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Growth of Scenedesmus obliquus under artificial flue gas with a high sulphur concentration neutralized with oil shale ash.

Abstract. Oil shale is the main energy resource in Estonia, which generates large amounts of C[O.sub.2] and waste oil shale ash. Flue gas from oil shale combustion can also contain large amounts of S[O.sub.2]. Microalgae can be used for biological sequestration of carbon from flue gas. In this research, green algae Scenedesmus obliquus were grown with 14% C[O.sub.2] in 1 L bioreactors. Sulphuric acid was added with a concentration of 500 ppm and 1000 ppm in order to imitate the dissolution of sulphur dioxide from flue gas into the growth medium. Oil shale ash was used to neutralize S[O.sub.2]. Biomass measurements of S. obliquus, carried out every 24 hours for 7 days, were used as a proxy for carbon fixation. The biomass yields of the untreated control and of the treatments were similar (maximum yield 2.9, 3.1, and 3.9 g [L.sup.-1] for the control, 500 ppm, and 1000 ppm treatment, respectively), suggesting that neither the sulphur nor the ash had an inhibitory effect on algal growth. In fact, the biomass yield was slightly higher in the treatments, which implies that minerals contained in waste ash could be utilized by algae. The calculated C[O.sub.2] fixation rate was 0.45 g [L.sup.-1] [d.sup.-1] for the control, and 0.62 and 0.83 g [L.sup.-1] [d.sup.-1] for 500 ppm and 1000 ppm treatment, respectively. Therefore, microalgae can be used for carbon sequestration from flue gas. Further research should be done in order to optimize the growth conditions and maximize carbon fixation.

Key words: microalgae, Scenedesmus obliquus, oil shale ash, flue gas, C[O.sub.2] sequestration.

1. INTRODUCTION

* Oil shale is the main energy resource used in Estonia with annual use approximately 15 million tonnes [1]. Oil shale is a low-grade fossil fuel with a mineral content of 40-50%; therefore approximately half of the oil shale used for generating electricity ends up as waste oil shale ash [2,3]. According to the report of Eesti Arengufond (Estonian Development Fund), 6.9 million tonnes of oil shale ash was generated in 2012 [4]. As with any fossil fuel, the combustion of oil shale generates high amounts of carbon dioxide: in 2014, 12.8 million tonnes of C[O.sub.2] was emitted by Eesti Energia (the largest energy provider in Estonia) [5]. Due to climate change concerns, measures are taken to mitigate the emissions of C[O.sub.2]. This can be achieved by decreasing energy consumption, increasing the proportion of renewable energy, and sequestering the carbon from flue gas. Sequestering methods vary depending on the storage time and quantity of the fixed C[O.sub.2]. In spite of the fact that C[O.sub.2] mitigation by biological systems is marginal in terms of storage time and global emission rates [6], it offers many advantages.

Although methods based on chemical (using metal oxides or aqueous amine solutions) and physical (e.g. geological sequestration) reactions often require special equipment, are energy-consuming and costly if scaled up for commercial use, and involve disposal problems [7,8], the end product of biological mitigation--biomass--can be refined into various products or converted into energy, thereby making it cost-efficient.

Agricultural and forest ecosystems are able to mitigate atmospheric C[O.sub.2]. However, autotrophic micro-algae can be used to sequester carbon straight from flue gas. Autotrophic microalgae are microscopic, mostly unicellular photosynthetic organisms. Many microalgae species exhibit a higher photosynthesis rate than terrestrial plants and thus can fixate larger quantities of C[O.sub.2] [9]. Algae have been shown to grow under an atmosphere with a C[O.sub.2] concentration as high as 80% and 100% [10]. Flue gas contains on average 10-20% C[O.sub.2] [11,12] and flue gas from oil shale combustion approximately 12% C[O.sub.2] (P. Rohumaa, personal communication). Additionally, bio-mitigation with microalgae can be combined with urban wastewater treatment to establish a cost-effective and sustainable biomass production [7,13].

