New Biotechnology Volume 32, Number 3 May 2015
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Research Paper
Dual purpose system that treats anaerobic effluents from pig waste and produce Neochloris oleoabundans as lipid rich biomass Eugenia J. Olguı´n, Omar S. Castillo, Anilu´ Mendoza, Karla Tapia, Ricardo E. Gonza´lez-Portela and Vı´ctor J. Herna´ndez-Landa Environmental Biotechnology Group, Institute of Ecology (INECOL), Carretera Antigua a Coatepec No. 351, Xalapa 91070, Veracruz, Mexico
Abstract
Dual purpose systems that treat wastewater and produce lipid rich microalgae biomass have been indicated as an option with great potential for production of biodiesel at a competitive cost. The aim of the present work was to develop a dual purpose system for the treatment of the anaerobic effluents from pig waste utilizing Neochloris oleoabundans and to evaluate its growth, lipid content and lipid profile of the harvested biomass and the removal of nutrients from the media. Cultures of N. oleoabundans were established in 4 L flat plate photobioreactors using diluted effluents from two different types of anaerobic filters, one packed with ceramic material (D1) and another one packed with volcanic gravel (D2). Maximum biomass concentration in D1 was 0.63 g L1 which was significantly higher than the one found in D2 (0.55 g L1). Cultures were very efficient at nutrient removal: 98% for N–NH4+ and 98% for PO43. Regarding total lipid content, diluted eflluents from D2 promoted a biomass containing 27.4% (dry weight) and D1 a biomass containing 22.4% (dry weight). Maximum lipid productivity was also higher in D2 compared to D1 (6.27 0.62 mg L1 d1 vs. 5.12 0.12 mg L1 d1). Concerning the FAMEs profile in diluted effluents, the most abundant one was C18:1, followed by C18:2 and C16:0. The profile in D2 contained less C18:3 (linolenic acid) than the one in D1 (4.37% vs. 5.55%). In conclusion, this is the first report demonstrating that cultures of N. oleoabundans treating anaerobic effluents from pig waste are very efficient at nutrient removal and a biomass rich in lipids can be recovered. The maximum total lipid content and the most convenient FAMEs profile were obtained using effluents from a digester packed with volcanic gravel. Introduction Microalgae have been considered as a potential raw material for the production of biofuels such as biodiesel, bioethanol, biomethane and biohydrogen [1,2]. However, several recent reports have indicated that there are various technological, market and policy constraints that need to be overcome in order to develop economically feasible processes for the production of biofuels from
Corresponding author. Tel.: +52 2288 42 18 48. Olguı´n, E.J. ([email protected], [email protected]) http://dx.doi.org/10.1016/j.nbt.2014.12.004 1871-6784/ß 2015 Elsevier B.V. All rights reserved.
microalgae. Furthermore, recent studies involving the use of Life Cycle Analysis have indicated that the use of wastewater as source of water and nutrients for the cultivation of microalgae could contribute strongly to decrease the cost of microalgae production. It is within this context, that dual purpose systems for the treatment of wastewater and production of valuable microalgae biomass have been proposed as one of the main strategies for the production of biodiesel from microalgae at a competitive cost [3]. On the other hand, pig manure is one of the most polluting wastes because of its high organic matter concentration, in terms
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Research Paper
of chemical oxygen demand (COD), nitrogen and phosphorous. The lack of suitable procedures for storing and treatment of purines and manures can lead to eutrophication of water bodies, underground streams and soil acidification [3,4]. It has been considered that anaerobic digestion is one of the most appropriate technologies to manage these wastes, not only because it helps to reduce the emission of greenhouse gases, but also because there is a valorization of the waste with the production of biogas. At a large scale, biogas can be further utilized to produce electricity [5]. However, it is important to take into account that there is the need of a post-treatment of the anaerobic effluents, due to their high content of nitrogen and phosphorous [3]. Concerning the cultivation of microalgae in dual purpose systems, one of the main advantages of using anaerobic effluents from animal waste is the presence of volatile fatty acids (VFA), which can be used as carbon source in mixotrophic cultures [3]. Under such conditions, microalgae use both, the light and the organic carbon added to the medium as their energy source [6]. Thus, during the day, the photosynthesis and the organic carbon assimilation occur simultaneously, and during the night, microalgae keep growing using the organic carbon added to the medium [7]. There are some reports that show that different microalgae strains reach higher biomass production in mixotrophic cultures than in heterotrophic conditions; e.g. Chlorella vulgaris [8], Chlorella protothecoides [9], Chlorella pyneroidosa [10]. However, the main carbon source used in microalgae cultures under mixotrophic conditions has been glucose, which is expensive [11]. In contrast, the use of anaerobic effluents of pig manure as source of nutrients for the cultivation of microalgae has been demonstrated since a decade ago at pilot plant scale for the successful cultivation of Spirulina (Arthrospira) [12]. Recent reports are related to the cultivation of various microalgae species using anaerobic effluents from agricultural wastes [13], cattle manure [14] and pig manure [6,15], among others. Nevertheless, the use of anaerobic effluents (AE) for microalgae cultivation involves important challenges such as the imbalance between organic and inorganic nutrients, the high turbidity and the presence of potentially competitive microorganisms. However, there is scanty Information in this respect [14]. On the other hand, Neochloris oleoabundans is a green microalgae that has the ability to accumulate large amounts of lipids under stress conditions such as nitrogen limitation [16–18], light intensity [19] and high temperature [20]. Although several research groups have adopted the strategy of exposing N. oleoabundans to stress conditions in order to increase the lipid content, recent reports [6] have demonstrated several cell damages during such nutrient starvation conditions such as chloroplast degeneration, pigment content decrease, alterations in the light harvesting complex II–photosystem II assembly and accumulation, apart from accumulation of a high amount of lipid globules inside the cells. One of the major attractive characteristics of N. oleoabundans, is that its fatty acid methyl esters (FAMEs) profile is adequate for biodiesel production [16]. Thus, it is important to develop sustainable and inexpensive alternatives for the cultivation of this valuable microalgae. The main FAMEs reported for N. oleoabundans cultivated in Bold’s basal medium (BBM) are C18:1, C16:0 and C18:2 [14,21]. However, such composition is not necessarily fixed since it has been demonstrated that the fatty acid profile in N. 388
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New Biotechnology Volume 32, Number 3 May 2015
oleoabundans changes according to the cultivation conditions [22]. These authors showed that the pH and the nitrogen depletion changed the fatty acid profile of these green microalgae: the major changes were observed in the relative percentage of C18:1 which increased significantly during nitrogen depletion in both tested conditions (pH 8.1 and 10). The aim of the present work was to develop a dual purpose system for the treatment of the anaerobic effluents from pig waste utilizing N. oleoabundans and simultaneously, to evaluate its growth, the lipid content and lipid profile of the biomass and the removal of total nitrogen (NTK), ammonium nitrogen (N– NH4+) and phosphorus.
Materials and methods N. oleoabundans cultivation in BBM and adaptation to BBMNH4 Growth and lipid productivity of N. oleoabundans (UTEX 1185) were examined in three different media: (a) modified Bold’s basal medium (BBM); (b) modified BBM (BBM-NH4), in which the nitrogen source was added as ammonium chloride NH4Cl, and (c) alternative culture medium (ACM) amended with anaerobic effluents. Cultures for inoculum preparation and control treatment were maintained in BBM (concentration in mg L1): 250NaNO3, 25CaCl22H2O, 75MgSO47H2O, 75K2HPO4, 175KH2PO4, 25NaCl, 11.42H3BO3, 8.82ZnSO47H2O, 1.44MnCl24H2O, 0.142NaMoO46H2O, 1.57CuSO45H2O, 0.49Co(NO3)26H2O, 50Na2EDTA, 3.1KOH, 4.98FeSO47H2O and 1 mL H2SO4 concentrated. In BBM-NH4, the composition was similar except that NaNO3 was substituted by NH4Cl (157.23 mg L1). This concentration corresponds to 41.18 mg L1 of ammonium nitrogen, which is the same concentration of nitrogen, as nitrate, in BBM medium. Flat plate photobioreactors (FPB), 31 cm (height) 52 cm (length) 7 cm (width) built with acrylic material and containing a volume of 4 L of medium were utilized for cultivation of N. oleoabundans and were maintained in a growth chamber, under the following conditions: 134 mmol m2 s1 and 35 1 8C; the light source during the incubation photoperiod (16 h light/8 h darkness) was provided by six 39 W ‘‘cool white’’ fluorescent lamps (PHILIPSß Model F48T12/D). Light intensity was measured with a Luxometer (Lutronß Model YK-10LX) and the conversion factor from lux units to equivalent photon flux density units (mmol m2 s1) was 0.0135 [23]. The selected incubation temperature (35 1 8C) was chosen because in the experiments performed at the lab level in an early work by the research group [19], N. oleoabundans showed a higher percentage of stained cells with Sudan III, (from 74.5 at day 1 to 93.2% at day 13) which is an indicator of lipids (triglycerides) when cultures were incubated at 33 2 8C. The continuous air flow rate in the FPBs was 0.375 vvm; it was provided by an aquarium air pump (HAGENß Model ELITE820). At the beginning, the pH of the cultures in BBM was adjusted to 6.6 and in cultures with BBM-NH4 to 7.3 to avoid a fast decrease of pH.
Diluted anaerobic effluents (DAE) from pig manure as an alternative culture medium Two anaerobic digesters (capacity 0.25 m3 each one, hydraulic retention time 25 days) were fed with pig manure. One of them (Digester 1) was packed with ceramic material and the other one
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growth and nutrient concentration. The loss of volume due to continuous aeration of the reactor was replenished with distilled water.
Algal cell dry weight was determined gravimetrically according to [12]: 10-mL culture aliquots were filtered through previously weighted microfiber glass filters (Whatmanß GF/C, pore size = 1.2 mm), washed with distilled water and allowed to dry in an oven at 100 8C, until reaching a constant weight. The corresponding specific growth rate (m) was calculated according to the following formula [24]: FIG. 1
Packing materials of anaerobic filters utilized to process the pig waste. Digestor 1 (D1) was packed with ceramic material and Digestor 2 (D2) was packed with volcanic gravel.
