Accepted Manuscript Fed-batch cultivation of Arthrospira and Chlorella in ammonia-rich wastewater: optimization of nutrient removal and biomass production Giorgos Markou PII: DOI: Reference:
S0960-8524(15)00862-7 http://dx.doi.org/10.1016/j.biortech.2015.06.071 BITE 15151
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 May 2015 13 June 2015 15 June 2015
Please cite this article as: Markou, G., Fed-batch cultivation of Arthrospira and Chlorella in ammonia-rich wastewater: optimization of nutrient removal and biomass production, Bioresource Technology (2015), doi: http:// dx.doi.org/10.1016/j.biortech.2015.06.071
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Fed-batch cultivation of Arthrospira and Chlorella in ammonia-rich wastewater: optimization of nutrient removal and biomass production
Giorgos Markou 1,2 1
Department of Agricultural Engineering, Institute of Soil and Water Resources, Hellenic Agricultural Organization-Demeter,
Leoforos Dimokratias 61, 13561, Athens, Greece 2
Department of Natural Resources Management and Agricultural Engineering, Agricultural University of Athens, Iera Odos
75, 11855 Athens, Greece * E-mail: [email protected], [email protected]
Abstract In the present work the cyanobacterium Arthrospira platensis and the microalga Chlorella vulgaris were fed-batch cultivated in ammonia-rich wastewater derived from the anaerobic digestion of poultry litter. Aim of the study was to maximize the biomass production along with the nutrient removal aiming to wastewater treatment. Ammonia and phosphorus removals were very high (>95%) for all cultures investigated. Both microorganisms were able to remove volatile fatty acids to an extent of >90%, indicating that they were capable for mixotrophic growth. Chemical oxygen demand and proteins were also removed in various degrees. In contrast, in all cultures carbohydrate concentration was increased. The biochemical composition of the microorganisms varied greatly and was influenced by the indicate that the nutrient availability. A. platensis accumulated carbohydrates (≈40%), while C. vulgaris accumulated lipids (≈50%), rendering them interesting for biofuel production.
Keywords: Ammonia toxicity; anaerobic digestion; biomass; cyanobacteria; microalgae; poultry litter; wastewater
1. Introduction Microalgal-based wastewater treatment (MBWT) has recently gained the interest worldwide, mainly because it displays a dual role, i.e. on the one hand the wastewater is treated rendering it more harmless to be disposed, and on the other hand producing valuable microalgal biomass for the generation of biofuels and other chemicals (Pittman et al., 2011; Zeng et al., 2015). MBWT can be considered as a biological treatment method, aiming to the reduction of both, inorganic (primarily N and P) and organic load of wastewater (Dickinson et al., 2015; Park et al., 2011). However, ammonia-rich wastewaters display a major limitation to be treated with MBWT, due to the occurrence of ammonia toxicity. When ammonia concentration exceeds a level in the aquatic surroundings, it acts inhibitory to the microalgal cells, reducing their growth rates or even causing their death (Abeliovich and Azov, 1976; Markou et al., 2014). Some organic substrates, such as poultry litter, due to the high content of nitrogenous compounds, have a very low carbon to nitrogen ratio, and during anaerobic digestion tend to accumulate ammonia. The total ammonia (TA) concentration on the liquor can exceed 5 g/L (Gangagni Rao et al., 2008; Yenigün and Demirel, 2013), which is almost two orders of magnitude higher than the TA concentration that microalgae could tolerate (Abeliovich and Azov, 1976; Peccia et al., 2013). Therefore, the only strategy to avoid ammonia toxicity is the dilution of the wastewater, adapting ammonia concentration below the toxic limits (Peccia et al., 2013). Ammonia-rich wastewater though, should first be strongly diluted, resulting as well to a high dilution of other essential nutrients (such as P and K) and
their low final concentration with a consequence of acting limiting to microalgal growth. Moreover, limiting conditions can affect the removal rate of the nutrients (Beuckels et al., 2015), decreasing the treatment efficiency of the MBWT system. Hence, for an unhindered biomass production it is required to apply the nutrients in such a way that neither ammonia toxicity or nutrient limitation will occur. A strategy that has been proven successful for avoiding ammonia toxicity is the fed-batch cultivation mode (Ji et al., 2015; Rodrigues et al., 2010), by which ammonia is added (fed) gradually to the cultivation medium. Generally, fed-batch mode is superior to the batch one, regarding the gradually addition of toxic substances, but both have some limitations in scaledup systems. A major disadvantage is the need to prevent contamination during the initial inoculation and early growth period due to low initial cell density. Especially when dealing with wastewater the contamination of the culture is more possible. However, batch and fedbatch modes, display some advantages regarding their simplicity and flexibility and are frequently used (Lavens and Sorgeloos, 1996). In any case, little attention is given to the fedbatch cultivation mode regarding the ammonia-rich wastewater treatment. Consequently, aim of this study was to investigate the capability of using the fed-batch cultivation mode in order to facilitate the simultaneously wastewater treatment and production of biomass.
2. Material and methods
2.1 Microorganisms and cultivation conditions The cyanobacterium Arthrospira platensis SAG 21.99 and the green microalga Chlorella vulgaris SAG 211-11b used in the study were obtained from SAG (Sammlung von Algenkulturen der Universität Göttingen, Germany). Arthrospira and Chlorella are two species
that are cultivated on commercial large scale worldwide and were therefore selected as model organisms for this study. The inoculum of A. platensis was prepared using Zarrouk medium with the following composition: 16.8 g/L NaHCO3, 2.5 g/L NaNO3, 0.5 g/L KH2PO4, 1.0 g/L K2SO4, 1.0 g/L NaCl, 40 mg/L CaCl2, 80 mg/L Na2EDTA, 200 mg/L MgSO4·7H2O, 10 mg/L FeSO4·7H2Oand 1.0 ml of trace elements stock solutions: 2.86 g/L H3BO3, 20 mg/L (NH4)6Mo7O24, 1.8 g/L MnCl2·4H2O, 80 mg/L CuSO4 and 220 mg/L ZnSO4·7H2O. Inoculum of the green alga C. vulgaris was prepared using a modified BG-11 medium (initial pH 7) with the following composition: 20 mg/L Na2CO3, 2.5 g/L NaNO3, 40 mg/L KH2PO4, 0.2 g/L K2SO4, 36 mg/L CaCl2, 75 mg/L MgSO4·7H2O, 6 mg/L citric acid, 6 mg/L ammonium ferric citrate green, 1 mg/L Na2EDTA and 1.0 ml of trace elements stock solutions as above. The above cultivation media served as the control cultures as well. The experiments were carried out in 400 mL glass bubble photobioreactors, with working volume of 250 mL. The cultures were aerated with filtered air provided by a membrane air pump. The cultivation was carried out in a growth chamber and culture temperature was kept constant at 30°C (± 2°C). Light intensity (measured in the middle of the photobioreactors) was set at 9 klux and was provided through two 57W cool fluorescence tube-lamps on the one side of the photobioreactors. The photoperiod light/dark was set at 16h/8h. Cultures were carried out in duplicates. Feeding of substrate was performed every day.
