Microalgae have recently drawn a lot of attention as promising candidates for CO2
neutral biofuel production. However, highly competitive crude oil prices, as well as the high expenses related to biomass-to-biofuel processing, are hindering its economic sustainability [1
]. Efforts to overcome problematic economical aspects of the biofuel production from microalgae are directed towards (i) the increase in biomass yield and cellular lipid content by transgenic engineering [2
], (ii) optimization of cogeneration process including the use of the excess heat, water and flue gases from the industry and (iii) usage of the effluent water after phycoremediation [3
] in agriculture and other purposes.
Some selected wild strains of microalgae can naturally produce a high proportion of valuable products such as lipids, carbohydrates or proteins, whereas recent advances in genetic engineering opened the possibilities to produce an even greater variety of valuable molecules useful for food, chemical and pharmaceutical industries [1
]. Although it is rarely possible to achieve as high a proportion of valuable compounds in a wild strain, as in a genetically modified one there are many advantages in the cultivation of non-transgenic, indigenous algal flora [4
]. Mass production of indigenous microalgae avoids the possible risks associated with large-scale propagation of genetically modified microorganisms and legal constraints related to their use [6
Cyanobacteria have large surface/volume ratio due to relatively small cell size (picocyanobacteria and nanocyanobacteria), high nutrient uptake efficiency and high reproduction rates. However, these traits are strongly dependent on the environmental conditions as well as on the species individual phenotypic adaptability. Cyanobacteria vary in preference for light intensity, temperature, nutrient concentrations, salinity and pH, as well as in capabilities to tolerate different ammonia, heavy metals and other toxic substances concentrations [7
]. Many studies exist on municipal wastewater phycoremediation [8
] and some authors explore the growth of cyanobacteria on swine [10
] or poultry litter effluent as well as the paper industry [11
] or carpet industry [12
]. However, the viability of cyanobacteria in industrial wastewater is still a challenge. The untreated petrochemical industry effluent is, thus, rich in ammonium and because of the presence of various growth inhibiting and toxic substances (such as toluene, xylene, benzene, thiols, phenols, sulfides, cyanides, heavy metals, ammonia, etc.) may represent a hostile environment for microalgal growth [13
]. However, we hypothesize that due to their high stoichiometric flexibility, high tolerance to salinity stress, photoheterotrophic metabolism, as well as capabilities in the degradation of crude oil [14
] cyanobacteria make good candidates for growth on heavily polluted and toxic oil refinery wastewaters. Moreover, if present in trace amounts, the metals such as Cu, Zn, Ni, etc. may represent micronutrients for growing algae [9
Most of the preselected potentially oleaginous species [15
] have not yet been systematically evaluated for the capacities in fatty acid production [17
], and/or growth on wastewater. Since nutrient quality can severely affect growth rates and biochemical composition of biomass feedstock, optimization is required in order to produce a high proportion of lipids along with the fast growth. In cyanobacteria, fatty acids are doubly important, as membrane lipids and as constituents of glycolipids, which form the thylakoid membranes where photosynthesis takes place. Cyanobacteria often contain significant quantities of some essential polyunsaturated fatty acids (PUFAs) such as C18 linoleic (18:2ω6) and α-linolenic (18:3ω3) acids whose production can reach up to 20% of the cellular dry weight via genetic engineering [18
]. The species rich in saturated fatty acids (SFAs) are good candidates for biofuel production whereas species rich in PUFAs are considered more appropriate for production of nutrition supplements, animal feed, etc. [19
]. In cyanobacteria lipid content rarely exceeds 20% of the dry algal biomass though under certain growth conditions it can reach as high as 85%, bringing the exploitation closer to its economic sustainability [20
]. Regulating the changes in lipid content is of utmost importance to the protection of the cellular metabolic functions, and a response to environmental stress. By regulating the saturation degree of the structural fatty acid profile, cyanobacteria maintain the membrane integrity/fluidity under pressures such as desiccation, heat-shock, salinity changes or the presence of toxic substances. There is also the dependence of desiccation tolerance on the accumulation of sucrose, trehalose or more complex carbohydrate molecules in cyanobacteria, i.e., in Synechococcus
sp., etc. [21
]. The physiological stress of nutrient starvation leads to lipid biosynthesis and accumulation in the cells. However, the photosynthesis process is highly protein bound and P-dependent and may result in lower biomass production and overall lipid yield.