However, there are some constraints in using microalgae to mitigate C[O.sub.2] straight from flue gas. Firstly, the temperature of flue gas is usually 100-120 [degrees]C, which is unsuitable for algal growth. Therefore, the flue gas has to be cooled to an adequate temperature. This may be achieved with different heat-exchange systems. Secondly, flue gas contains additional gases such as sulphur and nitrogen oxides, which together with carbon dioxide decrease the pH of the growth medium to pH 2-3, and thereby inhibit algal growth. It has been argued, however, that besides lowering the pH, N[O.sub.x] does not have a direct negative impact on microalgal growth because NO for example is transformed into N[O.sub.2] in the culture medium and serves as a nitrogen source for microalgae [14,15]. On the other hand, high amounts of sulphur have been found to be toxic to many algal species [16]. Flue gas from oil shale combustion contains on average 550 ppm and maximally up to 1500 ppm of S[O.sub.2] (P. Rohumaa, personal communication). For this reason the effect of N[O.sub.x] was not studied in the current study and the focus was placed on the possible inhibitory impact of S[O.sub.2] on algae. Although S[O.sub.2] emissions need to be reduced according to the set limits (for Estonia, 100 kt by 2010 and 10 kt by 2020) [17] and desulphurization units are being integrated with power plants, flue gas may still have quite high S[O.sub.2] concentrations in regard to algae. In 2014, Eesti Energia emitted 24.2 kt of S[O.sub.2] [5].

For pH adjustment of acidic conditions, NaOH and CaC[O.sub.3] are widely used with microalgae. In this research, oil shale ash was used for pH adjustment. Oil shale ash, as a waste product of oil shale based energy generation, is readily available in Estonia. It is water-soluble and its pH is 12-13. Therefore, it can be used for the neutralization of acidic conditions caused by high C[O.sub.2] and S[O.sub.2] concentrations. Additionally, oil shale ash contains traces of Ca, Fe, K, Mg, etc. [18,19], which are important elements in algal nutrition. On the other hand, shale ash contains variable quantities of such metals and polyaromatic hydrocarbons that may, depending on their concentration, inhibit algal growth [20-22]. Oil shale fly ash eluate has been shown to be toxic to green algae Pseudokirchneriella subcapitata, while green algae Selenastrum capricornutum, used in Algaltoxkit F[TM], exhibited low toxicity to ash-heap leacheates [23,24]. Although oil shale ash has been used for liming substrates of agricultural crops, to the best of our knowledge no research papers cover the use of oil shale ash for the cultivation of microalgae.

The aim of our study was to investigate the growth of the microalga Scenedesmus obliquus in conditions of high carbon dioxide supply and sulphur concentration. This species was chosen as a test organism because it has been reported to grow under 80% C[O.sub.2] conditions with a maximum cell concentration of 10-20% C[O.sub.2]. Oil shale ash was used as a neutralizing agent and the content of four heavy metals in the biomass was analysed.

2. MATERIALS AND METHODS

2.1. Microalgae cultures and medium composition

Cultures of S. obliquus were obtained from the University of Gottingen (Georg-August-Universitat Gottingen Algae Collection). The cultures were pre-grown in Bold Basal Medium (BBM) [25] containing NaN[O.sub.3] (2.5 mg [L.sup.-1]), Ca[Cl.sub.2] * 2[H.sub.2]O (0.17 mg [L.sup.-1]), MgS[O.sub.4] * 7[H.sub.2]O (0.30 mg [L.sup.-1]), [K.sub.2]HP[O.sub.4] (0.43 mg [L.sup.-1]), K[H.sub.2]P[O.sub.4] (0.13 mg [L.sup.-1]), NaCl (0.43 mg [L.sup.-1]), EDTA (0.17 mg [L.sup.-1]) + KOH (0.55 mg [L.sup.-1]), FeS[O.sub.4]*7[H.sub.2]O (0.02 mg [L.sup.-1]) + [H.sub.2]S[O.sub.4], [H.sub.3]B[O.sub.3] (0.19 mg [L.sup.-1]), ZnS[O.sub.4]*7[H.sub.2]O (0.003 mg [L.sup.-1]), Mn[Cl.sub.2]*4[H.sub.2]O (0.0007 mg [L.sup.-1]), Mo[O.sub.3] (0.0005 mg [L.sup.-1]), CuS[O.sub.4]*5[H.sub.2]O (0.0006 mg [L.sup.-1]), Co[(N[O.sub.3]).sub.2]*6[H.sub.2]O (0.0002 mg [L.sup.-1]). The cultures were pre-grown with 2% C[O.sub.2] under cool fluorescent lamps with an irradiation of 2500 lux (measured with TES 1335). The ambient temperature was 24-25 [degrees]C.