2.2 Ammonia-rich wastewater As an ammonia-rich wastewater model, effluents of the anaerobic mono-digestion of poultry litter were used. Anaerobic digestion was performed using moderate diluted poultry litter with total solids of about 20% and hydraulic retention time 30 days. The digestion was conducted using semi-continuous mode under mesophilic temperature (35-36 °C). The
effluents (liquor) of the anaerobic digestion were centrifuged at 5000 rpm for 10 min and the supernatant was used for the experiments. Note, that due to high TA concentration, the anaerobic digestion process was to some extent inhibited and VFA were accumulated in high concentrations. Some selected physicochemical characteristics of the wastewater are: pH 7.85; Electrical conductivity (mS/cm) 9.06; Total dissolved solids 4.53 g/L; PO4 -P 83 ± 3 mg P/L; Total P 96 ± 5 mg P/L; NH4+-N 4315 ± 834 mg N/L; Volatile fatty acids 13958 ± 375 mg/L; COD 25821 ± 1659 mg O2/L; Carbohydrates 310 ± 27 mg/L; Proteins 4879 ± 194 mg/L; K 2590 ± 74 mg/L; Na 261 ± 11 mg/L; Mg 10.43 ± 0.14 mg/L; Fe 4.33 ± 0.34 mg/L; Mn 471 ± 20 μg/L; Zn 134 ± 1 μg/L; Ni 421 ± 34 μg/L; Cu 585 ± 75 μg/L; Pb 150 ± 47 μg/L, and Cd >3 μg/L.
2.3 Experimental set-up The cultivation was performed using the fed-batch cultivation technique, by which the substrate is being enriched gradually during the cultivation period. As the main investigation subject of the present study was the ammonia toxicity, the addition of the ammonia-rich wastewater was performed in the basis of total ammonia (TA) concentration. Both microorganisms, were cultivated applying wastewater in amounts that corresponded to the daily addition of 5, 10, 20 and 30 mg-N/(L d). To start the cultures, 100-fold diluted wastewater was used as the initial cultivation medium. For the dilution of wastewater tap water was used. Because A. platensis requires high concentrations of sodium and alkalinity, in the media for its cultivation, 10 g/L NaHCO3 was additionally added. In Table 1, the final concentrations of selected compounds are listed. Note, that due to the evaporation of water (about 2.5-5%) during the cultivation, the addition of the wastewater and fresh (deionized) water to fill the evaporation losses, was performed in such way so that the final culture volume was fixed for all cultures equal to 250 mL, i.e. although the added volume of
wastewater corresponding to each culture was different, at the end of each addition all cultures were of 250 mL.
2.4 Analytical methods Dry algal biomass was measured indirectly by spectrophotometry at 750 nm. Proteins were determined according to the Lowry method using bovine albumin as standard, total lipids according to the sulfo-phospho-vanillin reaction method, using corn-oil as the standard, carbohydrates by the phenol-sulfuric acid method using D-glucose as standard, and pigments were extracted with hot 90% methanol, while phycocyanin with phosphate buffer by freezing and thawing the samples (for references see Markou et al. (2014)). All biomass composition analyses were performed after the washing of the samples for several times with DI water. Total ammonia was measured with the phenate method, orthophosphate with the ascorbic acid method, and chemical oxygen demand (COD) with the closed reflux method according to standard methods (APHA, 1995). Volatile fatty acids (VFA) were determined according to a modified method of Montgomery et al. (1962), using acetic acid as the standard. Briefly, in 50 μL of sample, 170 μL of acidic ethylene glycol reagent was added, heated for 3 min in boiling water, cooled down followed by the addition of 250 μL of hydroxylamine reagent. After 1-2 minutes 1000 μL of acidic ferric chloride reagent was added. The modification allowed to increase the sensitivity of the essay; the detection limit of VFA was 7 mg/L. All spectrophotometric determinations were carried out on a spectrophotometer Dr. Lange, Cadas 50 (Germany). Potassium was measured by flame-photometer (Sherwood Scientific, model 400). Micro-nutrients and heavy metals were measured by flame atomic absorption spectrometry (Varian AA-200). The results are given as the average of six values (n=6; analyses were carried in triplicates for each replicate). Statistical analysis was performed using ANOVA.
3. Results and discussion
3.1 Biomass production Fig. 1 illustrates the biomass production during the cultivation of A. platensis and C. vulgaris using four different fed-batch TA levels (5, 10, 20 and 30 mg-N/(L d). A. platensis displayed a growth without lag phase (Fig. 1a), and the final biomass density was gradually increased as the TA addition level increased. However, the final biomass density in cultures with 20 and 30 mg-N/(L d), was not statistically significant different (p>0.05), showing that both cultures reached the maximum possible biomass density determined by the specific cultivation parameter, such as light intensity and temperature. The macroscopic appearance of the cultures with 5 and 10 mg-N/(L d) was yellowish, with a strong tendency of the cells to bioflocculate. This indicated that these cultures were under some nutrient limitation (see section 3.2). Final biomass density reached 829 ± 90, 1009 ± 117, 1627 ± 82, and 1519 ± 38 mg/L, for 5, 10, 20 and 30 mg-N/(L d), respectively. In contrast, C. vulgaris displayed a lag phase until day 4 (Fig. 1b). After day 4, its growth was unhindered and all cultures had the same growth pattern, having an overall sigmoid growth curve. Final biomass density was almost the same for all TA addition levels, except 5 mg-N/(L d), reaching 1407 ± 8, 1526 ± 6, 1522 ± 42, and 1433 ± 98 mg/L, for 5, 10, 20 and 30 mg-N/(L d), respectively. The addition of wastewater to the cultivation medium, results to a decrease of the light penetration, and hence to a decrease of photosynthetic rate (Depraetere et al., 2013). In Fig. 2a the light absorption of the cultivation medium (without cells) at the end of the cultivation period (day 9 and 12 for A. platensis and C. vulgaris, respectively) is shown. As the fed-batch
level increased, increased also the light absorption of the medium due to the turbidity and the dissolved colored compounds of the wastewater. However, in the case of A. platensis with 5 and 10 mg-N/(L d) the effect of the light absorption was not strong, probably because the limiting factor of these cultures was the nutrient starvation, defining the final biomass production. In the cultures with 20 and 30 mg-N/(L d), as in all of C. vulgaris the biomass density was not much different showing that the light absorption in these cultures did not have a significant influence. This perhaps is due to the presence of organic compounds (mainly VFA), which can be taken up by the cells mixotrophically. The myxotrophic growth, induced the biomass synthesis by providing energy from the VFA instead of photosynthesis (Chojnacka and Marquez-Rocha, 2004). Uptake of VFA perhaps mitigated the negative effect of the lower light penetration due to light absorption.