(Digester 2) with volcanic gravel (Fig. 1). Table 1 shows the nutrient concentration in the concentrated AE and in diluted anaerobic effluents (DAE) that were prepared as an alternative culture medium. In order to obtain 41.18 mg L1 of N–NH4+ in the alternative culture media, concentrated AE were added in the following percentages: 7.40% (v/v) from Digester 1 (D1) and 7.60% (v/v) from Digester 2 (D2) to a final volume of 4 L of distilled water, including 20% (v/v) of inoculum culture. The initial pH values for the two diluted effluents were 7.88 for DAE-D1 and 7.81 for DAED2. The concentration of total carbohydrates was determined with the colorimetric method of phenol–sulfuric acid according to Dubois (1956) cited by Zhou et al. [32]. In the undiluted effluents from D2, the concentration found was 1.947 g L1. No data was available for effluents from D1.
Cultivation of N. oleoabundans in synthetic and alternative culture medium Flat Plate 4 L reactors were prepared. Control cultures were used to analyze the growth of N. oleoabundans utilizing synthetic medium BBM and modified medium BBM-NH4. The controls were inoculated with cultures of N. oleoabundans of 7 days of growth in 4 L flat plate reactors. While in treatments (DAE-D1 and DAE-D2), the inoculum was obtained from N. oleoabundans 300 mL flask cultures (7 days of growth) already acclimatized in this type of media. Such reactors were kept 21 days in a growth chamber exposed at a photon flux density of 134 mmol m2 s1, photoperiod of 16 h light/8 h darkness, temperature of 35 1 8C and continuous air flow rate. Every 48 h, an aliquot of 290 mL was taken for measuring
m¼
lnðN t 2 =N t 1 Þ ðln N t 2 ln N t 1 Þ ¼ ; Dt ðt 2 t 1 Þ
where N t 1 and N t 2 are the biomass amounts at the beginning and the end of a specific time interval, respectively, and Dt is the length of the time interval. On the other hand, digital images (100) of N. oleoabundans cultures were obtained with an optic microscope (Leicaß Model DM750).
Consumption of nutrients in N. oleoabundans cultures Every 48 h, a sample (250 mL) was centrifuged for 12 min at 4500 rpm and the supernatant was used for analyzing N–NH4+, TKN, PO43 and alkalinity. These parameters were measured using standard spectrophotometric procedures with HACHß analytical kits and a HACHß DR4000 spectrophotometer. The removal percentage (%R) was calculated according to the formula: Ci C f 100; %R ¼ Ci where Ci and Cf are the initial and final concentrations of the analyte, respectively. The pellet obtained by centrifugation was washed twice with distilled water and stored for a further neutral lipids analysis.
Nile red staining for qualitative monitoring of neutral lipids presence Samples from the culture medium were taken every 48 h and 1.5 mL of them were centrifuged during 4 min at 12,000 rpm. The neutral lipids stain with Nile red (Sigma-Aldrichß) (9-diethylamina-5H-benzo[a]phenoxazine-5-one) was performed according to [25]: 10 mL of concentrated sample (pellet) were added to 584 mL of an aqueous solution (25% v/v.) of dimethylsulphoxide (DMSO).
TABLE 1
Partial chemical characterization of concentrated anaerobic effluents and of diluted anaerobic effluents (DAE). Parameter
COD N–NH4+ N–NO3 PO43 SO42 VFA
Concentration in anaerobic effluents (mg L1)
Concentration in diluted anaerobic effluents (mg L1)
Digester 1 (D1)
Digester 2 (D2)
DAE-D1 (packed with ceramic material)
DAE-D2 (packed with volcanic gravel)
5100 556 40 212.7 1355 131
3200 542 50 142 797 171
377.40 41 3 16 100 9.70
243.20 41 4 11 61 13.00
COD: chemical oxygen demand; VFA: volatile fatty acids.
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Quantification of N. oleoabundans growth
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Then, 6 mL of a solution of 50 mg L1 of Nile red in acetone were added. The mix was vortexed 10 s to 1200 rpm and incubated in darkness 10 min to 40 8C. Finally, the images of the stained samples were obtained using an optical microscope (Leicaß Model DM750) adapted with a fluorescence lamp and camera. The excitation and emission wavelengths used were 410 and 580 nm, respectively.
