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Testing of Chlorella/Scenedesmus microalgae consortia for remediation of wastewater, C[O.sub.2] mitigation and algae biomass feasibility for lipid production.

Reference to this paper should be made as follows: Koreiviene, J.; Valciukas, R.; Karosiene, J.; Baltrenas, P. 2014. Testing of Chlorella/Scenedesmus microalgae consortia for remediation of wastewater, C[O.sub.2] mitigation and algae biomass feasibility for lipid production, Journal. of Environmental Engineering and Landscape Management 222 (022): 105- 114. http://dx.doi.org/10.3846/16486897.2013.911182

Introduction

The demands for energy and freshwater resources are increasing with the growing human population in the world. Industry, transport, unsustainable agriculture increase the quantity of wastewater as well as the release of nutrients and emission of carbon dioxide that promotes eutrophication of the waters and global climate change IChiu et al 2009; Pittman et al. 2011; Olaizola et al. 2004; Rawat et al. 2011). Photosynthesizing algae are important organisms that: can help to control undesirable processes in the ecosystems. Currently, the green technologies are one of the most growing sectors 0[degrees] the world. Algae biomass m ay be used for alternative application such as biofuel, natural fertilizers production, additives to animal nutrition, etc. (Munoz, Guieysse 2006; Mata et al. 2010; Misevicius, Baltrenas 2011). Applications of algae for hum an needs are considered to have many benefits such as increasing sustainability reduction of greenhouse gas emissions, regional development, social structure, agriculture and security of supply (Reijnders 22006).

The wastewater is a suitable resource for microalgae, because it contains large quantities of nitrogen and phosphorus that are the key elements for tire algae growth (Li et al. 2011; Park et al. 2011; Pittman et al. 20111. Although lralrCg:e-2scale production of algal biofuels using wastewater treatment was first proposed by Oswald and Golueke in 1960 (Park et al. 2011 and references therein), the use of microalgae in the wastewater industry is still fairly limited (Pittman et al. 2011; Rawat et al. 2011). The application of microalgae for phycoremediation and sustainable biofuels production could become economically feasible (Rawat et al. 2011). The algal biomass from wastewater treatment systems could be converted to biofuels: by anaerobic digestion to biogas, transesterification of lipids to biodiesel, fermentation of carbohydrate to bioethanol and high temperature conversion to bio-crude oil (Pittman et al. 2011; Craggs et al. 2011). Under controlled growth conditions, microalgae can accumulate up to 80% of oil in dry cell mass (Pittman et al. 2011). Particularly, Chlorella species may accumulate 14-63% and Scenedesmus 6-55% of oil under various conditions of cultivation (Li et al. 2008; Gouveia, Oliveira 2009; Mutanda et al. 2011 and references therein). However, lipid accumulation in wastewater grown microalgae is much lower and ranges from low (<10% DW) to moderate (25-30% DW) lipid content (Halim et al. 2012).

Various algae species have different ecological requirements for their development. The highly variable composition of wastewater may limit the growth of some algae, whereas the others will flourish (Bhatnagar et al. 2011). So, it is essential to select algal species or their consortia capable of growing in particular wastewater under changing climatic conditions that will define the success of the treatment process. Unicellular fast growing green algae are very tolerant to many wastewater conditions and efficient at accumulating nutrients from it (Ruiz-Marin et al. 2010). Chlorella (C. ellipsoidea, C. minutissima, C. vulgaris) and Scenedesmus (S. bijuga, S. quadricauda, S. obliquus) are the most studied algae for their application in the wastewater treatment (Lau et al. 1995; Gouveia, Oliveira 2009; Ruiz-Marin et al. 2010; Johnson, Wen 2010; Wang et al. 2010; Bhatnagar et al. 2011; Praepilas, Kaewkannetra 2011; Yang et al. 2011a, b). Pate et al. (2011) emphasized that autotrophic microalgae productivity is strongly dependent on environmental conditions most suitable for growth, especially high solar resource and temperatures, which are characteristic of particular geographical region. Thus, the actual values of daily biomass productivity and neutral lipid content that can be achieved in practice will depend on a complex combination of algal strain, cultivation system and local growing conditions.