3.2 Removal of wastewater constituents
Fig.3 and Fig. 4 illustrate the removal capacity of A. platensis and C. vulgaris of selected wastewater constituents. The removal corresponds to measures performed at the end of each cultivation period (day 9 and 12 for A. platensis and C. vulgaris, respectively). A. platensis removed almost completely (>99%) the NH4+-N, while C. vulgaris had also high removal rates (>95%) in all fed-batch TA addition levels. The N taken up by the cells was calculated based on the protein content divided by 6.25, corresponded to a intracellular content of about 47, 59, 90, and 128 mg-N/g, and 59, 89, 97, and 112 mg-N/g for 5, 10, 20 and 30 mg-N/(L d), and A. platensis and C. vulgaris, respectively. Based on the mass of TA added to the cultures, the uptake efficiency was 53%, 44%, 40%, and 41% and 60%, 58%, 37%, and 30% for 5, 10, 20 and 30 mg-N/(L d), and A. platensis and C. vulgaris, respectively, a fact that indicates that a
significant portion of the ammonia was lost to the atmosphere due to its volatilization under high pH values. Till now, little attention is given to the ammonia losses during the cultivation. The most significant factors that affects ammonia losses is the pH of the medium and the initial ammonia concentration. Increasing pH and ammonia concentration the ammonia losses increase too (Markou et al., 2014). However, the quantity of ammonia loss is not only determined by the pH of the culture and the ammonia concentration but also by the agitation type used. Aeration of cultures results to higher ammonia losses than stirring (Pouliot et al., 1989). From an economic and environmental point of view, this fact should be taken into consideration during the design of MBWT systems treating wastewaters with high ammonia concentration. A. platensis was capable to remove almost completely (>99%) PO4-P, while TP was removed >96%. The taken up by the cells P was 2.26, 2.81, 2.92, and 4.39 mg-P/g dry biomass, showing that there was a gradual increase of the intracellular P as the fed-batch TA addition level increased. Intracellular P content of 3 mg-P/g and lower in A. platensis could be considered as limiting (Markou, 2012). C. vulgaris had almost the same removal capacity as A. platensis (>99% and >97%, for PO4-P and TP, respectively) and the intracellular P was calculated to be 1.48, 2.14, 3.68, and 5.54 mg-P/g for 5, 10, 20 and 30 mg-N/(L d), respectively. However there are no available data yet for C. vulgaris to judge on which intracellular P concentration indicates that the growth is limited. P is frequently reported as the limiting factor in various wastewaters investigated (Cai et al., 2013; Wang et al., 2010). P removal and recovery displays particular significance, because P is associated with eutrophication and environmental issues and because it is not renewable and its rock deposits will be exhausted in the future (Elser, 2012).
It is well known that the nutrient limitation causes growth and biochemical composition alterations (Markou et al., 2012). Mg and Fe are frequently reported as limiting nutrients, while K is a significant nutrient and it is one of the most abundant in the microalgal biomass (Tokuşoglu and Ünal, 2003). In Table 2 the residual concentrations of K, Mg, and Fe are tabulated. K residual concentration is abundant indicating that the cultures were not K limited. Regarding Mg and Fe it is not clear whether they were limited, because their residual concentrations were very low, but their removal were not as high as should be expected under limiting conditions. Some microalgae can grow on organic molecules, such as monosaccharides (glucose fructose etc), organic acids (acetate etc.), amino acids, glycerol etc. (Heredia-Arroyo et al., 2011). Among the organic acids, the short-change fatty acids up to 6 carbons (or volatile fatty acids-VFA), are of great interest because they can serve as a cheap energy and carbon source for microalgae growth. Moreover, they are frequently contained in organic wastewater and could be produced through the biochemical hydrolysis of organic matter (Hu et al., 2013; Mohan and Devi, 2012). Both microorganisms used in the present study could severe remove VFA from the wastewater (>93% and >90% for A. platensis and C. vulgaris, respectively), indicating that both microorganisms could grow on the VFA contained in the wastewater. However, because the cultures were aerated and enriched with atmospheric CO2, the effect of the different VFA concentrations on growth and biomass production was not manifested. It is demonstrated in various works that microalgae and cyanobacteria are capable to take up and assimilate amino acids however, the degree of their removal depends on the microalgal and amino acid species (Perez-Garcia et al., 2011). The specific wastewater used for the experiments, originated from the anaerobic digestion of poultry litter and therefore was rich in proteins and amino acids. Between the two microalgae used, A. platensis in the
cultures with 5 and 10 mg-N/(L d) was not able to remove proteins; in contrast the protein content in the cultivation medium increased, indicating that some additional proteins were released to it. As shown in Fig. 1a in these two cultures the biomass density became to decrease. Perhaps this decrease corresponded to the phase of death and cell lysis (Li et al., 2011), causing protein and amino leaching to the cultivation medium. In the cultures with 20 and 30 mg-N/(L d) the protein removal was low (less than 20%). In contrast, C. vulgaris had significant higher protein removal (49-58%) capacity than A. platensis. Regarding the removal of carbohydrates, in both microorganisms the removal was negative, indicating that carbohydrates (mainly polysaccharides) were released to the medium (De Philippis et al., 2001; Maksimova et al., 2004). Among the two species, A. platensis released more carbohydrates in the medium than C. vulgaris, because A. platensis being a cyanobacterium tends to excrete high amounts of polysaccharides. COD expresses the overall organic load (dissolved and suspended matter) of the wastewater. From the point of view of wastewater treatment, higher COD removals and hence lower residual organic loads are preferable. In general, the COD removal was higher as the fed-batch TA addition level increased. Among the two species, C. vulgaris displayed a higher COD removal. The highest COD removals reached around 75% for both species.
3.3 Biomass composition
In Table 3 the biochemical biomass composition of A. platensis and C. vulgaris is tabulated. A. platensis displayed different biochemical compositions in the four fed-batch TA addition levels. A general observation was that increasing the TA addition level, proteins, lipids, phycocyanin, chlorophyll α, and total carotenoids increased, while carbohydrates decreased.
This behavior is due to the gradually nutrient starvation, which occurred. It has been documented that under nutrient starvation A. platensis tends to accumulate carbohydrates, while nitrogenous compounds like proteins, chlorophyll and phycocyanin are synthesized less resulting to their lower biomass content (Markou, 2012). Same overall behavior was observed also for C. vulgaris. The most significant change in C. vulgaris biomass was the very high lipid accumulation (about 50%) with 5 mg-N/(L d). It is however, notable that the biomass density in the cultures with 5 mg-N/(L d) was not significant lower, showing that in these cultures a simultaneous maximum biomass production along with the accumulation of lipids occurred. It is well documented that in some microalgae, the nutrient starvation, and in particular the nitrogen starvation induces the accumulation of lipids (Hu et al., 2008). High accumulation of carbohydrates or lipids is of particular interest because the potential of using microalgal biomass for the production of biofuels increases (Breuer et al., 2012; Markou et al., 2012). Besides the biofuel production, both microorganisms investigated displayed a high protein and lipid content, which could also be used to produce feed supplements or other chemicals. Some major concerns about microalgal and cyanobacterial cultivation is their low economic feasibility, especially when dealing with low value products such as energy and biofuels, and their environmental impact and sustainability, mainly due to high energy water consumption in the various production stages. However, microalgal cultivation systems are a relative new technology, which is expected to be improved in the future bringing them closer to be feasible and sustainable (Acién et al., 2012; Taelman et al., 2015).
4. Conclusions
The present study suggests that the fed-batch cultivation mode is a successful tool for using ammonia-rich wastewaters, for their simultaneously treatment and the production of biomass. Ammonia and phosphorus removals from the wastewater were very high (>95%) for all cultures investigated, while chemical oxygen demand and proteins were removed in various degrees. Both microorganisms were capable to grow on volatile fatty acids, indicating that they were capable for mixotrophic growth. A. platensis accumulated carbohydrates (≈40%), while C. vulgaris accumulated lipids (≈50%), making them interesting feedstock for bioethanol and biodiesel, respectively.
Acknowledgments This research project is funded under the Action “Research & Technology Development Innovation projects (AgroETAK)”, MIS 453350, in the framework of the Operational Program “Human Resources Development”. It is co-funded by the European Social Fund and by National Resources through the National Strategic Reference Framework 2007-2013 (NSRF 2007-2013) coordinated by the Hellenic Agricultural Organisation "DEMETER" (Institute of Soil and Water Resources / Scientific supervisor: Dr. Dimitris Oiconomou).