2.2. Preparation of treatment solutions

Treatment solutions were prepared with sulphuric acid (96% [H.sub.2]S[O.sub.4]). Since algal cells take up sulphur in the form of sulphate, we consider for simplicity that all of the [H.sub.2]S[O.sub.4] dissociates into sulphate and is thus bioavailable to microalgae. For dilute solutions, 1 ppm is considered equal to 1 mg [L.sup.-1]. In order to achieve a 500 ppm concentration, 0.9615 mL/L of [H.sub.2]S[O.sub.4] is required. Because the working volume of the reactor was 0.8 L, ~0.77 mL was used for the preparation of the 500 ppm treatment solution and twice the amount for the 1000 ppm treatment solution. Sulphuric acid was dissolved in 20 mL of BBM. Treatment solutions were prepared in triplicate. Oil shale ash obtained from the Polevkivi Kompetentsikeskus (Estonian Oil Shale Competence Centre) was added for the neutralization of the pH. Around 3.1 g and 6 g of oil shale ash was used for the neutralization of the 500 ppm and 1000 ppm treatment solutions, respectively (the exact amount varied between the replicates and was adjusted depending on the pH, which was measured constantly). The paste was transferred quantitatively to a vacuum pump and filtered through 2 [micro]m glass microfibre filters (Whatman, GF/F) into 100 mL of BBM. The pH of the solution was measured with a pH meter (SevenCompact[TM] pH/Ion S220, Mettler Toledo) and the pH was adjusted with 1 M HCl or 1 M NaOH, as necessary. The final pH was 6.5-6.7. As the control, BBM with a similar pH (6.2-6.5) was used.

2.3. Operation of the photobioreactor

The experiments were operated in batch mode. As bio-reactors 1 L Schott Duran HPLC high pressure glass bottles with ports were used. The culture of S. obliquus with an initial concentration of ~0.8 g [L.sup.-1] was added to the neutralized solutions and BBM was added to the final volume of 800 mL. A mixture of air and C[O.sub.2] (14% of total), serving as the carbon source, was sparged through 2 [micro]m filters (Whatman, FP30/0.2 CA-S) into the culture medium with a flow rate of 1 L [mi.sup.-1] (set using gas flow meters); at the same time the culture was mixed. The bioreactors were placed under cool fluorescent lamps with an irradiation of 2500 lux under a continuous light cycle.

2.4. Algal biomass measurements

The experiment lasted for 7 days. Measurements were carried out every 24 hours. Optical density (OD) measurements were made with a spectrophotometer (Spectronic 200, Termo Scientific) at 650 nm ([OD.sub.650]) with proper dilutions to give an absorbance in the range 0.1-1.0 in case of OD greater than 1.0, and were used as a proxy for determining algal biomass. A calibration scale using [OD.sub.650] measurements and dry biomass weight was established beforehand. The dry biomass weight was obtained by filtering 50 mL algal culture through pre-weighed and predried (100 [degrees]C, 24 h) filters with a 2 [micro]m pore size (Whatman, GF/F), and dried at 100 [degrees]C for 24 h or until the weight was invariable. Then the filters loaded with biomass were weighed on an analytical scale (ME204, Mettler Toledo) and the weight of the blank dried filters was subtracted to obtain the weight of the dried biomass. The biomass was calculated from the following equation:

M = 0.01207 + 0.0876469 x abs, (1)

where M is biomass (g [L.sup.-1]) and abs is the optical density measured at 650 nm.