In Lithuania with the population of about 3 mln., approximately 170 mln. [m.sup.3] of domestic-industrial wastewater are produced yearly (http://gamta.lt/cms/index). The release of wastewater around Vilnius city is about one third of the total amount in the country. Study of Gudas, Povilaitis (2013) has revealed that wastewater factor is prominent for water quality in small rivers downstream larger towns. Studies of combined use of microalgae for the treatment and biomass production are very scarce in the country. Makareviciene et al. (2011) tested separately Chlorella and Scenedesmus species preference to nitrogen resource utilisation and algae biomass as the feedstock for biofuel at temperatures higher than are natural to Lithuania.

The aim of our experiments was to investigate feasibility of Chlorella/Scenedesmus microalgae consortia to treat local municipal wastewater and reduce greenhouse gas. We also seeked to evaluate biomass yield of the tested algae and its suitability for biofuel production under the conditions close to climate in Lithuania during summer.

1. Materials and methods

1.1. Algae cultures and growing media for the experiments

The strains of freshwater green algae Chlorella sp. and Scenedesmus sp. were isolated from Lithuanian lakes. Both cultures were grown in the BG-11 media at 20 [degrees]C and 1600 Lux illumination prior to the experiment.

Experimental studies with the mixture of Chlorella and Scenedesmus cultures were carried out in mechanically cleaned wastewater collected from the water supply and wastewater treatment company "Vilniaus vandenys". Fine soil particles, organic and mineral additives were removed from the solution in laboratory passing the water through the plankton net (pore diameter 20 um) and additionally filtering through several layers of filter paper. Wastewater was sterilized by autoclave at 120-130[degrees]C 1 atm. pressure for 30 min. for the elimination of bacteria. Bacteria may influence the experiment results of nutrients removal by algae due to decomposition of the organic matter in the solution. Conductivity, pH and chemical analysis of the prepared wastewater media (P[O.sub.4], N[O.sub.3], N[O.sub.2], N[H.sub.4] and [BOD.sub.4]) were performed prior the beginning of the experiment.

The experiment was carried out in 250 ml sterile Erlenmeyer flasks covered with cotton-gauze plugs coated with the plastic. Four types of media with different concentrations of wastewater were used to test the optimal growth conditions for selected green algae species. Wastewater was diluted with distilled water to reach appropriate concentration: M100--100% of wastewater; M75--75% of wastewater; M50--50% of wastewater and M25--25% of wastewater in the final solution. BG-11 medium was used as a control. All experiments were carried out in triplicate.

1.2. Experiment design

The generalized experiment scheme is presented in Fig 1. Each variant of 230 ml of prepared growth media was inoculated with 5 ml mixture of Chlorella/Scenedesmus species taken from the stock algal cultures. The concentration of algae at the beginning of the experiment was 39 x [10.sup.3] cells/ml (Chlorella sp. 31 x [10.sup.3] cells/ml and Scenedesmus sp. 8 x [10.sup.3] cells/ml). Algae were counted in Fuchs-Rosenthal chamber using light microscope.

[FIGURE 1 OMITTED]

Climatic conditions close to the natural in Lithuania in June-August were selected to maintain algae in specialized cultivation chamber Percival Intellus.

The flasks were incubated at 16:8 day-night cycle with the average 2080 Lux illumination during light period. Day temperature was +20 [+ or -] 1[degrees]C and night temperature -+15 [+ or -] 1[degrees]C. The mixing of the algae cultures by air pumping (aeration intensity 2 L/min) maintained an equal gas balance and kept algae in the suspension.

Algae growth curves were developed by measuring absorption of samples at 680 nm using spectrophotometer Libra S32PC every second day on the course of 21 days experiment. Algae biomass was evaluated from cell abundance and cell volume calculated based on Olrik et al. (1998) recommended formula for ellipsoid with circular cross section algae cells:

V = [[pi]/6] x [d.sup.2] x l, (1)

where: V--the algal cell volume, [micro][m.sup.3]; d--cell diameter, um; l--cell length, um.

Cell volume of [10.sup.6] [micro][m.sup.3] was considered as equal to 1 mg of biomass.

1.3. Nutrients and C[O.sub.2] removal

For the evaluation of nutrients removal by algae, the chemical analysis of P[O.sub.4], N[O.sub.3], N[O.sub.2], N[H.sub.4] concentrations were determined in accordance with standard methods (LST EN ISO 10304; LST EN ISO 14911) in each variant before and after the experiment. After the experiment, the solution was centrifuged 5000 rpm for 1 minute for the separation of algal biomass and media. The remaining water was filtered through 0.9 [micro]m pore diameter filter. Nutrients residues were evaluated. C[O.sub.2] consumed and incorporated into algae biomass via photosynthesis was recalculated using formula suggested by Buehner et al. (2009):

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], (2)

where: [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] consumed by algae, [micro][m.sup.3]; V--algae cell volume, [micro][m.sup.3].