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Table and figure captions. Table 1. Final concentration of the major chemical compounds. The final concentrations corresponds to the initial concentration of the 100-fold diluted wastewater plus its daily addition for 9 and 12 days for A. platensis and C. vulgaris, respectively. Table 2. Residual concentrations of K, Mg and Fe. Inside the brackets their removal is indicated. Table 3. Biomass composition of A. platensis and C. vulgaris
Fig. 1. Biomass production of (a) A. platensis and (b) C. vulgaris cultivated with the addition of wastewater corresponding to 5, 10, 20 and 30 mg-N/(L d). Fig. 2. Light absorption of the four different cultivation media derived by the gradual addition of wastewater. The light absorption corresponds to the cultivation media on day 9 and 12 for A. platensis (a) and C. vulgaris (b). Fig. 3. Removal of selected constituents of the wastewater in cultures of A. platensis Fig. 4. Removal of selected constituents of the wastewater in cultures of C. vulgaris
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Fig. 1. Biomass production of (a) A. platensis and (b) C. vulgaris cultivated with the addition of wastewater corresponding to 5, 10, 20 and 30 mg-N/(L d).
a) 0.8 5 mg-N/(L d) 10 mg-N/(L d) 20 mg-N/(L d) 30 mg-N/(L d)
Absorption
0.6 0.4 0.2 0.0
350 400 450 500 550 600 650 700 750 Wavelenght (nm)
b) 0.8 5 mg-N/(L d) 10 mg-N/(L d) 20 mg-N/(L d) 30 mg-N/(L d)
Absorption
0.6 0.4 0.2 0.0
350 400 450 500 550 600 650 700 750 Wavelenght (nm)
Fig. 2. Light absorption of the four different cultivation media derived by the gradual addition of wastewater. The light absorption corresponds to the cultivation media on day 9 and 12 for A. platensis (a) and C. vulgaris (b).
PO4 -P removal (%)
100
80 60
3-
40
+
NH4 -N removal (%)
100
20
80 60 40 20
0
0
VFA removal (%)
5
10 20 30 Fed-batch level
5
10 20 30 Fed-batch level
100 80 60 40 20 0 10 20 30 Fed-batch level
40 20 0 -20 -40 -60 5
10 20 30 Fed-batch level
Carbohydrates removal (%)
Proteins removal (%)
5
0 -200 -400 -600 -800 5
10 20 30 Fed-batch level
COD removal (%)
100 80 60 40 20 0 5
10 20 30 Fed-batch level
Fig. 3. Removal of selected constituents of the wastewater in cultures of A. platensis
PO4 -P removal (%)
100
80 60
80 60 40
3-
40
+
NH4 -N removal (%)
100
20
20
0
0
VFA removal (%)
5
10 20 30 Fed-batch level
5
10 20 30 Fed-batch level
100 80 60 40 20 0 10 20 30 Fed-batch level
60 40 20 0 5
10 20 30 Fed-batch level
Carbohydrates removal (%)
Proteins removal (%)
5
100 0 -100 -200 -300 -400 -500 5
10 20 30 Fed-batch level
COD removal (%)
100 80 60 40 20 0 5
10 20 30 Fed-batch level
Fig. 4. Removal of selected constituents of the wastewater in cultures of C. vulgaris
Table 1. Final concentration of the major chemical compounds. The final concentrations corresponds to the initial concentration of the 100-fold diluted wastewater plus its daily addition for 9 and 12 days for A. platensis and C. vulgaris, respectively. + NH4 -N
(mg-N/L)
-
PO4 P (mg-P/L) TP (mg-P/L) VFA (mg/L) COD (mg/L)
A. platensis C. vulgaris A. platensis C. vulgaris A. platensis C. vulgaris A. platensis C. vulgaris A. platensis C. vulgaris
5 mg-N/(L d) 88 98 1.69 1.88 1.95 2.18 285 317 528 587
10 mg-N/(L d) 138 153 2.56 2.94 2.95 3.40 431 495 797 917
20 mg-N/(L d) 223 263 4.28 5.84 4.95 5.84 722 851 1335 1575
30 mg-N/(L d) 313 373 6.01 8.28 6.94 8.28 1013 1207 1874 2333
Table 2. Residual concentrations of K, Mg and Fe. Inside the brackets their removal is indicated. K (mg/L) Mg (μg/L) Fe (μg/L)
A. platensis C. vulgaris A. platensis C. vulgaris A. platensis C. vulgaris
5 mg-N/(L d) 43 ± 1 (17.9%) 44 ± 2 (26%) 199 ± 1 (7%) 68 ± 24 (71%) 29 ± 21 (67%) 41 ± 1 (58%)
10 mg-N/(L d) 63 ± 1 (21.8%) 72 ± 4 (22%) 190 ± 11 (41%) 94 ± 12 (75%) 109 ± 30 (19%) 47 ± 1 (70%)
20 mg-N/(L d) 103 ± 1 (22.7%) 123 ± 1 (22.2%) 176 ± 17 (67%) 78 ± 11 (88%) 199 ± 2 (11%) 117 ± 21 (56%)
30 mg-N/(L d) 142 ± 2 (24.5%) 184 ± 2 (17.8%) 184 ± 7 (76%) 81 ± 6 (91%) 268 ± 16 (15%) 215 ± 23 (43%)
Table 3. Biomass composition of A. platensis and C. vulgaris
C. vulgaris
A. platensis
Species Proteins (%) Carbohydrates (%) Lipids (%) Phycocyanin (%) Chlorophyll α (%) Carotenoids (%) Proteins (%) Carbohydrates (%) Lipids (%) Chlorophyll α (%) Chlorophyll β (%) Carotenoids (%)
Fed-batch TA addition 10 mg-N/(L d) 5 mg-N/(L d) 28.4 ± 2.4 36.6 ± 2.9 41.2 ± 2.8 34.5 ± 4.2 23.6 ± 2.6 22.0 ± 3.1 4.77 ± 0.98 5.02 ± 1.81 0.78 ± 0.15 0.81 ± 0.06 0.17 ± 0.02 0.17 ± 0.01 26.3 ± 2.4 36.3 ± 4.0 24.8 ± 2.3 25.0 ± 1.6 50.1 ± 1.1 41.7 ± 2.7 0.63 ± 0.11 1.11 ± 0.12 0.60 ± 0.16 0.90 ± 0.06 0.24 ± 0.07 0.31 ± 0.03
20 mg-N/(L d) 34.4 ± 3.0 38.5 ± 1.9 21.8 ± 1.2 4.55 ± 1.01 1.01 ± 0.4 0.18 ± 0.0.2 40.0 ± 1.7 23.5 ± 1.4 36.5 ± 2.6 1.76 ± 0.13 1.35 ± 0.13 0.33 ± 0.06
30 mg-N/(L d) 52.6 ± 1.8 16.9 ± 1.0 31.0 ± 2.6 12.1 ± 3.21 1.33 ± 0.24 0.42 ± 0.03 48.8 ± 1.3 21.0 ± 1.7 33.9 ± 2.1 2.14 ± 0.09 1.70 ± 0.10 0.33 ± 0.02
Highlights • • • •
Ammonia-rich wastewater derived from anaerobic digestion of poultry litter Fed-batch cultivation of Arthrospira platensis and Chlorella vulgaris Successful treatment of wastewater with high N and P removals Optimized biomass production with accumulated carbohydrates or lipids
S0960-8524(15)00862-7 http://dx.doi.org/10.1016/j.biortech.2015.06.071 BITE 15151
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 May 2015 13 June 2015 15 June 2015
Please cite this article as: Markou, G., Fed-batch cultivation of Arthrospira and Chlorella in ammonia-rich wastewater: optimization of nutrient removal and biomass production, Bioresource Technology (2015), doi: http:// dx.doi.org/10.1016/j.biortech.2015.06.071
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Fed-batch cultivation of Arthrospira and Chlorella in ammonia-rich wastewater: optimization of nutrient removal and biomass production
Giorgos Markou 1,2 1
Department of Agricultural Engineering, Institute of Soil and Water Resources, Hellenic Agricultural Organization-Demeter,
Leoforos Dimokratias 61, 13561, Athens, Greece 2
Department of Natural Resources Management and Agricultural Engineering, Agricultural University of Athens, Iera Odos
75, 11855 Athens, Greece * E-mail: [email protected], [email protected]
Abstract In the present work the cyanobacterium Arthrospira platensis and the microalga Chlorella vulgaris were fed-batch cultivated in ammonia-rich wastewater derived from the anaerobic digestion of poultry litter. Aim of the study was to maximize the biomass production along with the nutrient removal aiming to wastewater treatment. Ammonia and phosphorus removals were very high (>95%) for all cultures investigated. Both microorganisms were able to remove volatile fatty acids to an extent of >90%, indicating that they were capable for mixotrophic growth. Chemical oxygen demand and proteins were also removed in various degrees. In contrast, in all cultures carbohydrate concentration was increased. The biochemical composition of the microorganisms varied greatly and was influenced by the indicate that the nutrient availability. A. platensis accumulated carbohydrates (≈40%), while C. vulgaris accumulated lipids (≈50%), rendering them interesting for biofuel production.