We tested Synechococcus sp. MK568070 isolated from the Adriatic Sea for growth on the oil refinery wastewater. Potential of the tested strain for production of biofuel was assessed through the analysis of lipid content and composition, as well as changes in fatty acids, carbohydrates and protein concentrations along with the growth dynamics monitored during the uptake of nutrients from wastewater. Herein we present the first results of marine Synechococcus species growth and nutrient sequestration in a heavily polluted, oil refinery wastewater.
3.1. NH4 and Salinity Tolerance Testing
In the microwell plates we tested the Synechococcus
sp. MK568070 growth potential in a range of NH4+
concentrations (2–3.2 mM) corresponding to annual fluctuation of NH4+
in the refinery wastewaters. The working medium in microwells was WW3 adjusted by dilution or amendment of nutrients as described in Section 2.3
of the materials and methods section. The results are presented on Figure 2
Cultures demonstrated identically ascending growth pattern in the whole tested range of NH4+ concentrations. The highest cell densities were achieved at ammonium concentration of 2.8 mM, with maximum OD690 of 0.150 after seven days of growth. Although the biomass yield of Synechococcus sp. MK568070 growth on WW3 followed the increase in initial NH4+ concentrations from 2 mM to 2.8 mM, there was a decrease in yield at maximum NH4+ concentration of 3.2 mM.
Salinity test in microwells was performed over Synechococcus
sp. MK568070 and the results are presented on Figure 3
sp. MK568070 was incubated for a week in a standard growth medium with a range of salinity from 0 to 35 and OD690
was measured daily. High cell densities were reached in the range of salinities from 11 to 25, whereas salinities below 11 and above 25 inhibited cellular growth (Figure 3
The highest biomass yield occurred at a salinity of 19, corresponding to ~1/2 of the common salinity in the surface waters of the Adriatic Sea. This enables us to consider diluting the heavily polluted, untreated refinery wastewater, normally having salinity values <1, with seawater, which in reality is easily available for industrial plants situated at the coast. Such a concept was tested in PBR3, and applied in PBR4 where the biochemical aspects of Synechococcus sp. MK568070 growth on WW were explored. In addition, the effect of higher CO2 inflow to the overall yield was tested by constant blowing of air/CO2 mixture in 97:3 (vol:vol) proportion.
3.2. Effect of N-Source on Growth of Synechococcus sp. MK568070
The influence of N-source on growth performance and biomass yield of Synechococcus sp. (MK568070) was tested by growth in PBRs 1–3 under the controlled conditions in three different waste waters: WW1, WW2 and WW3 (diluted with ASW in 1:1 (v/v)).
The growth curves of experiments in PBRs 1–3 are presented in Figure 4
. The lowest growth performance was achieved in WW1 containing predominantly NH4+
as a source of nitrogen with an initial DIN concentration of ≈0.8 mM (Table 1
). Final biomass yield in WW1 was 152 mg/L of dry weight.
The wastewaters provided herein are sparse in phosphorous. In an attempt to satisfy the need for N and P by a costless source we used WW2 already containing some of the sanitary effluent. WW2 being richer in organic and inorganic phosphorous introduced as well significant amounts of NO3− into the cultivation medium. Although the final concentration of DIN 0.8 mM, was the same as in PBR1, the final biomass yield in PBR2 was 240 mg/L, substantially higher than in PBR1. Finally, the highest biomass yield 390 mg/L was obtained by growth on the WW3 (diluted with ASW) containing high concentration of NH4+ (1.3 mM).
3.3. Biocheimcal Aspects of Synechococcus sp. MK568070 Growth on WW
In PBR4 Synechococcus
sp. MK568070 was grown on WW3:ASW (1:1, vol/vol) at salinity 19. The optimum salinity for growth was determined based on the salinity tolerance findings (Figure 3
). The initial concentration of NH4+
was 1.3 mM, DIN/DIP was set to 8 and 3% CO2
was continuously added through air/CO2
bubbling system. The nutrient uptake is represented in Figure 5
. The majority of DIN (>99%) was depleted in seven days, while 20% of SRP remained unutilized by the end of experiment on day 15 (Figure 5
sp. MK568070 showed lower P-needs for the build up of biomass in respect to the initial experimental set point (N/P = 8), which resulted in higher residual concentration of SRP at day 15 (0.02 mmol L−1
). During the first six days of the growth (day 1 to day 7) Synechococcus
sp. MK568070 consumed 1.3 mmol DIN L−1
and 0.1 mmol SRP L−1
, resulting in the average DIN/SRP uptake of 13. From day 7 towards the end of experiment the growth was N-limited.