1.4. Oil accumulation study

Algae start to accumulate oil and other storage resources at critical for their development conditions. The analysis of oil accumulation in the algal cells was performed twice: a) at the end of the experiment when the culture reached stationary phase of growth; b) after growth in nitrogen deprived conditions. In order to create shock conditions and initiation of oil accumulation in the studied green algae cells, 5 ml of algae suspension of each variant was centrifuged (2000 rpm for 5 min.) to separate algae from the growth media. Algae biomass was resuspended into BG-11 medium without nitrogen compounds for three days under the same conditions as during the experiment.

Epifluorescence microscopy was applied to evaluate lipid accumulation in the algae. To stain lipid in the algae cells, BODIPI 505/515 (4,4-difluro-1,3,5,7-tetramethyl-4-bora-3a, 4adiaza-S-indacene; Invitrogen, USA) dyes were added to algae suspension according to Govender et al. (2012). Stock solution was prepared by dissolving 1 mg BODIPI 505/515 dyes into 10 ml of DMSO (0.2%). Five [micro]l of prepared dyes was added to 6.25 ml of the sample with Chlorella/Scenedesmus culture (dye concentration 0.08 [micro]g/ ml) and incubated for 5 min. in the dark at 20[degrees]C. Lipids stained with BODIPI 505/515 dyes fluoresce green in algae cells and were investigated with Nikon Eclipse Ni epifluorescence microscope using a 515 nm emission wavelength filter. Images of algae were taken with Nikon DS-Ri1 camera and analyzed in the NIS-Elements BR software.

1.5. Statistical analysis

All values are expressed as mean [+ or -] standard deviation (SD). The data were checked for normality and analyzed by one-way ANOVA followed by Tukey's post-hoc test. The significant differences were considered at p < 0.05.

2. Results and discussion

2.1. Characteristics of the wastewater

The physico-chemical parameters of untreated municipal wastewater collected from Vilnius wastewater treatment plant are given in Table 1. The nutrient concentrations of the wastewater fell into the range from medium to strong level according to Rawat et al. (2011). Generally, untreated wastewater is characterized by very high concentrations of nutrients and toxic metals (Pittman et al. 2011). Total N (TN) and total P (TP) concentrations can be found at values of 10-100 mg/L in municipal wastewater and >1000 mg/L in agricultural effluent (Noue et al. 1992). The conductivity values that reach 2.68 mS/cm indicate high amount of various salts. Biochemical Oxygen Demand ([BOD.sub.5]) shows high amount of easy degradable organic matter in the wastewater. In agreement, light microscopy revealed high abundance of bacteria and scarce number of heterotrophic flagellates, colourless protists.

2.2. Growth rate of microalgae

The mixed culture of Chlorella and Scenedesmus showed similar pattern of their biomass development under various concentrations of wastewater and considerably differed from the control with BG-11 medium (Fig. 2A). Duration of the lag period was 5 days followed by the exponential growth during the next 8-12 days. Intense photosynthesis processes during the exponential phase also indicated the highest pH values in the 5-13 days (Fig. 2B). Culture slowly entered stationary phase in the 15-19 days of the experiment.

The biomass yield of mixed algae cultivated in BG-11 medium was the highest 3.7 g/L (Fig. 3). There were little differences of the algae biomass yield between concentrated (mean 2.75-3.04 g/L for M75 and M100) and diluted wastewater (mean 2.1-1.8 g/L for M25 and M50). Moreover, the growth of tested algae in concentrated wastewater was of the similar magnitude as in the chemical media, where the strains were cultured prior the experiment, indicating the wastewater as a perfect source to grow species separately or as their mixture. It seems that our results are similar to biomasses of Chlorella and Scenedesmus species recorded by the other researchers (Table 2). Nevertheless, it is difficult to compare the results from different studies due to variations of media, culturing time and regime. Chlorella minutussima cultured photoheterotrophically built biomass up to 0.38 g/L after 10 days growth (Pittman et al. 2011). Strain was defined as a good candidate for high biomass productivity in a municipal wastewater. Biomass yield of our studied Chlorella/Scenedesmus culture was 4.5 times higher that recorded by Pittman et al. (2011) even in the lowest biomass variant (mean 1.8 g/L) showing good perspectives of the consortia.