Keywords: Ammonia toxicity; anaerobic digestion; biomass; cyanobacteria; microalgae; poultry litter; wastewater
1. Introduction Microalgal-based wastewater treatment (MBWT) has recently gained the interest worldwide, mainly because it displays a dual role, i.e. on the one hand the wastewater is treated rendering it more harmless to be disposed, and on the other hand producing valuable microalgal biomass for the generation of biofuels and other chemicals (Pittman et al., 2011; Zeng et al., 2015). MBWT can be considered as a biological treatment method, aiming to the reduction of both, inorganic (primarily N and P) and organic load of wastewater (Dickinson et al., 2015; Park et al., 2011). However, ammonia-rich wastewaters display a major limitation to be treated with MBWT, due to the occurrence of ammonia toxicity. When ammonia concentration exceeds a level in the aquatic surroundings, it acts inhibitory to the microalgal cells, reducing their growth rates or even causing their death (Abeliovich and Azov, 1976; Markou et al., 2014). Some organic substrates, such as poultry litter, due to the high content of nitrogenous compounds, have a very low carbon to nitrogen ratio, and during anaerobic digestion tend to accumulate ammonia. The total ammonia (TA) concentration on the liquor can exceed 5 g/L (Gangagni Rao et al., 2008; Yenigün and Demirel, 2013), which is almost two orders of magnitude higher than the TA concentration that microalgae could tolerate (Abeliovich and Azov, 1976; Peccia et al., 2013). Therefore, the only strategy to avoid ammonia toxicity is the dilution of the wastewater, adapting ammonia concentration below the toxic limits (Peccia et al., 2013). Ammonia-rich wastewater though, should first be strongly diluted, resulting as well to a high dilution of other essential nutrients (such as P and K) and
their low final concentration with a consequence of acting limiting to microalgal growth. Moreover, limiting conditions can affect the removal rate of the nutrients (Beuckels et al., 2015), decreasing the treatment efficiency of the MBWT system. Hence, for an unhindered biomass production it is required to apply the nutrients in such a way that neither ammonia toxicity or nutrient limitation will occur. A strategy that has been proven successful for avoiding ammonia toxicity is the fed-batch cultivation mode (Ji et al., 2015; Rodrigues et al., 2010), by which ammonia is added (fed) gradually to the cultivation medium. Generally, fed-batch mode is superior to the batch one, regarding the gradually addition of toxic substances, but both have some limitations in scaledup systems. A major disadvantage is the need to prevent contamination during the initial inoculation and early growth period due to low initial cell density. Especially when dealing with wastewater the contamination of the culture is more possible. However, batch and fedbatch modes, display some advantages regarding their simplicity and flexibility and are frequently used (Lavens and Sorgeloos, 1996). In any case, little attention is given to the fedbatch cultivation mode regarding the ammonia-rich wastewater treatment. Consequently, aim of this study was to investigate the capability of using the fed-batch cultivation mode in order to facilitate the simultaneously wastewater treatment and production of biomass.
2. Material and methods
2.1 Microorganisms and cultivation conditions The cyanobacterium Arthrospira platensis SAG 21.99 and the green microalga Chlorella vulgaris SAG 211-11b used in the study were obtained from SAG (Sammlung von Algenkulturen der Universität Göttingen, Germany). Arthrospira and Chlorella are two species
that are cultivated on commercial large scale worldwide and were therefore selected as model organisms for this study. The inoculum of A. platensis was prepared using Zarrouk medium with the following composition: 16.8 g/L NaHCO3, 2.5 g/L NaNO3, 0.5 g/L KH2PO4, 1.0 g/L K2SO4, 1.0 g/L NaCl, 40 mg/L CaCl2, 80 mg/L Na2EDTA, 200 mg/L MgSO4·7H2O, 10 mg/L FeSO4·7H2Oand 1.0 ml of trace elements stock solutions: 2.86 g/L H3BO3, 20 mg/L (NH4)6Mo7O24, 1.8 g/L MnCl2·4H2O, 80 mg/L CuSO4 and 220 mg/L ZnSO4·7H2O. Inoculum of the green alga C. vulgaris was prepared using a modified BG-11 medium (initial pH 7) with the following composition: 20 mg/L Na2CO3, 2.5 g/L NaNO3, 40 mg/L KH2PO4, 0.2 g/L K2SO4, 36 mg/L CaCl2, 75 mg/L MgSO4·7H2O, 6 mg/L citric acid, 6 mg/L ammonium ferric citrate green, 1 mg/L Na2EDTA and 1.0 ml of trace elements stock solutions as above. The above cultivation media served as the control cultures as well. The experiments were carried out in 400 mL glass bubble photobioreactors, with working volume of 250 mL. The cultures were aerated with filtered air provided by a membrane air pump. The cultivation was carried out in a growth chamber and culture temperature was kept constant at 30°C (± 2°C). Light intensity (measured in the middle of the photobioreactors) was set at 9 klux and was provided through two 57W cool fluorescence tube-lamps on the one side of the photobioreactors. The photoperiod light/dark was set at 16h/8h. Cultures were carried out in duplicates. Feeding of substrate was performed every day.