The biomass yield and overall lipid productivity of the experimental culture grown in PBR4 are shown in Figure 6
. The culture reached stationary phase after 14 days obtaining the biomass yield of 767 mg/L.
The structure of the fatty acid (FAME) profile of Synechococcus
sp. MK568070 was analyzed and results are presented in Table 2
. The FAME profile was dominated by C16 saturated and monounsaturated fatty acids, followed by C14 saturated and C18 monounsaturated FAME. The highest proportion of total lipids was achieved on the 2nd day of experiment by 41% of the dry weight. After the 2nd day, during the early exponential phase the contribution of lipids in the dry biomass was descending, whereas in the mid-exponential phase an increase in the lipid proportion was observed up to 24%. In the late exponential and stationary phase lipid content oscillated again between 14% and 16%. The final lipid content in the dry biomass was 21.4% in the stationary phase of the Synechococcus
sp. MK568070 growth.
The most abundant fatty acid was C16:0 with a 43.04% mass fraction of total fatty acids at the end of the experiment in the stationary growth phase. Due to the high percentage of other saturated fatty acids (C14 and C18) profiles are characterized with an overall unsaturation index (UND) lower than 1. The lowest UND of 0.45 was observed at the end of the experiment. The proportion of summarized C16 FA maintained high relative values, with decreasing dynamics in the exponential phase of the experiment and then again increasing the proportion towards the end, finally reaching the final 66.06%.
3.4. Carbohydrates and Proteins
Carbohydrate and protein content were measured daily during the first seven days of experiment, and later on every 2nd day. The results are presented in Figure 7
Proteins percentage in dry biomass was observed to increase from 3% to 21% during the first six days of the experiment from 3% to 21% of the dry biomass. At the same time the NH4+ values were continuously declining from 1.3 mM to <0.01 mM. During the rest of the exponential phase protein content continued to slowly decrease until remaining at the constant average value of 15.1% towards the end of the experiment.
After the immediate decline upon inoculation, carbohydrate content was observed to continuously increase from 9.8% to 34.5% by the 11th
day of experiment roughly corresponding to the end of the exponential growth. Towards the end of the experiment, the decline in carbohydrate to 18.9% occurred, whereas at the same point, the lipids, in particular saturated FAME, started to accumulate within the cells, as shown in Figure 5
To evaluate the metabolic plasticity of Synechococcus
sp. MK568070 and its response to stressing growth conditions, the FTIR analysis of the dry biomass was performed. Most of the absorption variation among the spectra was observed in the region 1200–1400 cm−1
, at 1655 cm−1
and at 1745 cm−1
. Major absorption bands in the IR spectra of the microalgae are presented in Table 3
. The bands selected as discriminating tool between protein and lipid functional group abundance were: 1655 cm−1
band for Amid1 and at 1745 cm−1
for vibrational stretching of Carboxylic C=O ester bond. Although two regions, methyl and methylene at 2800–3000 cm−1
, are commonly used to determine lipid content by FTIR [32
], in this study we used the vibrational stretching of ester bond C=O at 1740 cm−1
because it is considered to be exclusively related to ester bonds of fatty acids and avoids overlapping with functional groups present in more than one compound within the microalgae, as recommended by Mayers Flynn and Shields [34
The spectra were integrated and quantified and the abundance of Amid1 and Carboxylic functional groups were assessed, as proxies for protein and fatty acid, respectively. The ratio between Amid1 and Carboxylic group in Synechococcus
sp. MK568070 during the course of the experiment is presented in Figure 8
. The increase in proportion indicates accumulation of lipids with respect to proteins.