[FIGURE 2 OMITTED]

2.3. Chlorella, Scenedesmus algae density and biomass variations

Control of algae species is still not achieved in wastewater treatment ponds and algal dominance, various species interactions are still poorly understood (Park et al. 2011). Our experiment revealed species interactions in the consortia depending on the concentration of nutrients. Scenedesmus showed reverse pattern of growth compared to Chlorella in the gradient of wastewater concentration (p < 0.05) (Fig. 3). Concentrated wastewater (M100) stimulated the growth of Scenedesmus algae giving up to 5.5 times higher biomass augmentation compared to the most diluted (M25) variant (p < 0.05). Scenedesmus cells density increased hundreds to thousands of times by the end of the experiment: up to 4.53 [+ or -] 1.01 x [10.sup.6] - 29.23 [+ or -] 3.68 x [10.sup.6] cells/mL in M25 and M100 variants, accordingly.

The small algae cells have larger ratio of surface area and volume what enabled a quicker uptake of nutrients (Hein et al. 1995). The average volume of Scenedesmus cells was nine times lower than that of Chlorella. It may determine the faster growth of Scenedesmus at higher concentrations of wastewater. Similarly, Ruiz-Marin et al. (2010) found that Scenedesmus obliquus grew better in municipal wastewater compared to Chlorella vulgaris.

Generally, Chlorella growth was suppressed by concentrate wastewater. Species growth in most diluted wastewater variants showed the highest values (Figs 3, 4A, B). Species density in M25 and M50 variants reached 2.72 [+ or -] 3.68 x [10.sup.6] cells/ml and was three times lower in the most concentrated wastewater (0.88 [+ or -] 0.28 x [10.sup.6] cells/ml). Our results coincide with Ras et al. (2011) data for C. vulgaris which density varied from 3.0 x [10.sup.6] to 5.3 x [10.sup.6] cells/ ml. The maximum values were stated as high density cultures.

The increase of Chlorella density was lower up to 28-88 times only compared to Scenedesmus. Possibly the weakest competition for the resources with Scenedesmus in the most diluted variants determined the success of Chlorella. Contrary to our results, Johnson, Wen (2010) showed that wastewater was feasible for biomass production of Chlorella sp. with pure wastewater producing the highest biomass yield. However, the species of even the same genus have different requirements and growth characteristics (Cha et al. 2011).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Algae size is important for the biomass harvesting and desirable products extraction from the cells within the processing of the biomass (Rawat et al. 2011). Chlorella size varied less if compared to Scenedesmus and did not show significant differences between wastewater concentrations as well as before and after nitrogen shock application (p > 0.05) (Fig. 5). Chlorella mean cell volume increased in all variants from the beginning towards the end of the experiment and at nitrogen deprivation conditions.

Chlorella cells volume was over 30% higher in M50 wastewater variant and coincided with the lowest species culture biomass. It might be because species used a lot of energy to build up large cells slowing down the reproduction processes, what consequently lead to less culture biomass in the variant.

[FIGURE 5 OMITTED]

Contrary to Chlorella, the mean cell size of Scenedesmus diminished during the course of experiment and at nitrogen deprivation conditions without a clear tendency between the variants (p > 0.05) (Fig. 5). Scenedesmus built large cells and formed typical to species four-celled coenobia in diluted M25 wastewater. It is in agreement with O'Donnell et al. (2013) data that increase of nutrients concentration promoted more intense formation of one-celled Scenedesmus instead of four-cell coenobia.

2.4. Nutrients and C[O.sub.2] removal

Various species of Chlorella and Scenedesmus can remove from 80% to almost 100% of ammonia, nitrate and phosphate from treated wastewater (Pittman et al. 2011), however, the treatment efficiency varies depending on wastewater type and to some extent on climate peculiarities. Phosphorus is particularly difficult to remove from wastewater. Inorganic phosphorus (IP) concentration in the wastewater media variants of the experiment varied from 1.04 to 4.17 mg/L and was slightly higher in the control (BG-11 medium) (Fig. 6).