2.5. Determination of kinetic parameters of growth

Biomass productivity was calculated from the change in biomass concentration (g [L.sup.-1]) within a cultivation time according to Eq. (2):

P = [[x.sub.1]-[x.sub.0]/[t.sub.1]-[t.sub.0]], (2)

where P is biomass productivity (g [L.sup.-1] [d.sup.1]), x1 is the final biomass determined in grams per litre, [x.sub.0] the initial biomass, [t.sub.0] is the starting time of the experiment, and [t.sub.1] is the end time in days. The maximum biomass concentration achieved in the bioreactors is denoted [P.sub.max] and calculated from the maximum biomass yield ([x.sub.max]) on the day.

Specific growth rate of the microalgae was calculated using the following equation:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (3)

The maximum fixed carbon dioxide (FCO2) in a single bioreactor was calculated according to the adjusted equation from de Morais and Costa [26]:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (4)

where [x.sub.max] is the maximum biomass yield, [x.sub.0] is the initial biomass, [M.sub.cbm] is the fraction of carbon in the biomass (g [g.sup.1]), derived from the typical molecular formula of microalgal biomass C[O.sub.0.48][H.sub.1.83][N.sub.0.11][P.sub.0.01] proposed by Chisti [27], [V.sub.p] is the working volume of the bioreactor, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] is the molar mass of C[O.sub.2], and [m.sub.C] is the molar mass of carbon.

Biomass productivity was used as a proxy for calculating C[O.sub.2] fixation by applying the following equation:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (5)

where [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] is C[O.sub.2] fixation (g [L.sup.-1] -d.sup.-1]) and P is biomass productivity.

At the end of the experiments the biomass from the 1000 ppm treatments was submitted to the Estonian Veterinary and Food Laboratory for heavy metal analyses. The results were compared to the standard threshold of heavy metals in animal feed and mean heavy metal concentrations in organic composts used as agricultural fertilizers. The results were analysed using R [28].

3. RESULTS AND DISCUSSION

3.1. Biomass productivity and C[O.sub.2] fixation

During the experiments, algal biomass was monitored for 7 days. The results are summarized in Table 1. Repeated measures ANOVA was used to analyse the data. The results show that in general the biomass yields for the control and treatments were similar (p > 0.05). This suggests that neither the added sulphur nor the oil shale ash had an inhibiting effect on the algae. A linear model was fitted to confirm if there was any difference in biomass yield between the treatments and control in regard to the experiment day. The results showed that although the biomass yield was time-dependent, the group-means were not significantly different (adjusted [R.sup.2] = 0.84 and p < 0.05). The biomass yields were similar up until the last day of the experiment, when the biomass yields became notably different (Fig. 1).

[FIGURE 1 OMITTED]

The initial decrease in biomass as shown in Fig. 1 could be explained by the sudden change in environmental conditions: the change in C[O.sub.2] concentration from 2% in the pre-growth phase to 14% in the experimental conditions. After acclimatization, the algae entered an exponential growth stage. The control and the 500 ppm treatment reached their maximum on day 6, the 1000 ppm treatment on day 7. Although the biomass yields did not differ significantly, the biomass yield was slightly higher in the treatments (Fig. 1). It is likely that the minerals contained in oil shale ash (Ca, Fe, K, Mg) were utilized in algal nutrition. While Ca and Fe are micronutrients, K and Mg are macronutrients and are essential in algal nutrition. For example, [Mg.sup.2+] controls the distribution of excitation energy between photosystems and regulates the activity of the enzyme RuBisCo, which regulates carbon fixation [29]. It is also required for nitrogenase activity [7]. Potassium is important in growth and photosynthesis processes; potassium-deficient cells show higher rates of respiration [30]. Iron is involved in the chlorophyll manufacturing process and is contained in different proteins such as ferrodoxin, which is used for electron transfer in a range of metabolic reactions [31]. It is also involved in the electron flow from [H.sub.2]O to [NADP.sup.+] [7]. Further, sulphur is crucial in algal biochemical processes. It is an imperative component in the composition of the amino acids cysteine and methionine, and plays an important part in protein biosynthesis as well as in the repair cycle of photo-synthetic system PSII [7,32,33]. Sulphur metabolism is also linked with RuBisCo--in sulphur-deprived cells the amount of RuBisCo declines, C[O.sub.2] exchange rate and quantum efficiency decrease and thus C[O.sub.2] fixation also declines [32,34,35]. It has been established that a moderate level of S[O.sub.x] and N[O.sub.x] (up to 150 ppm) is tolerated by many microalgal species [7]. However, Li et al. [36] found no inhibition on the growth of Scenedesmus sp. grown with 18% C[O.sub.2], 150 ppm N[O.sub.x], and 200 ppm S[O.sub.x]. In fact, they suggest that N[O.sub.x] and S[O.sub.x] can be effectively used as nutrients for microalgae growth. When sparged through water (or algal culture medium), S[O.sub.2] dissolves according to the (simplified) equation: [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. The sulphuric acid further dissociates into sulphate and hydrogen ions according to the following steps [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. Sulphate, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], is the main bioavailable form of sulphur that is taken up by microalgae [33].