2.2 Ammonia-rich wastewater As an ammonia-rich wastewater model, effluents of the anaerobic mono-digestion of poultry litter were used. Anaerobic digestion was performed using moderate diluted poultry litter with total solids of about 20% and hydraulic retention time 30 days. The digestion was conducted using semi-continuous mode under mesophilic temperature (35-36 °C). The
effluents (liquor) of the anaerobic digestion were centrifuged at 5000 rpm for 10 min and the supernatant was used for the experiments. Note, that due to high TA concentration, the anaerobic digestion process was to some extent inhibited and VFA were accumulated in high concentrations. Some selected physicochemical characteristics of the wastewater are: pH 7.85; Electrical conductivity (mS/cm) 9.06; Total dissolved solids 4.53 g/L; PO4 -P 83 ± 3 mg P/L; Total P 96 ± 5 mg P/L; NH4+-N 4315 ± 834 mg N/L; Volatile fatty acids 13958 ± 375 mg/L; COD 25821 ± 1659 mg O2/L; Carbohydrates 310 ± 27 mg/L; Proteins 4879 ± 194 mg/L; K 2590 ± 74 mg/L; Na 261 ± 11 mg/L; Mg 10.43 ± 0.14 mg/L; Fe 4.33 ± 0.34 mg/L; Mn 471 ± 20 μg/L; Zn 134 ± 1 μg/L; Ni 421 ± 34 μg/L; Cu 585 ± 75 μg/L; Pb 150 ± 47 μg/L, and Cd >3 μg/L.
2.3 Experimental set-up The cultivation was performed using the fed-batch cultivation technique, by which the substrate is being enriched gradually during the cultivation period. As the main investigation subject of the present study was the ammonia toxicity, the addition of the ammonia-rich wastewater was performed in the basis of total ammonia (TA) concentration. Both microorganisms, were cultivated applying wastewater in amounts that corresponded to the daily addition of 5, 10, 20 and 30 mg-N/(L d). To start the cultures, 100-fold diluted wastewater was used as the initial cultivation medium. For the dilution of wastewater tap water was used. Because A. platensis requires high concentrations of sodium and alkalinity, in the media for its cultivation, 10 g/L NaHCO3 was additionally added. In Table 1, the final concentrations of selected compounds are listed. Note, that due to the evaporation of water (about 2.5-5%) during the cultivation, the addition of the wastewater and fresh (deionized) water to fill the evaporation losses, was performed in such way so that the final culture volume was fixed for all cultures equal to 250 mL, i.e. although the added volume of
wastewater corresponding to each culture was different, at the end of each addition all cultures were of 250 mL.
2.4 Analytical methods Dry algal biomass was measured indirectly by spectrophotometry at 750 nm. Proteins were determined according to the Lowry method using bovine albumin as standard, total lipids according to the sulfo-phospho-vanillin reaction method, using corn-oil as the standard, carbohydrates by the phenol-sulfuric acid method using D-glucose as standard, and pigments were extracted with hot 90% methanol, while phycocyanin with phosphate buffer by freezing and thawing the samples (for references see Markou et al. (2014)). All biomass composition analyses were performed after the washing of the samples for several times with DI water. Total ammonia was measured with the phenate method, orthophosphate with the ascorbic acid method, and chemical oxygen demand (COD) with the closed reflux method according to standard methods (APHA, 1995). Volatile fatty acids (VFA) were determined according to a modified method of Montgomery et al. (1962), using acetic acid as the standard. Briefly, in 50 μL of sample, 170 μL of acidic ethylene glycol reagent was added, heated for 3 min in boiling water, cooled down followed by the addition of 250 μL of hydroxylamine reagent. After 1-2 minutes 1000 μL of acidic ferric chloride reagent was added. The modification allowed to increase the sensitivity of the essay; the detection limit of VFA was 7 mg/L. All spectrophotometric determinations were carried out on a spectrophotometer Dr. Lange, Cadas 50 (Germany). Potassium was measured by flame-photometer (Sherwood Scientific, model 400). Micro-nutrients and heavy metals were measured by flame atomic absorption spectrometry (Varian AA-200). The results are given as the average of six values (n=6; analyses were carried in triplicates for each replicate). Statistical analysis was performed using ANOVA.
3. Results and discussion
3.1 Biomass production Fig. 1 illustrates the biomass production during the cultivation of A. platensis and C. vulgaris using four different fed-batch TA levels (5, 10, 20 and 30 mg-N/(L d). A. platensis displayed a growth without lag phase (Fig. 1a), and the final biomass density was gradually increased as the TA addition level increased. However, the final biomass density in cultures with 20 and 30 mg-N/(L d), was not statistically significant different (p>0.05), showing that both cultures reached the maximum possible biomass density determined by the specific cultivation parameter, such as light intensity and temperature. The macroscopic appearance of the cultures with 5 and 10 mg-N/(L d) was yellowish, with a strong tendency of the cells to bioflocculate. This indicated that these cultures were under some nutrient limitation (see section 3.2). Final biomass density reached 829 ± 90, 1009 ± 117, 1627 ± 82, and 1519 ± 38 mg/L, for 5, 10, 20 and 30 mg-N/(L d), respectively. In contrast, C. vulgaris displayed a lag phase until day 4 (Fig. 1b). After day 4, its growth was unhindered and all cultures had the same growth pattern, having an overall sigmoid growth curve. Final biomass density was almost the same for all TA addition levels, except 5 mg-N/(L d), reaching 1407 ± 8, 1526 ± 6, 1522 ± 42, and 1433 ± 98 mg/L, for 5, 10, 20 and 30 mg-N/(L d), respectively. The addition of wastewater to the cultivation medium, results to a decrease of the light penetration, and hence to a decrease of photosynthetic rate (Depraetere et al., 2013). In Fig. 2a the light absorption of the cultivation medium (without cells) at the end of the cultivation period (day 9 and 12 for A. platensis and C. vulgaris, respectively) is shown. As the fed-batch
level increased, increased also the light absorption of the medium due to the turbidity and the dissolved colored compounds of the wastewater. However, in the case of A. platensis with 5 and 10 mg-N/(L d) the effect of the light absorption was not strong, probably because the limiting factor of these cultures was the nutrient starvation, defining the final biomass production. In the cultures with 20 and 30 mg-N/(L d), as in all of C. vulgaris the biomass density was not much different showing that the light absorption in these cultures did not have a significant influence. This perhaps is due to the presence of organic compounds (mainly VFA), which can be taken up by the cells mixotrophically. The myxotrophic growth, induced the biomass synthesis by providing energy from the VFA instead of photosynthesis (Chojnacka and Marquez-Rocha, 2004). Uptake of VFA perhaps mitigated the negative effect of the lower light penetration due to light absorption.
3.2 Removal of wastewater constituents
Fig.3 and Fig. 4 illustrate the removal capacity of A. platensis and C. vulgaris of selected wastewater constituents. The removal corresponds to measures performed at the end of each cultivation period (day 9 and 12 for A. platensis and C. vulgaris, respectively). A. platensis removed almost completely (>99%) the NH4+-N, while C. vulgaris had also high removal rates (>95%) in all fed-batch TA addition levels. The N taken up by the cells was calculated based on the protein content divided by 6.25, corresponded to a intracellular content of about 47, 59, 90, and 128 mg-N/g, and 59, 89, 97, and 112 mg-N/g for 5, 10, 20 and 30 mg-N/(L d), and A. platensis and C. vulgaris, respectively. Based on the mass of TA added to the cultures, the uptake efficiency was 53%, 44%, 40%, and 41% and 60%, 58%, 37%, and 30% for 5, 10, 20 and 30 mg-N/(L d), and A. platensis and C. vulgaris, respectively, a fact that indicates that a
significant portion of the ammonia was lost to the atmosphere due to its volatilization under high pH values. Till now, little attention is given to the ammonia losses during the cultivation. The most significant factors that affects ammonia losses is the pH of the medium and the initial ammonia concentration. Increasing pH and ammonia concentration the ammonia losses increase too (Markou et al., 2014). However, the quantity of ammonia loss is not only determined by the pH of the culture and the ammonia concentration but also by the agitation type used. Aeration of cultures results to higher ammonia losses than stirring (Pouliot et al., 1989). From an economic and environmental point of view, this fact should be taken into consideration during the design of MBWT systems treating wastewaters with high ammonia concentration. A. platensis was capable to remove almost completely (>99%) PO4-P, while TP was removed >96%. The taken up by the cells P was 2.26, 2.81, 2.92, and 4.39 mg-P/g dry biomass, showing that there was a gradual increase of the intracellular P as the fed-batch TA addition level increased. Intracellular P content of 3 mg-P/g and lower in A. platensis could be considered as limiting (Markou, 2012). C. vulgaris had almost the same removal capacity as A. platensis (>99% and >97%, for PO4-P and TP, respectively) and the intracellular P was calculated to be 1.48, 2.14, 3.68, and 5.54 mg-P/g for 5, 10, 20 and 30 mg-N/(L d), respectively. However there are no available data yet for C. vulgaris to judge on which intracellular P concentration indicates that the growth is limited. P is frequently reported as the limiting factor in various wastewaters investigated (Cai et al., 2013; Wang et al., 2010). P removal and recovery displays particular significance, because P is associated with eutrophication and environmental issues and because it is not renewable and its rock deposits will be exhausted in the future (Elser, 2012).