The experiment revealed that more than 99% of IP was eliminated by growing Chlorella/Scenedesmus culture within three weeks period (Fig. 7A). The elimination of inorganic nitrogen (IN) compounds from the wastewater was 88.6-96.4% indicating that the main limiting element for algae growth was IP. According to Makareviciene et al. (2011), the removal efficiency of nitrogen and phosphorus of Chlorella sp. and Scenedesmus sp. reached relatively high values: TN - 91% (for both algae), TP - 94.7% and 95.6%, respectively. Similarly, Woertz et al. (2009) laboratory experiments showed nutrient removals of >98% for ammonium and >96% for phosphate with mixed culture microalgae grown on C[O.sub.2] supplemented primary wastewater effluent. Chlorella ellipsoidea eliminated 99% of nitrogen and 90% of phosphorus from the secondary effluent (Yang et al. 2011a). Johnson, Wen (2010) recorded slightly lower values for Chlorella sp. removing up to 90% of the TP and 79% of the TN contained within the wastewater.

Usually NH4-N and PO4-P predominate as the nutrients in wastewater, thus algae having high productivity and able to utilize these resources are ideal species for the treatment and biomass production (Park et al. 2011). IN concentration in the wastewater variants of the experiment media varied from 7.3 to 29.0 mg/L with dominating ammonium (Fig. 6). The concentration of IN in the control media was about ten times higher with domination of nitrate. It should be noted that ammonium from wastewater was eliminated much easier by Chlorella/ Scenedesmus culture compared to nitrates showing that cultures are suitable for application in wastewater treatment. Makareviciene et al. (2011) recorded the best Chlorella and Scenedesmus biomass productivity using urea as a nitrogen source or modified growing medium BG-11 with decreased concentration of NaN[O.sub.3]. Chaichalerm et al. (2012) showed the lowest biomass yield of six chlorophyte species in the BG-11 medium compared to other three tested media (N-8, Kuhl, 3NBBM). They concluded that BG-11 medium has unbalanced concentrations of IN and IP, e.g. nitrogen concentration is high and phosphorous concentration is too low. Conversely, Phukan et al. (2011) obtained the highest biomass yield of Chlorella sp. (824 mg/L) in BG-11 medium compared to the other tested media (Basal, BBM, Chu-13). Li et al. (2008) experimentally showed that nitrate was the most favourable nitrogen source for Neochloris oleoabundans among the three tested nitrogen compounds, i.e. sodium nitrate, urea and ammonium bicarbonate. Even the same genus species have different preference for the nutrients. Cha et al. (2011) observed different growth pattern of Chlorella vulgaris (strain UMT-M1) and C. sorokiniana (strain KSMB2) cultures at various nitrate concentrations.

The results of our experiment showed that the mixture of the culture is suitable for the effective elimination of nutrients from the wastewater. It is in agreement with Bhatnagar et al. (2011) that algal consortia grown in wastewater offer high production of renewable biomass for various applications, because in consortia the substantial loss of population of one algae may be compensated by the growth of the other. Chinnasamy et al. (2010) demonstrated that a consortium of 15 native algal isolates removed >96% of nutrients in treated wastewater.

Microalgae contain approximately 50% of carbon dry weight, thus about 1.8 kg of C[O.sub.2] is required to generate 1 kg of algal biomass (Yang et al. 2011b). Chlorella/ Scenedesmus culture was tested for C[O.sub.2] elimination by evaluating biomass increase after three weeks of incubation. The mixture of culture growing on the wastewater incorporated from atmosphere into their biomass on average up to 1.37 g of C[O.sub.2]/L per day without any significant differences between wastewater concentrations (p > 0.05). The values were up to two times lower if compare with the control (p < 0.05) (Fig. 7B). Usually room air contains 0.04% of C[O.sub.2] (Cheng et al. 2006). The experiments of the other researchers indicate that elevated concentrations of C[O.sub.2] may enhance the algae biomass yields by several times (Olaizola et al. 2004; Ota et al. 2009; Yoo et al. 2010; Jiang et al. 2011). Chinnasamy et al. (2009) found that C. vulgaris culture fixed 18.3 and 38.4 mg C[O.sub.2]/L per day at ambient and elevated C[O.sub.2] (6%) levels.