In the current study no inhibition of algal growth was noted; therefore, it is likely that sulphur is not directly toxic to S. obliquus once the pH has been adjusted. On the contrary, it is possible that the biomass yield and the C[O.sub.2] fixation were slightly higher in the treatments due to a higher sulphur availability, among other nutrients described earlier. The amount of sulphur used in the study can be considered slightly higher than would dissolve from flue gas, because the solubility of S[O.sub.2] is temperature dependent with more S[O.sub.2] dissolving at lower temperatures. Hence it is likely that the amount of S[O.sub.2] that would dissolve from flue gas into the growth medium would be lower, because flue gas would not be cooled to room temperature prior to sparging through the culture medium.

The maximum biomass yield achieved in the bioreactors was 2.9 g [L.sup.-1] for the controls (on day 6), 3.1 g [L.sup.-1] for the 500 ppm treatment (on day 6), and 3.9 g [L.sup.-1] for the 1000 ppm treatment (on day 7). The maximum biomass productivity ([P.sub.max]) was 0.36, 0.39, and 0.44 g [L.sup.-1] [d.sup.-1], respectively. These results are comparable with similar studies. Tang et al. [37] achieved a [P.sub.max] 0.155 g [L.sup.-1] [d.sup.-1] with S. obliquus grown with 10% C[O.sub.2] and 0.134 g [L.sup.-1] [d.sup.-1] when grown with 20% C[O.sub.2]. Ho et al. [38] achieved a [P.sub.max] of 0.28 g [L.sup.-1] [d.sup.-1] and 0.38 g [L.sup.-1] [d.sup.-1] with 10% C[O.sub.2] for S. obliquus strains isolated in Taiwan. Compared to these studies, the maximum biomass productivity of S. obliquus in given experiments can be considered high. However, it should be noted that because the growth conditions and the duration of experiments were different in the studies, these results should be interpreted with caution.

A high biomass yield implies good carbon fixation. It is estimated that per gram of assimilated biomass, 1.88 grams of C[O.sub.2] is fixed [7]. Carbon contained in different molecules (carbohydrates, lipids, etc.) makes up circa 50% of the dry weight of the biomass [27]. Studies of S. obliquus grown with 12% C[O.sub.2] at 30 [degrees]C had a C[O.sub.2] fixation rate of 0.26 g [L.sup.-1] [d.sup.-1] [26]. Tang et al. [37] calculated a C[O.sub.2] fixation rate of 0.288 g [L.sup.-1] [d.sup.-1] for 10% C[O.sub.2] and 0.246 g [L.sup.-1] [d.sup.-1] for 20% C[O.sub.2]. The calculated fixation rates of carbon dioxide in this study were 0.45 g [L.sup.-1] [d.sup.-1], 0.62 g [L.sup.-1] [d.sup.-1], and 0.83 g [L.sup.-1] [d.sup.-1] for controls, 500 ppm and 1000 ppm treatment, respectively. The mean C[O.sub.2] fixed per litre was 6.08, 8.40, and 11.39 grams for the control, 500 ppm and 1000 ppm treatments, respectively.