It is well known that the nutrient limitation causes growth and biochemical composition alterations (Markou et al., 2012). Mg and Fe are frequently reported as limiting nutrients, while K is a significant nutrient and it is one of the most abundant in the microalgal biomass (Tokuşoglu and Ünal, 2003). In Table 2 the residual concentrations of K, Mg, and Fe are tabulated. K residual concentration is abundant indicating that the cultures were not K limited. Regarding Mg and Fe it is not clear whether they were limited, because their residual concentrations were very low, but their removal were not as high as should be expected under limiting conditions. Some microalgae can grow on organic molecules, such as monosaccharides (glucose fructose etc), organic acids (acetate etc.), amino acids, glycerol etc. (Heredia-Arroyo et al., 2011). Among the organic acids, the short-change fatty acids up to 6 carbons (or volatile fatty acids-VFA), are of great interest because they can serve as a cheap energy and carbon source for microalgae growth. Moreover, they are frequently contained in organic wastewater and could be produced through the biochemical hydrolysis of organic matter (Hu et al., 2013; Mohan and Devi, 2012). Both microorganisms used in the present study could severe remove VFA from the wastewater (>93% and >90% for A. platensis and C. vulgaris, respectively), indicating that both microorganisms could grow on the VFA contained in the wastewater. However, because the cultures were aerated and enriched with atmospheric CO2, the effect of the different VFA concentrations on growth and biomass production was not manifested. It is demonstrated in various works that microalgae and cyanobacteria are capable to take up and assimilate amino acids however, the degree of their removal depends on the microalgal and amino acid species (Perez-Garcia et al., 2011). The specific wastewater used for the experiments, originated from the anaerobic digestion of poultry litter and therefore was rich in proteins and amino acids. Between the two microalgae used, A. platensis in the
cultures with 5 and 10 mg-N/(L d) was not able to remove proteins; in contrast the protein content in the cultivation medium increased, indicating that some additional proteins were released to it. As shown in Fig. 1a in these two cultures the biomass density became to decrease. Perhaps this decrease corresponded to the phase of death and cell lysis (Li et al., 2011), causing protein and amino leaching to the cultivation medium. In the cultures with 20 and 30 mg-N/(L d) the protein removal was low (less than 20%). In contrast, C. vulgaris had significant higher protein removal (49-58%) capacity than A. platensis. Regarding the removal of carbohydrates, in both microorganisms the removal was negative, indicating that carbohydrates (mainly polysaccharides) were released to the medium (De Philippis et al., 2001; Maksimova et al., 2004). Among the two species, A. platensis released more carbohydrates in the medium than C. vulgaris, because A. platensis being a cyanobacterium tends to excrete high amounts of polysaccharides. COD expresses the overall organic load (dissolved and suspended matter) of the wastewater. From the point of view of wastewater treatment, higher COD removals and hence lower residual organic loads are preferable. In general, the COD removal was higher as the fed-batch TA addition level increased. Among the two species, C. vulgaris displayed a higher COD removal. The highest COD removals reached around 75% for both species.
3.3 Biomass composition
In Table 3 the biochemical biomass composition of A. platensis and C. vulgaris is tabulated. A. platensis displayed different biochemical compositions in the four fed-batch TA addition levels. A general observation was that increasing the TA addition level, proteins, lipids, phycocyanin, chlorophyll α, and total carotenoids increased, while carbohydrates decreased.
This behavior is due to the gradually nutrient starvation, which occurred. It has been documented that under nutrient starvation A. platensis tends to accumulate carbohydrates, while nitrogenous compounds like proteins, chlorophyll and phycocyanin are synthesized less resulting to their lower biomass content (Markou, 2012). Same overall behavior was observed also for C. vulgaris. The most significant change in C. vulgaris biomass was the very high lipid accumulation (about 50%) with 5 mg-N/(L d). It is however, notable that the biomass density in the cultures with 5 mg-N/(L d) was not significant lower, showing that in these cultures a simultaneous maximum biomass production along with the accumulation of lipids occurred. It is well documented that in some microalgae, the nutrient starvation, and in particular the nitrogen starvation induces the accumulation of lipids (Hu et al., 2008). High accumulation of carbohydrates or lipids is of particular interest because the potential of using microalgal biomass for the production of biofuels increases (Breuer et al., 2012; Markou et al., 2012). Besides the biofuel production, both microorganisms investigated displayed a high protein and lipid content, which could also be used to produce feed supplements or other chemicals. Some major concerns about microalgal and cyanobacterial cultivation is their low economic feasibility, especially when dealing with low value products such as energy and biofuels, and their environmental impact and sustainability, mainly due to high energy water consumption in the various production stages. However, microalgal cultivation systems are a relative new technology, which is expected to be improved in the future bringing them closer to be feasible and sustainable (Acién et al., 2012; Taelman et al., 2015).
4. Conclusions
The present study suggests that the fed-batch cultivation mode is a successful tool for using ammonia-rich wastewaters, for their simultaneously treatment and the production of biomass. Ammonia and phosphorus removals from the wastewater were very high (>95%) for all cultures investigated, while chemical oxygen demand and proteins were removed in various degrees. Both microorganisms were capable to grow on volatile fatty acids, indicating that they were capable for mixotrophic growth. A. platensis accumulated carbohydrates (≈40%), while C. vulgaris accumulated lipids (≈50%), making them interesting feedstock for bioethanol and biodiesel, respectively.
Acknowledgments This research project is funded under the Action “Research & Technology Development Innovation projects (AgroETAK)”, MIS 453350, in the framework of the Operational Program “Human Resources Development”. It is co-funded by the European Social Fund and by National Resources through the National Strategic Reference Framework 2007-2013 (NSRF 2007-2013) coordinated by the Hellenic Agricultural Organisation "DEMETER" (Institute of Soil and Water Resources / Scientific supervisor: Dr. Dimitris Oiconomou).