Total lipids quantification and FAMEs profile analysis
Research Paper
The analysis of total lipids was performed by gravimetric methods [26–28]. For the total lipids quantification, the lipids of 50 mg of dry and ground biomass were extracted with chloroform, methanol and water (2:4:1.8). The chloroform phase was separated and the chloroform was evaporated. Total lipids were quantified after drying in the oven at 60 8C, according to [26] until constant weight. FAMEs were obtained by trans-esterification [27,28]: 1 mL of methanolic hydrochloric acid 1N (methanolic HCl 3N, Supelcoß 33050-U) was added to a sub-sample of the total lipids extracted as previously described. The solution was vortexed and placed in a thermo-block 1 h at 65 8C. Immediately afterwards, 200 mL of distilled water were added to stop the reaction and 1 mL of hexane was added. Finally, gas chromatography (GC) analysis was performed with a GC Agilent 7890A (Agilent Technologiesß) using a flame ionization detector (FID). The sample volume injected was 1 mL (split ratio 0.1:1) onto a DB23 capillary column (60 m 0.25 mm ID 0.25 mm film) using nitrogen as the carrier gas (column flow 41.1 mL min1). Temperatures of injector and detector were 225 8C and 230 8C, respectively. The temperature in column oven was programmed as follows: initial 50 8C, then to 170 8C with a heating rate of 30 8C min1, and the final temperature was 225 8C for 15 min with a heating rate of 4 8C min1. The FAMES identification was made by comparison of retention times with those of a FAME Mix (Supelcoß 47885-U) standard. The FAMES quantification was made by correlating the individual peak areas to the calibration curves previously obtained with the proper dilutions of the FAME Mix standard.
Results and discussion Kinetics of growth of N. oleoabundans cultivated in diluted anaerobic effluents (DAE) Growth of N. oleoabundans was quantified as dry weight (Fig. 2) in DAE from two digesters containing different packing substrates and in two control synthetic media. As expected, biomass density (measured as dry weight) in modified BBM after 21 days of cultivation was significantly higher compared to the one registered in any of both DAE cultures. Likewise, the trend of increase in biomass density was very different, since it showed a quick increase in both DAE cultures during the first 5–7 days (showing an specific growth rate m = 0.213 d1 and m = 0.250 d1 for DAE-1 and DAE-2, respectively), followed by a very slow or null increase afterwards. In contrast, biomass density in the modified BBM showed a clear increase from the initial zero time to the fifth day (m = 0.361 d1) and kept growing until day 9. It showed a second peak of increase from day 14 to day 17 but at a lower specific growth rate (m = 0.077 d1). It is interesting to note that biomass density in the modified BBM-NH4, showed a similar trend that the one observed in the two DAE during the first 5 days decreasing afterwards. Since this later culture was observed to be 390
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FIG. 2
Biomass density (g L1) of N. oleoabundans in Ctrl BBM, Ctrl BBM-NH4, DAED1 and DAE-D2 culture media, throughout the experimental period. Each data represents the average of two replicates.
heavily contaminated with a fungi after 9 days (Fig. 3, panel L), it was decided to eliminate this treatment. The maximum biomass density registered for the DAE-D1 (packed with ceramic material) was 0.63 g L1 and for the DAED2 (packed with volcanic gravel) was 0.55 g L1 after 14 days of cultivation. In both cases, the N/P ratio, being 8.46 for Neo-DAED1 and 12.59 for Neo-DAE-D2, falls within the nitrogen-to-phosphorus stoichiometry encountered in phytoplankton (8–45), according to [29]. Thus, the difference between both cultures could be explained on the basis of a higher content of additional nutrients indicated by a higher COD value in the effluents from D1 (Table 1). On the other hand, the presence of organic matter most probably facilitated the occurrence of mixotrophic growth. The biomass density reached in these two cultures is in a similar range of other reports of cultivation of various microalgae using diluted anaerobic effluents from animal waste (Table 2). Furthermore, the average maximum biomass density found in this work for N. oleoabundans (0.59 g L1), is higher than the one reported for another green microalgae, Desmodesmus sp. cultivated also in anaerobic effluents (10%) from pig waste (0.41 g L1) after 14 days [30]. An additional important remark after comparing the various reports summarized in Table 2, is that the anaerobic effluents utilized in this work were no subjected to any special pre-treatment apart from dilution with non-sterilized water, as it was the case with the rest of reports. The resulting obvious advantage is that cultures in this work can be easily scaled up at a lower cost and lower energy input compared with other processes involving various kind of pre-treatment. Regarding the pH changes throughout the experimental period (Fig. 4A), all treatments except the BBM-NH4, showed a clear tendency toward alkalinity during the first 3 days. The BBM culture remained in a pH around 9 and those in DAE maintained their pH around 7.5–8 for the rest of the experimental period. However, the BBM-NH4 culture showed a clear tendency toward
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FIG. 3
Micrographs (100) of N. oleoabundans in Ctrl BBM, Ctrl BBM-NH4, DAE-D1 and DAE-D2 culture media, at different cultivation days. Treatments are shown in columns and growing days in rows.
TABLE 2
Biomass production and lipid content of various microalgae species cultivated in diluted anaerobic effluents from animal waste. Type of effluent
Dilution of Pretreatment the effluent
Anaerobic effluents from pig manure
7.4% (D1) 7.6% (D2) Anaerobic effluents from pig manure 10% Anaerobic effluent from dairy manure 1:50 Anaerobic effluent from pig manure N.R.