2.5. Oil accumulation in the algae cells

Stockenreiter et al. (2013) found a clear correlation between light use and lipid production in functional diverse algae communities. It is considered as a powerful and cost-effective way to improve biofuel production. Nitrogen starvation was shown as one of the important factors to start oil accumulation by algae (Rodolfi et al. 2009; Brennan, Owende 2010; Deng et al. 2011; Pruvost et al. 2011). However, Stephenson et al. (2010) found that the maximal triacylglyceride productivity in Chlorella vulgaris was achieved by allowing the cells to deplete the nitrogen naturally instead of transferring cells to a medium without nitrogen. During our experiment period Chlorella/Scenedesmus culture reached the stationary phase in all variants of media, however, the nitrogen deprivation conditions were not achieved. The oil accumulation in Chlorella and Scenedesmus cells was additionally studied after inoculation of the algae biomass into nitrogen starvation conditions.

[FIGURE 7 OMITTED]

Under the nitrogen limitation, the growth of algae culture mixture from high concentration wastewater variants was suppressed more compared to diluted variants (Fig. 8). The experiment showed that Chlorella species is less susceptible to the N limitation than Scenedesmus. In the control variant Chlorella biomass increased, whereas Scenedesmus diminished. Nitrogen starvation might be not the best method to increase oil accumulation in the cells of particular algae, but application of high light or salt shock may give a positive result. It is also difficult to apply nitrogen starvation conditions at large operational scale to algae biomass grown in wastewater treatment plant.

[FIGURE 8 OMITTED]

Conclusions

1. Chlorella/Scenedesmus algae culture effectively eliminated inorganic phosphorus from the wastewater by removing up to 99.7-99.9% and inorganic nitrogen by 88.6 to 96.4% of the initial concentrations after three weeks of growth. Nitrogen effectively was eliminated in the form of ammonium, which predominated in the wastewater. Nitrate elimination was less effective.

2. The maximum biomass of algal culture consortium up to 3.04 g/L was in the concentrated wastewater. Its dilution almost twice reduced algal biomass.

3. The intensity of Chlorella algae growth was higher in the more diluted, while Scenedesmus algae - in the concentrated wastewater solutions. The consortia of Chlorella/ Scenedesmus algae treat the wastewater more effectively than the single species, since the chemical composition of wastewater is highly variable over the time.

4. Chlorella/Scenedesmus algae culture accumulated on average 0.65-1.37 g of C[O.sub.2]/L per day in their biomass.

5. The tested Chlorella and Scenedesmus algae have no ability to accumulate oil in the cells under nitrogen starvation conditions.

6. Chlorella/Scenedesmus algae consortium is a promising tool for elimination of nitrogen and phosphorus compounds from local wastewater resources as well as for diminishing C[O.sub.2] in the atmosphere under climatic conditions specific to Lithuania.

doi: 10.3846/16486897.2013.911182

Caption: Fig. 1. The general scheme of the experiment

Caption: Fig. 2. The growth pattern of Chlorella/Scenedesmus algae culture under different wastewater concentrations (A) and the variation of the pH values (B) during the course of experiment

Caption: Fig. 3. Biomass yield of Chlorella and Scenedesmus species in wastewater at different concentrations

Caption: Fig. 4. The mixture of Chlorella and Scenedesmus species in the stationary growth phase at different concentrations of wastewater

Caption: Fig. 5. Variations of Chlorella (A) and Scenedesmus (B) species cell size in wastewater at different concentrations

Caption: Fig. 7. Elimination of inorganic nitrogen and inorganic phosphorus (A) and the average amount of C[O.sub.2] incorporated into algae biomass via photosynthesis (B) at the different concentrations of wastewater

Caption: Fig. 8. Biomass yield of Chlorella and Scenedesmus species in wastewater at different concentrations after three days under nitrogen starvation conditions

Acknowledgements

Authors are indebted to V. Ptasekiene for correcting the English. The research was partly supported by the EU (European Regional Development Fund) through the Baltic Sea Region Programme project, Sustainable Uses of Baltic Marine Resources (SUBMARINER No. 055).

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Judita KOREIVIENE. Researcher at the Institute of Botany of the Nature Research Centre. Doctor of Biomedicine (Botany) sciences of Vilnius University and Institute of Botany, 2005. Author of 15 scientific papers. Scientific interests: cyanobacteria and algae flora, morphology, ecology and distribution; trophic interactions in hydroecosystems, harmful algae blooms and cyanotoxins; invasive species; cyanobacteria and algae isolation; algae application in biotechnology.

Robertas VALCIUKAS. Master of Environment Engineering, Vilnius Gediminas Technical University. Scientific interests: environmental protection, application of algae in the environment engineering.