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Table and figure captions. Table 1. Final concentration of the major chemical compounds. The final concentrations corresponds to the initial concentration of the 100-fold diluted wastewater plus its daily addition for 9 and 12 days for A. platensis and C. vulgaris, respectively. Table 2. Residual concentrations of K, Mg and Fe. Inside the brackets their removal is indicated. Table 3. Biomass composition of A. platensis and C. vulgaris
Fig. 1. Biomass production of (a) A. platensis and (b) C. vulgaris cultivated with the addition of wastewater corresponding to 5, 10, 20 and 30 mg-N/(L d). Fig. 2. Light absorption of the four different cultivation media derived by the gradual addition of wastewater. The light absorption corresponds to the cultivation media on day 9 and 12 for A. platensis (a) and C. vulgaris (b). Fig. 3. Removal of selected constituents of the wastewater in cultures of A. platensis Fig. 4. Removal of selected constituents of the wastewater in cultures of C. vulgaris
a)
Biomass (mg/L)
2000 5 mg-N/(L d) 10 mg-N/(L d) 20 mg-N/(L d) 30 mg-N/(L d)
1500 1000 500 0
0
2
4
6
8
10
Time (d)
b)
Biomass (mg/L)
2000 5 mg-N/(L d) 10 mg-N/(L d) 20 mg-N/(L d) 30 mg-N/(L d)
1500 1000 500 0 0
2
4
6
8
10
12
14
Time (d)
Fig. 1. Biomass production of (a) A. platensis and (b) C. vulgaris cultivated with the addition of wastewater corresponding to 5, 10, 20 and 30 mg-N/(L d).
a) 0.8 5 mg-N/(L d) 10 mg-N/(L d) 20 mg-N/(L d) 30 mg-N/(L d)
Absorption
0.6 0.4 0.2 0.0
350 400 450 500 550 600 650 700 750 Wavelenght (nm)
b) 0.8 5 mg-N/(L d) 10 mg-N/(L d) 20 mg-N/(L d) 30 mg-N/(L d)
Absorption
0.6 0.4 0.2 0.0
350 400 450 500 550 600 650 700 750 Wavelenght (nm)
Fig. 2. Light absorption of the four different cultivation media derived by the gradual addition of wastewater. The light absorption corresponds to the cultivation media on day 9 and 12 for A. platensis (a) and C. vulgaris (b).
PO4 -P removal (%)
100
80 60
3-
40
+
NH4 -N removal (%)
100
20
80 60 40 20
0
0
VFA removal (%)
5
10 20 30 Fed-batch level
5
10 20 30 Fed-batch level
100 80 60 40 20 0 10 20 30 Fed-batch level
40 20 0 -20 -40 -60 5
10 20 30 Fed-batch level
Carbohydrates removal (%)
Proteins removal (%)
5
0 -200 -400 -600 -800 5
10 20 30 Fed-batch level
COD removal (%)
100 80 60 40 20 0 5
10 20 30 Fed-batch level
Fig. 3. Removal of selected constituents of the wastewater in cultures of A. platensis
PO4 -P removal (%)
100
80 60
80 60 40
3-
40
+
NH4 -N removal (%)
100
20
20
0
0
VFA removal (%)
5
10 20 30 Fed-batch level
5
10 20 30 Fed-batch level
100 80 60 40 20 0 10 20 30 Fed-batch level
60 40 20 0 5
10 20 30 Fed-batch level
Carbohydrates removal (%)
Proteins removal (%)
5
100 0 -100 -200 -300 -400 -500 5
10 20 30 Fed-batch level
COD removal (%)
100 80 60 40 20 0 5
10 20 30 Fed-batch level
Fig. 4. Removal of selected constituents of the wastewater in cultures of C. vulgaris
Table 1. Final concentration of the major chemical compounds. The final concentrations corresponds to the initial concentration of the 100-fold diluted wastewater plus its daily addition for 9 and 12 days for A. platensis and C. vulgaris, respectively. + NH4 -N
(mg-N/L)
-
PO4 P (mg-P/L) TP (mg-P/L) VFA (mg/L) COD (mg/L)
A. platensis C. vulgaris A. platensis C. vulgaris A. platensis C. vulgaris A. platensis C. vulgaris A. platensis C. vulgaris
5 mg-N/(L d) 88 98 1.69 1.88 1.95 2.18 285 317 528 587
10 mg-N/(L d) 138 153 2.56 2.94 2.95 3.40 431 495 797 917
20 mg-N/(L d) 223 263 4.28 5.84 4.95 5.84 722 851 1335 1575
30 mg-N/(L d) 313 373 6.01 8.28 6.94 8.28 1013 1207 1874 2333
Table 2. Residual concentrations of K, Mg and Fe. Inside the brackets their removal is indicated. K (mg/L) Mg (μg/L) Fe (μg/L)
A. platensis C. vulgaris A. platensis C. vulgaris A. platensis C. vulgaris
5 mg-N/(L d) 43 ± 1 (17.9%) 44 ± 2 (26%) 199 ± 1 (7%) 68 ± 24 (71%) 29 ± 21 (67%) 41 ± 1 (58%)
10 mg-N/(L d) 63 ± 1 (21.8%) 72 ± 4 (22%) 190 ± 11 (41%) 94 ± 12 (75%) 109 ± 30 (19%) 47 ± 1 (70%)
20 mg-N/(L d) 103 ± 1 (22.7%) 123 ± 1 (22.2%) 176 ± 17 (67%) 78 ± 11 (88%) 199 ± 2 (11%) 117 ± 21 (56%)
30 mg-N/(L d) 142 ± 2 (24.5%) 184 ± 2 (17.8%) 184 ± 7 (76%) 81 ± 6 (91%) 268 ± 16 (15%) 215 ± 23 (43%)
Table 3. Biomass composition of A. platensis and C. vulgaris
C. vulgaris
A. platensis
Species Proteins (%) Carbohydrates (%) Lipids (%) Phycocyanin (%) Chlorophyll α (%) Carotenoids (%) Proteins (%) Carbohydrates (%) Lipids (%) Chlorophyll α (%) Chlorophyll β (%) Carotenoids (%)
Fed-batch TA addition 10 mg-N/(L d) 5 mg-N/(L d) 28.4 ± 2.4 36.6 ± 2.9 41.2 ± 2.8 34.5 ± 4.2 23.6 ± 2.6 22.0 ± 3.1 4.77 ± 0.98 5.02 ± 1.81 0.78 ± 0.15 0.81 ± 0.06 0.17 ± 0.02 0.17 ± 0.01 26.3 ± 2.4 36.3 ± 4.0 24.8 ± 2.3 25.0 ± 1.6 50.1 ± 1.1 41.7 ± 2.7 0.63 ± 0.11 1.11 ± 0.12 0.60 ± 0.16 0.90 ± 0.06 0.24 ± 0.07 0.31 ± 0.03
20 mg-N/(L d) 34.4 ± 3.0 38.5 ± 1.9 21.8 ± 1.2 4.55 ± 1.01 1.01 ± 0.4 0.18 ± 0.0.2 40.0 ± 1.7 23.5 ± 1.4 36.5 ± 2.6 1.76 ± 0.13 1.35 ± 0.13 0.33 ± 0.06
30 mg-N/(L d) 52.6 ± 1.8 16.9 ± 1.0 31.0 ± 2.6 12.1 ± 3.21 1.33 ± 0.24 0.42 ± 0.03 48.8 ± 1.3 21.0 ± 1.7 33.9 ± 2.1 2.14 ± 0.09 1.70 ± 0.10 0.33 ± 0.02
Highlights • • • •
Ammonia-rich wastewater derived from anaerobic digestion of poultry litter Fed-batch cultivation of Arthrospira platensis and Chlorella vulgaris Successful treatment of wastewater with high N and P removals Optimized biomass production with accumulated carbohydrates or lipids