Anaerobic effluent from pig manure Anaerobic effluent from pig manure
20% 20 Fold
Microalga specie
0.63 (D1) 0.55 (D2) Filtered with microfibers Desmodesmus sp. 0.41 N.R. Neochloris oleoabundans 0.97 Filtered and sterilized Chlamydomonas mexicana 0.56 Scenedesmus obliquus 0.53 Chlorella vulgaris 0.49 Filtered with microfibers Chlorella vulgaris
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Research Paper
Dual purpose system that treats anaerobic effluents from pig waste and produce Neochloris oleoabundans as lipid rich biomass Eugenia J. Olguı´n, Omar S. Castillo, Anilu´ Mendoza, Karla Tapia, Ricardo E. Gonza´lez-Portela and Vı´ctor J. Herna´ndez-Landa Environmental Biotechnology Group, Institute of Ecology (INECOL), Carretera Antigua a Coatepec No. 351, Xalapa 91070, Veracruz, Mexico
Abstract
Dual purpose systems that treat wastewater and produce lipid rich microalgae biomass have been indicated as an option with great potential for production of biodiesel at a competitive cost. The aim of the present work was to develop a dual purpose system for the treatment of the anaerobic effluents from pig waste utilizing Neochloris oleoabundans and to evaluate its growth, lipid content and lipid profile of the harvested biomass and the removal of nutrients from the media. Cultures of N. oleoabundans were established in 4 L flat plate photobioreactors using diluted effluents from two different types of anaerobic filters, one packed with ceramic material (D1) and another one packed with volcanic gravel (D2). Maximum biomass concentration in D1 was 0.63 g L1 which was significantly higher than the one found in D2 (0.55 g L1). Cultures were very efficient at nutrient removal: 98% for N–NH4+ and 98% for PO43. Regarding total lipid content, diluted eflluents from D2 promoted a biomass containing 27.4% (dry weight) and D1 a biomass containing 22.4% (dry weight). Maximum lipid productivity was also higher in D2 compared to D1 (6.27 0.62 mg L1 d1 vs. 5.12 0.12 mg L1 d1). Concerning the FAMEs profile in diluted effluents, the most abundant one was C18:1, followed by C18:2 and C16:0. The profile in D2 contained less C18:3 (linolenic acid) than the one in D1 (4.37% vs. 5.55%). In conclusion, this is the first report demonstrating that cultures of N. oleoabundans treating anaerobic effluents from pig waste are very efficient at nutrient removal and a biomass rich in lipids can be recovered. The maximum total lipid content and the most convenient FAMEs profile were obtained using effluents from a digester packed with volcanic gravel. Introduction Microalgae have been considered as a potential raw material for the production of biofuels such as biodiesel, bioethanol, biomethane and biohydrogen [1,2]. However, several recent reports have indicated that there are various technological, market and policy constraints that need to be overcome in order to develop economically feasible processes for the production of biofuels from
Corresponding author. Tel.: +52 2288 42 18 48. Olguı´n, E.J. ([email protected], [email protected]) http://dx.doi.org/10.1016/j.nbt.2014.12.004 1871-6784/ß 2015 Elsevier B.V. All rights reserved.
microalgae. Furthermore, recent studies involving the use of Life Cycle Analysis have indicated that the use of wastewater as source of water and nutrients for the cultivation of microalgae could contribute strongly to decrease the cost of microalgae production. It is within this context, that dual purpose systems for the treatment of wastewater and production of valuable microalgae biomass have been proposed as one of the main strategies for the production of biodiesel from microalgae at a competitive cost [3]. On the other hand, pig manure is one of the most polluting wastes because of its high organic matter concentration, in terms
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Research Paper
of chemical oxygen demand (COD), nitrogen and phosphorous. The lack of suitable procedures for storing and treatment of purines and manures can lead to eutrophication of water bodies, underground streams and soil acidification [3,4]. It has been considered that anaerobic digestion is one of the most appropriate technologies to manage these wastes, not only because it helps to reduce the emission of greenhouse gases, but also because there is a valorization of the waste with the production of biogas. At a large scale, biogas can be further utilized to produce electricity [5]. However, it is important to take into account that there is the need of a post-treatment of the anaerobic effluents, due to their high content of nitrogen and phosphorous [3]. Concerning the cultivation of microalgae in dual purpose systems, one of the main advantages of using anaerobic effluents from animal waste is the presence of volatile fatty acids (VFA), which can be used as carbon source in mixotrophic cultures [3]. Under such conditions, microalgae use both, the light and the organic carbon added to the medium as their energy source [6]. Thus, during the day, the photosynthesis and the organic carbon assimilation occur simultaneously, and during the night, microalgae keep growing using the organic carbon added to the medium [7]. There are some reports that show that different microalgae strains reach higher biomass production in mixotrophic cultures than in heterotrophic conditions; e.g. Chlorella vulgaris [8], Chlorella protothecoides [9], Chlorella pyneroidosa [10]. However, the main carbon source used in microalgae cultures under mixotrophic conditions has been glucose, which is expensive [11]. In contrast, the use of anaerobic effluents of pig manure as source of nutrients for the cultivation of microalgae has been demonstrated since a decade ago at pilot plant scale for the successful