If we consider the mean C[O.sub.2] fixation value per litre from the experiments (the overall mean from control and treatments was 8.62 g [L.sup.-1] per week and the mean from treatments 9.90 g [L.sup.-1] per week), then we can say that roughly 0.45-0.51 tonnes [m.sup.-3] of C[O.sub.2] could be fixed annually. According to the results of biomass productivity, 123-140 kg [m.sup.-3] of biomass could be produced and harvested annually. Because C[O.sub.2] emissions are taxed, reduction in C[O.sub.2] emissions means lower costs for C[O.sub.2]-emitting companies.

If waste ash could be used as an additional nutrient source for algal cultivation, it could lead to savings as a result of minimizing the use of chemicals in cultivation. Fly ash from municipal solid waste and bituminous coal combustion has been used in agriculture for the enrichment of soils with phosphorus, potassium, and several micronutrients (Ni, Co, Mo, Se, etc.), and additionally as a liming agent for acidity reduction in acidic soils [39,40]. It is also common practice to use oil shale ash for agricultural liming in Estonia, and attempts have been made to use oil shale ash for the treatment of damaged pine forests growing on acid sandy soil in order to reduce the acidity of the soil and improve the health of pine stands [41,42]. According to Hasanen et al. [19], oil shale ash sampled from different parts of different oil shale power plants contains (per 20-100 mg of ash) 3.7-7.7 ppm Co, 8.1-17.9 ppm Cu, 335-700 ppm Mn, 2.8-18.6 ppm Mo, 18.9-44.5 ppm Ni, and 27.7-380 ppm Zn. All these are essential micronutrients for algae. Most of these elements are soluble in water (except Mo, which is soluble if bound with oxygen as Mo[O.sup.2-], and Mn, which is soluble in dilute acid or as Mn[Cl.sub.2]). Additionally oil shale ash contains up to 2800 ppm phosphorus [19], which could be utilized by algae. Therefore oil shale ash could be used as a substitute for commonly used reagents or as fertilizer in culture medium preparation, thereby reducing cultivation costs. For further cost reductions, wastewater could be used as a source of nitrogen and phosphorus, which are the main macronutrients required by algae. Urban wastewaters may contain up to 100 mg [L.sup.-1] N and P, while the concentration in agricultural wastewater may be as high as 1000 mg [L.sup.-1] [43]. The cultivation of microalgae in wastewater has been studied extensively [13,44-47].

However, because oil shale ash needs to be filtered out as it increases turbidity and thus the permeation of light through the culture medium, ash could be replaced by oil shale ash leachate, which is a clear liquid with high pH values originating in oil shale ash deposits. Further research should establish the elemental composition of the leachate and its usability for algae nutrition.

3.2. Heavy metal content and potential use of biomass

The biomass from the 1000 ppm treatment was analysed in order to determine the concentration of arsenic, mercury, lead, and cadmium to evaluate its usability as animal feed or aquafeed or as organic agricultural fertilizer. The results are summarized in Table 2.

Microalgal biomass is typically rich in nitrogen and phosphorus, which makes it suitable for use as organic fertilizer. The biomass is also rich in minerals and typically has a sufficient protein content to be used as an inexpensive addition to traditional animal feed and aquafeed. However, the protein content in microalgae is species specific. For example, the protein content in the dry biomass of Scenedesmus almeriensis can reach 55% [50]. Heavy metals and other pollutants can limit the use of algal biomass as agricultural fertilizer or feed. The concentrations of As, Hg, Pb, and Cd in the experimental biomass were much lower than the European accepted thresholds. This enables the use of biomass as fertilizer or feed; however, a full analysis of nutrient content should be carried out beforehand.

Another way to utilize algal biomass from the C[O.sub.2] sequestration process is to turn it into energy--ferment into bioethanol or turn into methane through anaerobic digestion [51]. Therefore the biomass can be sold to biorefineries. Profits from biomass sales as well as cost reductions associated with lower C[O.sub.2] emissions should, in the long-term, offset the initial investment costs of establishing an algae farm. This implies, however, the investment and operating costs of an algae farm to be minimal, which requires process optimization and operating cost reductions (e.g. using wastewater as discussed previously).

