaxanthin production from H. pluvialis in Shenzhen, China, for a hypothetically scaled-up plant with a production capacity of about 900 kg astaxanthin per year. They estimated about USD 163∙kg-1 CAPEX and USD 555∙kg1
OPEX respectively, and a total production cost of USD 718∙kg-1 astaxanthin.
For the present study, the proposed process could be integrated into an established bioethanol production plant, with an existing infrastructure, thereby reducing the capital investment on the infrastructure required, land procurement and wastewater (medium) supply station, as well as operational expenditures such as procurement of media, carbon dioxide, and
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sterilization of media. Hence, using the cost analysis data from the work of Li et al. (2011), an integrated process would save a minimum of 24% in CAPEX (savings on land acquisition and improvement, medium supply station for bioreactors, and CO2 storage tank, while maintaining building area and construction costs) and 22% in OPEX (savings on potassium nitrate, disodium hydrogen phosphate, sodium bicarbonate, carbon dioxide, water and wastewater treatment). Regarding wastewater treatment, the ethanol plant utilizes ozonation to presently treat its wastewater, so no additional cost is expected by the integration. The total production cost could thus be reduced by USD 159∙kg-1 astaxanthin. 5. Conclusions A high density (4.37 ± 0.07 g/l) H. pluvialis culture was obtained using the bioethanol production waste stream as the growth media, in an air-lift bioreactor supplemented with 5% CO2. The residual Haematococcus biomass showed potential to be used as a future bioenergy feedstock. This microalga can make use of a simple and inexpensive nutrient medium, wastewater, to grow. Also, H. pluvialis resulted in 91.67 % total nitrogen and 100 % total phosphorous removal. The FTIR analysis confirmed the presence of carbohydrates and lipids, and the thermal analysis presented a useful decomposition profile of the biomass. Hence, the assessment of the biomass with respect to bioenergy production proves it to be a potential feedstock. H. pluvialis cultivation can be coupled with CO2 capture from bioethanol production, wastewater reutilization, and production of high-value astaxanthin and microalgal biomass. This proposed process should be more environmentally friendly and economical, compared to conventional astaxanthin production processes, due to integrating microalgal culture in an exisiting bioethanol plant and utilizing the waste product generated
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in the plant. The processing costs may be further reduced with advances of separation technologies and optimization of culturing processes. Acknowledgements This research was financially supported by the Ontario Ministry of Agriculture, Food and Rural Affairs (LAAIR2014-5089). The author would like to thank Greenfield Ethanol Plant, Chatham, Ontario, for providing the wastewaters, and Joanne Ryks, Ryan Smith, and Michael Speagle, from University of Guelph, for their help.
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Figure 1. a) Draft tube design, b) Experimental set-up.
31
5.0 4.5
Biomass Density (g/l)
4.0 3.5 3.0
2.5 2.0 1.5 1.0 0.5 0.0 1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time (days)
Air
2.5% CO2
5% CO2
10% CO2
No aeration
Figure 2: Biomass density at different CO2 gas supply.
32
15
16
Figure 3: FTIR Spectrum of astaxanthin-rich algae and residual biomass.
33
Figure 4: The TGA-DTG curve of residual biomass at 10 °C/min heating flow rate.
34
Figure 5: FTIR Spectrum of the gasses evolved during the TGA process.
35
Figure 6: Integrated H. pluvialis cultivation in bioethanol waste effluent.
36
Table 1: pH change, specific growth rate and TAC at different CO2 concentrations. CO2 Initial pH Tmax DWmax Concentration pH (day) (g/l) Lag Exponential Stationary phase phase Phase
µ (day -1) TAC (mg/g DW)
Air
7.00
7.91
8.42
9.64
8
0.236
0.523 ± 0.02
2.5%
7.00
7.33
7.93
8.22
13
0.280
0.721 ± 0.019
5%
7.00
7.84
6.61
6.32
11
0.317
1.109 ± 0.009
10%
7.00
4.13
4.09
3.91
3
0.013
-
37
2.028 ± 0.09 2.836 ± 0.03 4.37 ± 0.07 0.259 ± 0.02
Table 2: Wastewater analysis and nutrient removal. Analysis (g/l)
TS
PC
Modified TS
Modified M1B5
pH
3.82
3.11
7
6.74
6.61
6.32
Nutrient Removal (%) NA
TN
1.52
0.017
0.025
0.025
0.012
0.001
91.7
TOC
23.1
1.28
0.385
0.03
0.882
0.291
67.0
TP
1.79
0.0015
0.031
0.026
0.004
0
100
Acetic Acid
0.861
0.336
0.0144
NA
0.215
0.124
42.3
38
GroAst Residual media media
Table 3: Microcystin concentration in the H. pluvialis sample. Compound
% Bo
Microcystin Concentration ppb H. pluvialis cultured in modified M1B5 media Green Cells 93.05 Negative Red Cells 90.79 Negative H. pluvialis cultured in GroAst media Green Cells 92.01 Negative Red cells 92.08 Negative
39
Table 4: Analysis of residual Haematococcus Biomass. Properties
Haematococcus pluvialis 22.78 ± 0.24
HHV (MJ/kg) of astaxanthin-rich algal biomass HHV (MJ/kg) of residual biomass Empirical formula (on ash free basis) H/C molar ratio (on ash free basis) O/C molar ratio (on ash free basis) Elemental analysis (wt. %)
Carbon Hydrogen Nitrogen Sulphur Oxygen Moisture Volatile matter Fixed Carbon Ash
Proximate analysis (wt. %)
40
15.60 ± 0.01 C3.63 H6.26 N0.14 S0.01 O3 1.72 0.83 43.57 ± 0.61 6.26 ± 0.54 1.98 ± 0.16 0.47 ± 0.03 48.04 ± 1.5 7.93 ± 0.12 71.28 ± 0.27 15.36 ± 0.56 5.43 ± 0.25
Table 5: Selection matrix of suitable biofuel conversion technology (“-”= Not favorable, “+” = Favorable) Process
Operating conditions
Liquefaction
250-330 °C, 5-20 MPa (Amaro et al., 2011) 300-600 °C, 0.1-0.5 MPa (Patil et al., 2008) 700-1400°C, > 0.1 MPa (Patil et al., 2008) 500-1300°C, > 0.1 MPa (Boer et al., 2012) 25-30 °C (Amin, 2009)
Pyrolysis Combustion Gasification Fermentation
Transesterification 60-120 °C (Amin, 2009) Hydrothermal 280 – 370 °C, 10 – 25 MPa liquefaction (López Barreiro et al., 2013)
41
Drying Remarks Required? No High Pressure is undesirable Yes High VM
Decision Preference -
Not essential Yes
High Temperature High VM
+
No
Integration with Bioethanol plant possible. Carbohydrates present Lipids Present Lipids Present
+++
No No
+
+
++ ++