cultivation of Spirulina (Arthrospira) [12]. Recent reports are related to the cultivation of various microalgae species using anaerobic effluents from agricultural wastes [13], cattle manure [14] and pig manure [6,15], among others. Nevertheless, the use of anaerobic effluents (AE) for microalgae cultivation involves important challenges such as the imbalance between organic and inorganic nutrients, the high turbidity and the presence of potentially competitive microorganisms. However, there is scanty Information in this respect [14]. On the other hand, Neochloris oleoabundans is a green microalgae that has the ability to accumulate large amounts of lipids under stress conditions such as nitrogen limitation [16–18], light intensity [19] and high temperature [20]. Although several research groups have adopted the strategy of exposing N. oleoabundans to stress conditions in order to increase the lipid content, recent reports [6] have demonstrated several cell damages during such nutrient starvation conditions such as chloroplast degeneration, pigment content decrease, alterations in the light harvesting complex II–photosystem II assembly and accumulation, apart from accumulation of a high amount of lipid globules inside the cells. One of the major attractive characteristics of N. oleoabundans, is that its fatty acid methyl esters (FAMEs) profile is adequate for biodiesel production [16]. Thus, it is important to develop sustainable and inexpensive alternatives for the cultivation of this valuable microalgae. The main FAMEs reported for N. oleoabundans cultivated in Bold’s basal medium (BBM) are C18:1, C16:0 and C18:2 [14,21]. However, such composition is not necessarily fixed since it has been demonstrated that the fatty acid profile in N. 388
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oleoabundans changes according to the cultivation conditions [22]. These authors showed that the pH and the nitrogen depletion changed the fatty acid profile of these green microalgae: the major changes were observed in the relative percentage of C18:1 which increased significantly during nitrogen depletion in both tested conditions (pH 8.1 and 10). The aim of the present work was to develop a dual purpose system for the treatment of the anaerobic effluents from pig waste utilizing N. oleoabundans and simultaneously, to evaluate its growth, the lipid content and lipid profile of the biomass and the removal of total nitrogen (NTK), ammonium nitrogen (N– NH4+) and phosphorus.
Materials and methods N. oleoabundans cultivation in BBM and adaptation to BBMNH4 Growth and lipid productivity of N. oleoabundans (UTEX 1185) were examined in three different media: (a) modified Bold’s basal medium (BBM); (b) modified BBM (BBM-NH4), in which the nitrogen source was added as ammonium chloride NH4Cl, and (c) alternative culture medium (ACM) amended with anaerobic effluents. Cultures for inoculum preparation and control treatment were maintained in BBM (concentration in mg L1): 250NaNO3, 25CaCl22H2O, 75MgSO47H2O, 75K2HPO4, 175KH2PO4, 25NaCl, 11.42H3BO3, 8.82ZnSO47H2O, 1.44MnCl24H2O, 0.142NaMoO46H2O, 1.57CuSO45H2O, 0.49Co(NO3)26H2O, 50Na2EDTA, 3.1KOH, 4.98FeSO47H2O and 1 mL H2SO4 concentrated. In BBM-NH4, the composition was similar except that NaNO3 was substituted by NH4Cl (157.23 mg L1). This concentration corresponds to 41.18 mg L1 of ammonium nitrogen, which is the same concentration of nitrogen, as nitrate, in BBM medium. Flat plate photobioreactors (FPB), 31 cm (height) 52 cm (length) 7 cm (width) built with acrylic material and containing a volume of 4 L of medium were utilized for cultivation of N. oleoabundans and were maintained in a growth chamber, under the following conditions: 134 mmol m2 s1 and 35 1 8C; the light source during the incubation photoperiod (16 h light/8 h darkness) was provided by six 39 W ‘‘cool white’’ fluorescent lamps (PHILIPSß Model F48T12/D). Light intensity was measured with a Luxometer (Lutronß Model YK-10LX) and the conversion factor from lux units to equivalent photon flux density units (mmol m2 s1) was 0.0135 [23]. The selected incubation temperature (35 1 8C) was chosen because in the experiments performed at the lab level in an early work by the research group [19], N. oleoabundans showed a higher percentage of stained cells with Sudan III, (from 74.5 at day 1 to 93.2% at day 13) which is an indicator of lipids (triglycerides) when cultures were incubated at 33 2 8C. The continuous air flow rate in the FPBs was 0.375 vvm; it was provided by an aquarium air pump (HAGENß Model ELITE820). At the beginning, the pH of the cultures in BBM was adjusted to 6.6 and in cultures with BBM-NH4 to 7.3 to avoid a fast decrease of pH.
Diluted anaerobic effluents (DAE) from pig manure as an alternative culture medium Two anaerobic digesters (capacity 0.25 m3 each one, hydraulic retention time 25 days) were fed with pig manure. One of them (Digester 1) was packed with ceramic material and the other one
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growth and nutrient concentration. The loss of volume due to continuous aeration of the reactor was replenished with distilled water.
Algal cell dry weight was determined gravimetrically according to [12]: 10-mL culture aliquots were filtered through previously weighted microfiber glass filters (Whatmanß GF/C, pore size = 1.2 mm), washed with distilled water and allowed to dry in an oven at 100 8C, until reaching a constant weight. The corresponding specific growth rate (m) was calculated according to the following formula [24]: FIG. 1
Packing materials of anaerobic filters utilized to process the pig waste. Digestor 1 (D1) was packed with ceramic material and Digestor 2 (D2) was packed with volcanic gravel.