4. CONCLUSIONS

We found that a high sulphur concentration did not have an inhibitory effect on algal growth when the pH drop caused by sulphuric acid was neutralized by oil shale ash. Additionally, the oil shale ash did not inhibit algal growth either. On the contrary, the biomass yield from the treatments was slightly higher, suggesting that a higher sulphur availability and minerals in the ash could serve as nutrients for algae. Considering the results, it can be estimated that 450-510 kg [m.sup.-3] of sulphur could be fixed and 123-140 kg [m.sup.-3] of sulphur biomass could be produced annually. Using flue gas as a carbon source and oil shale ash as an additional nutrient source for microalgae cultivation can provide inexpensive biomass, which can be used as fish feed or as an agricultural fertilizer. Further research needs to be carried out in order to optimize the growth conditions and C[O.sub.2] fixation, to study the possibility of using oil shale leachate as an additional nutrients source.

ACKNOWLEDGEMENTS

We gratefully acknowledge the financial support of the Environmental Investment Centre of Estonia, project No. 4910. We would also like to thank the Estonian Oil Shale Competence Centre for providing oil shale ash for the experiments. The publication costs of this article were covered by the Estonian Academy of Sciences.

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Lara Podkuiko (a,b*), Juri Olt (a), and Timo Kikas (a)

(a) Institute of Technology, Estonian University of Life Sciences, Fr. R. Kreutzwaldi 56/1, 51014 Tartu, Estonia (b) Department of Marine Systems, School of Science, Tallinn University of Technology, Ehitajate tee 5, 19086 Tallinn, Estonia

Received 26 October 2016, revised 26 January 2017, accepted 27 January 2017, available online 6 April 2017

[c] 2017 Authors. This is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/).

Rohevetika Scenedesmus obliquus kultiveerimine kunstliku suitsugaasi tingimustes polevkivituhaga neutraliseeritud suure vaavlisisalduse juures

Lara Podkuiko, Juri Olt ja Timo Kikas

Enim kasutatavaks energiatootmise kutuseks Eestis on polevkivi. See on madala kuttevaartuse ja suure mineraalaine-sisaldusega kutus, mille poletamisel tekib suurel hulgal aluselist polevkivituhka. Lisaks suurele susinikdioksiidi hulgale sisaldab polevkivienergeetikast saadav suitsugaas ka olulisel hulgal vaaveldioksiidi (S[O.sub.2]). Susihappegaasi seostatakse kliimamuutustega ja seetottu on voetud eesmargiks C[O.sub.2] heitme vahendamine. C[O.sub.2] sidumiseks suitsugaasist ja see-labi emissioonide vahendamist on voimalik teha mikrovetikate abil. Mitmed mikrovetikaliigid on voimelised kasvama vaga suure C[O.sub.2] kontsentratsiooni juures. Antud teadustoo eesmargiks oli kultiveerida rohevetikat Scenedesmus obliquus suure C[O.sub.2] ja vaavli sisalduse juures ning kasutada pH neutraliseerimiseks aluselist polevkivituhka.