Jurate KAROSIENE. Researcher at the Institute of Botany of the Nature Research Centre. Doctor of Biomedicine (Botany) sciences of Vilnius University and Institute of Botany, 2008. Author of 13 scientific papers. Scientific interests: algae and cyanobacteria diversity, distribution, ecology, species morphology, invasive species, harmful algae blooms, phytobentos functional activity, statistical analysis.

Pranas BALTRENAS. Dr Habil. Prof. and Head of the Department of Environmental Protection, Vilnius Gediminas Technical University (VGTU). Doctor Habil. of Science (air pollution), Leningrad Civil Engineering Institute (Russia), 1989. Doctor of Science (Air Pollution), Ivanov Textile Institute (Russia), 1975. Employment: Professor (1990), Associate Professor (1985), Senior Lecturer (1975), Vilnius Civil Engineering Institute (VISI, now VGTU). Publications: author of 13 monographs, 24 study guides, over 320 research papers and 67 inventions. Honorary awards and membership: prize-winner of the Republic of Lithuania (1994), a corresponding Member of the Ukrainian Academy of Technological Cybernetics, a full Member of International Academy of Ecology and Life Protection. Probation in Germany and Finland. Research interests: air pollution, pollutant properties, pollution control equipment and methods.

Judita Koreivien (a), Robertas Valciukas (b), Jurate Karosiene (a), Pranas Baltrenas (b)

(a) Nature Research Centre, Institute of Botany, Zaliuju Ezeru g. 49, 08604 Vilnius, Lithuania

(b) Department of Environ mental Protection, Faculty of Environmental Engineering,

Vilnius Gediminas Technical University, Sauletekio al. 11, 10223 Vilnius, Lithuania

Submitted 06 Dec. 2013; accepted 31 Mar. 2014

Corresponding author: Judita Koreiviene

E-mail: judita.koreiviaie@botanika.lt
Table 1. Physico-chemical parameters of municipal
wastewater collected from Vilnius wastewater treatment plant

Contaminants        Concentrations   untreated wastewater
                                            level *

Total nitrogen,         56.5         medium-strong
  mgN/L
Total phosphorus,        8.3         medium
  mgP/L
Inorganic                6.1         medium-strong
  phosphorus,
  mgP/L
[BOD.sub.5],             148         week-medium
  mg[O.sub.2]/L
Oxygen, mg/L
Temperature,            1.41
  [degrees]C
pH                      7.74
Conductivity,           2.68
  mS/cm
[H.sub.2]S, mg/l         0
Colour                 greyish

* according to Rawat et al. (2011).

Table 2. Biomass production of Chlorella and Scenedesmus
species based on references

Algae species    Biomass yield, g/L             Reference

Chlorella

C. pyrenoidosa   2.83 in 2 days          Li et al. 2011
C. minutissima   0.073-0.38 in 10 days   Bhatnagar et al. 2010
C. vulgaris      0.21                    Chinnasamy et al. 2009
C. vulgaris      av. 1.5, max 3.0        Gouveia, Oliveira 2009
C. ellipsoidea   0.43                    Yang et al. 2011a
Chlorella sp.    1.9 in 17 days          Rasoul-Amini et al. 2011
Chlorella sp.    1.47-1.71               Wang et al. 2010
Scenedesmus
S. obliquus      1.57 [+ or -] 0.67      Abou-Shanab et al. 2011
S. obliquus      av. 0.9, max 2.0        Gouveia, Oliveira 2009
S. acutus        0.82                    Chaichalerm et al. 2012

Fig. 6. The inorganic nitrogen (A) and inorganic phosphorus
(B) concentrations in the media before and after the
experiment

A Growing media

        before       after
        experiment   experiment

M 25       7.3          0.8
M 50      14.5          0.6
M 75      21.8          0.8
M 100     29.0          1.9
BG-11    227.0         155.7

B Growing media

         before       after
         experiment   experiment

M 25       1.0         0.003
M 50       2.1         0.003
M 75       3.1         0.003
M 100      4.2         0.003
BG-11      5.4         0.003

Note: Table made from bar graph.
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Author:Koreivien, Judita; Valciukas, Robertas; Karosiene, Jurate; Baltrenas, Pranas
Publication:Journal of Environmental Engineering and Landscape Management
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Date:Jun 1, 2014
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