(Digester 2) with volcanic gravel (Fig. 1). Table 1 shows the nutrient concentration in the concentrated AE and in diluted anaerobic effluents (DAE) that were prepared as an alternative culture medium. In order to obtain 41.18 mg L1 of N–NH4+ in the alternative culture media, concentrated AE were added in the following percentages: 7.40% (v/v) from Digester 1 (D1) and 7.60% (v/v) from Digester 2 (D2) to a final volume of 4 L of distilled water, including 20% (v/v) of inoculum culture. The initial pH values for the two diluted effluents were 7.88 for DAE-D1 and 7.81 for DAED2. The concentration of total carbohydrates was determined with the colorimetric method of phenol–sulfuric acid according to Dubois (1956) cited by Zhou et al. [32]. In the undiluted effluents from D2, the concentration found was 1.947 g L1. No data was available for effluents from D1.
Cultivation of N. oleoabundans in synthetic and alternative culture medium Flat Plate 4 L reactors were prepared. Control cultures were used to analyze the growth of N. oleoabundans utilizing synthetic medium BBM and modified medium BBM-NH4. The controls were inoculated with cultures of N. oleoabundans of 7 days of growth in 4 L flat plate reactors. While in treatments (DAE-D1 and DAE-D2), the inoculum was obtained from N. oleoabundans 300 mL flask cultures (7 days of growth) already acclimatized in this type of media. Such reactors were kept 21 days in a growth chamber exposed at a photon flux density of 134 mmol m2 s1, photoperiod of 16 h light/8 h darkness, temperature of 35 1 8C and continuous air flow rate. Every 48 h, an aliquot of 290 mL was taken for measuring
m¼
lnðN t 2 =N t 1 Þ ðln N t 2 ln N t 1 Þ ¼ ; Dt ðt 2 t 1 Þ
where N t 1 and N t 2 are the biomass amounts at the beginning and the end of a specific time interval, respectively, and Dt is the length of the time interval. On the other hand, digital images (100) of N. oleoabundans cultures were obtained with an optic microscope (Leicaß Model DM750).
Consumption of nutrients in N. oleoabundans cultures Every 48 h, a sample (250 mL) was centrifuged for 12 min at 4500 rpm and the supernatant was used for analyzing N–NH4+, TKN, PO43 and alkalinity. These parameters were measured using standard spectrophotometric procedures with HACHß analytical kits and a HACHß DR4000 spectrophotometer. The removal percentage (%R) was calculated according to the formula: Ci C f 100; %R ¼ Ci where Ci and Cf are the initial and final concentrations of the analyte, respectively. The pellet obtained by centrifugation was washed twice with distilled water and stored for a further neutral lipids analysis.
Nile red staining for qualitative monitoring of neutral lipids presence Samples from the culture medium were taken every 48 h and 1.5 mL of them were centrifuged during 4 min at 12,000 rpm. The neutral lipids stain with Nile red (Sigma-Aldrichß) (9-diethylamina-5H-benzo[a]phenoxazine-5-one) was performed according to [25]: 10 mL of concentrated sample (pellet) were added to 584 mL of an aqueous solution (25% v/v.) of dimethylsulphoxide (DMSO).
TABLE 1
Partial chemical characterization of concentrated anaerobic effluents and of diluted anaerobic effluents (DAE). Parameter
COD N–NH4+ N–NO3 PO43 SO42 VFA
Concentration in anaerobic effluents (mg L1)
Concentration in diluted anaerobic effluents (mg L1)
Digester 1 (D1)
Digester 2 (D2)
DAE-D1 (packed with ceramic material)
DAE-D2 (packed with volcanic gravel)
5100 556 40 212.7 1355 131
3200 542 50 142 797 171
377.40 41 3 16 100 9.70
243.20 41 4 11 61 13.00
COD: chemical oxygen demand; VFA: volatile fatty acids.
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Research Paper
Quantification of N. oleoabundans growth
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New Biotechnology Volume 32, Number 3 May 2015
Then, 6 mL of a solution of 50 mg L1 of Nile red in acetone were added. The mix was vortexed 10 s to 1200 rpm and incubated in darkness 10 min to 40 8C. Finally, the images of the stained samples were obtained using an optical microscope (Leicaß Model DM750) adapted with a fluorescence lamp and camera. The excitation and emission wavelengths used were 410 and 580 nm, respectively.
Total lipids quantification and FAMEs profile analysis
Research Paper
The analysis of total lipids was performed by gravimetric methods [26–28]. For the total lipids quantification, the lipids of 50 mg of dry and ground biomass were extracted with chloroform, methanol and water (2:4:1.8). The chloroform phase was separated and the chloroform was evaporated. Total lipids were quantified after drying in the oven at 60 8C, according to [26] until constant weight. FAMEs were obtained by trans-esterification [27,28]: 1 mL of methanolic hydrochloric acid 1N (methanolic HCl 3N, Supelcoß 33050-U) was added to a sub-sample of the total lipids extracted as previously described. The solution was vortexed and placed in a thermo-block 1 h at 65 8C. Immediately afterwards, 200 mL of distilled water were added to stop the reaction and 1 mL of hexane was added. Finally, gas chromatography (GC) analysis was performed with a GC Agilent 7890A (Agilent Technologiesß) using a flame ionization detector (FID). The sample volume injected was 1 mL (split ratio 0.1:1) onto a DB23 capillary column (60 m 0.25 mm ID 0.25 mm film) using nitrogen as the carrier gas (column flow 41.1 mL min1). Temperatures of injector and detector were 225 8C and 230 8C, respectively. The temperature in column oven was programmed as follows: initial 50 8C, then to 170 8C with a heating rate of 30 8C min1, and the final temperature was 225 8C for 15 min with a heating rate of 4 8C min1. The FAMES identification was made by comparison of retention times with those of a FAME Mix (Supelcoß 47885-U) standard. The FAMES quantification was made by correlating the individual peak areas to the calibration curves previously obtained with the proper dilutions of the FAME Mix standard.