S. obliquus valiti katseorganismiks seetottu, et ta on voimeline kasvama kuni 80% C[O.sub.2] sisaldusega atmosfaaris ja saavutab maksimaalse rakkude kontsentratisooni 10-20% C[O.sub.2] tingimustes. Sellesse vahemikku langeb ka polevkivi-suitsugaasi C[O.sub.2] keskmine sidaldus: ~12%. Katses kasvatati S. obliquus't 14% C[O.sub.2] kontsentratsiooni tingimustes bioreaktorites mahuga 1 L. Toitelahusesse lisati vaavelhapet kontsentratsioonis 500 ppm ja 1000 ppm, et jaljendada vaaveldioksiidi lahustumist suitsugaasist kasvulahusesse. Lahus neutraliseeriti aluselise polevkivituhaga. Seitsme-paevase katse kaigus moodeti iga 24 tunni jarel spektrofotomeetriliselt vetika biomassi, mida kasutati seotud C[O.sub.2] koguse arvutamiseks. Biomassi saagikus kahe tootluse puhul (maksimaalne saagikus 3,1 ja 3,9 g [L.sup.-1] vastavalt 500 ppm ja 1000 ppm puhul) oli sarnane tootlemata kontrollruhma saagikusega (vastavalt 2,9, 3,1 ja 3,9 g [L.sup.-1] kontrolli, 500 ppm ning 1000 ppm tootluse kohta), mis viitab sellele, et vaavlil ja polevkivituhal polnud katseorganismi kasvule parssivat moju. C[O.sub.2] sidumise kiirus oli vastavalt 0,45 g [L.sup.-1] [paev.sup.-1] kontrolli puhul, 0,62 g [L.sup.-1] [p.sup.-1] 500 ppm tootluse ja 0,83 g [L.sup.-1] [p.sup.-1] 1000 ppm tootluse puhul. Seega oleks aastas voimalik siduda 0,45 t [m.sup.-3] kuni 0,51 t [m.sup.-3] susihappegaasi, tootes seejuures 123-140 kg [m.sup.-3] biomassi. Kokkuvotteks voib oelda, et mikrovetikat S. obliquus on voimalik kasutada susihappegaasi sidumiseks suitsugaasist. Edasine uurimistoo peaks selgitama, kuidas antud protsessi optimeerida ja maksimeerida susihappegaasi sidumist.

(*) Corresponding author, lara.podkuiko@gmail.com
Table 1. Algae biomass productivity parameters and calculated
C[O.sub.2] fixation

Treatment   Maximum       Biomass
            biomass       productivity,
            yield,        g [L.sup.-1] [d.sup.-1]
            g [L.sup.-1]

Control     2.97 (a)      0.24
500 ppm S   3.13 (a)      0.33
1000 ppm S  3.91 (b)      0.44

Treatment   Maximum                 Specific
            biomass                 growth rate,
            productivity,           [d.sup.-1]
            g [L.sup.-1][d.sup.-1]

Control     0.36                    0.16
500 ppm S   0.39                    0.19
1000 ppm S  0.44                    0.23

Treatment   Average daily            Mean C[O.sub.2]
            C[O.sub.2] fixation      fixed per
            g [L.sup.-1] [d.sup.-1]  bioreactor, g (c)


Control     0.45                     4.89
500 ppm S   0.62                     6.72
1000 ppm S  0.83                     9.11

Treatment   Mean C[O.sub.2]
            fixed per litre,
            g


Control     6.08
500 ppm S   8.40
1000 ppm S  11.39

(a)  On day 6 of the experiment.
(b)  On day 7 of the experiment.
(c) Calculated per bioreactor with a working volume of 0.8 L.

Table 2. Comparison of heavy metal concentrations in algal biomass
from 1000 ppm treatment with heavy metal thresholds for animal feed
and mean concentrations in biowaste compost, sludge compost, and
compost from municipal solid waste

                      As        Hg       Pb      Cd       Reference

Concentration in      0.09      0.02     0.13    0.013
 algae biomass,
 mg [kg.sup.-1]
Maximal               2 (b)     0.1      10 (c)  1        [48]
 acceptable level
 in animal feed (a),
 mg [kg.sup.-1]
Mean concentration    3-10 (d)  0.2-2.5  40-750  0.4-5.3  [49]
 in bio-
 waste compost,
 mg [kg.sup.-1]

(a) Maximum content relative to a feed with a moisture content
of 12%.
(b) Seaweed meal and feed materials derived from seaweed have a higher
threshold: 40 (2) mg [kg.sup.-1] (ppm), while complementary and
complete feed for pet animals containing fish, other aquatic animals,
and products derived thereof and/or seaweed meal and feed materials
derived from seaweed have a threshold of 10 (2) mg [kg.sup.-1] (ppm).
(c) Feed materials derived from phosphates and calcareous marine algae
have a threshold of 15 mg [kg.sup.-1] (ppm).
(d) mg [kg.sup.-1] dm.
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Author:Podkuiko, Lara; Olt, Juri; Kikas, Timo
Publication:Proceedings of the Estonian Academy of Sciences
Date:Jun 1, 2017
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