Results and discussion Kinetics of growth of N. oleoabundans cultivated in diluted anaerobic effluents (DAE) Growth of N. oleoabundans was quantified as dry weight (Fig. 2) in DAE from two digesters containing different packing substrates and in two control synthetic media. As expected, biomass density (measured as dry weight) in modified BBM after 21 days of cultivation was significantly higher compared to the one registered in any of both DAE cultures. Likewise, the trend of increase in biomass density was very different, since it showed a quick increase in both DAE cultures during the first 5–7 days (showing an specific growth rate m = 0.213 d1 and m = 0.250 d1 for DAE-1 and DAE-2, respectively), followed by a very slow or null increase afterwards. In contrast, biomass density in the modified BBM showed a clear increase from the initial zero time to the fifth day (m = 0.361 d1) and kept growing until day 9. It showed a second peak of increase from day 14 to day 17 but at a lower specific growth rate (m = 0.077 d1). It is interesting to note that biomass density in the modified BBM-NH4, showed a similar trend that the one observed in the two DAE during the first 5 days decreasing afterwards. Since this later culture was observed to be 390
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FIG. 2
Biomass density (g L1) of N. oleoabundans in Ctrl BBM, Ctrl BBM-NH4, DAED1 and DAE-D2 culture media, throughout the experimental period. Each data represents the average of two replicates.
heavily contaminated with a fungi after 9 days (Fig. 3, panel L), it was decided to eliminate this treatment. The maximum biomass density registered for the DAE-D1 (packed with ceramic material) was 0.63 g L1 and for the DAED2 (packed with volcanic gravel) was 0.55 g L1 after 14 days of cultivation. In both cases, the N/P ratio, being 8.46 for Neo-DAED1 and 12.59 for Neo-DAE-D2, falls within the nitrogen-to-phosphorus stoichiometry encountered in phytoplankton (8–45), according to [29]. Thus, the difference between both cultures could be explained on the basis of a higher content of additional nutrients indicated by a higher COD value in the effluents from D1 (Table 1). On the other hand, the presence of organic matter most probably facilitated the occurrence of mixotrophic growth. The biomass density reached in these two cultures is in a similar range of other reports of cultivation of various microalgae using diluted anaerobic effluents from animal waste (Table 2). Furthermore, the average maximum biomass density found in this work for N. oleoabundans (0.59 g L1), is higher than the one reported for another green microalgae, Desmodesmus sp. cultivated also in anaerobic effluents (10%) from pig waste (0.41 g L1) after 14 days [30]. An additional important remark after comparing the various reports summarized in Table 2, is that the anaerobic effluents utilized in this work were no subjected to any special pre-treatment apart from dilution with non-sterilized water, as it was the case with the rest of reports. The resulting obvious advantage is that cultures in this work can be easily scaled up at a lower cost and lower energy input compared with other processes involving various kind of pre-treatment. Regarding the pH changes throughout the experimental period (Fig. 4A), all treatments except the BBM-NH4, showed a clear tendency toward alkalinity during the first 3 days. The BBM culture remained in a pH around 9 and those in DAE maintained their pH around 7.5–8 for the rest of the experimental period. However, the BBM-NH4 culture showed a clear tendency toward
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Research Paper
New Biotechnology Volume 32, Number 3 May 2015
FIG. 3
Micrographs (100) of N. oleoabundans in Ctrl BBM, Ctrl BBM-NH4, DAE-D1 and DAE-D2 culture media, at different cultivation days. Treatments are shown in columns and growing days in rows.
TABLE 2
Biomass production and lipid content of various microalgae species cultivated in diluted anaerobic effluents from animal waste. Type of effluent
Dilution of Pretreatment the effluent
Anaerobic effluents from pig manure
7.4% (D1) 7.6% (D2) Anaerobic effluents from pig manure 10% Anaerobic effluent from dairy manure 1:50 Anaerobic effluent from pig manure N.R.
Anaerobic effluent from pig manure Anaerobic effluent from pig manure
20% 20 Fold
Microalga specie
0.63 (D1) 0.55 (D2) Filtered with microfibers Desmodesmus sp. 0.41 N.R. Neochloris oleoabundans 0.97 Filtered and sterilized Chlamydomonas mexicana 0.56 Scenedesmus obliquus 0.53 Chlorella vulgaris 0.49 Filtered with microfibers Chlorella vulgaris