There was no significant change in (C=O/Amid I) ratio during active NH4+ consumption by Synechococcus sp. MK568070. After the 6th day, when majority of the DIN was sequestered from the wastewater, the ratio continuously increased during the exponential phase. The maximum (C=O/Amid I) value of 0.32 was achieved on the 14th day, when the culture reached the stationary phase.
As already reported for some oleaginous microalgae [30
] the increase in the ratio reflects the structural changes in the fatty acid profile during the exponential phase. The protein content during the exponential phase remained almost constant, whereas lipid content varied much more (Figure 6
). In the process of photosynthesis, a high proportion of protein is required for the energy bio-conversion process happening within the cells. This explains strong carbohydrate accumulation (Figure 6
) and biosynthesis of lipids reflected in high unsaturation and chain elongation during the exponential phase (Table 2
Most of the efforts studying bioremediation of wastewaters by microalgae are focused on freshwater species, and a substantial part of strains capable of growing under saline conditions needs jet to be explored. The results of this study have demonstrated that Synechococcus
sp. (MK568070), a cyanobacterium isolated from Adriatic coastal waters grows on industrial wastewater rich in ammonium, mercaptans, hydrocarbons and other potentially growth-inhibiting substances. Synechococcus
sp. MK568070 has demonstrated high tolerance to NH4+
concentration and strong dependence of biomass growth on wastewater quality. Although strong affinity towards NH4+
was determined, as already observed for Synechococcus
] the culture also grew well on the mixed source of nutrients, containing NH4+
and substantial amounts of organic phosphorus.
The studies on marine microalgae for wastewater remediation are rare because they are considered mostly metabolically adapted to the oligotrophic conditions and saline waters. However, Synechococcus
sp. MK568070 demonstrated good culturability in a wide range of salinities, achieving the highest biomass yield when grown at salinity 19. The tests of ammonium tolerance, as well as the growth in PBR1-3 suggest that all the concentrations used in this study allow for further increase. Most recent studies on wastewater phycoremediation use NO3−
as a source of N declaring optimum concentrations for lipid production to be 1.18–3.53 mM [37
]. Since the nitrogen deficiency is the primary trigger of lipids accumulation, the optimum growth conditions are always a trade-off between high biomass and high lipid yield. In this sense, the initial nutrient concentrations and wastewater quality used for cultivation of Synechococcus
sp. MK568070 were set up to support N-limited cellular growth but with suboptimal values for maximum biomass yield. Relying on the ability of Synechococcus
sp. (MK568070) to grow on industrial wastewater in a wide range of NH4+
concentrations and salinity it can be considered a good candidate for bioremediation of industrial wastewater of different origin and characteristics.
Cyanobacteria have a high potential in degrading dissolved organic matter, including hydrocarbons, as well as in dealing with high concentrations of heavy metals. Therefore, we tested an indigenous marine Synechococcus
strain from the Adriatic Sea regarding its growth potential and production of carbohydrates, proteins and most importantly lipids of the desired quality for biodiesel production during the process of remediation of oil refinery wastewater. Higher dry biomass yield, at the same concentration of nutrients, is possible in the case of more efficient dissolved organic matter usage. The Synechococcus
sp. MK568070 has shown high consistency of biomass growth in relation to NH4+
load and substantially higher proportion of lipids than most studied cyanobacteria [38
]. Whereas the Pacific Synechococcus
strains achieve lipid yield close to 11% of dry biomass weight, Synechococcus
sp. (MK568070) demonstrated yield of 21.40% of dry weight. The produced biomass of 761 mg/L corresponds to the yields obtained in other studies for cyanobacteria grown on commercial freshwater medium. Patel et al. [40
] have reported biomass yields close to 1 g/L for Synechococcus
species, but under a different light regime, higher starting N-concentration and with a combined NH4+
nutrient source. However, they observed lipid proportions that were significantly lower than those determined in our study.
In general, applying saline conditions for cyanobacteria growth may bring a disadvantage of slower division rate. However, there are some benefits such as microbial health control, resilience to pathogenic bacteria or opportunistic autotrophic invasions, better accumulation of lipids and independence of fresh water resources. The salinity of 19 used in our research corresponds to brackish conditions and is chosen as optimum salinity for Synechococcus sp. (MK568070) biomass productivity.
The cyanobacteria have evolutionary developed adaptive mechanisms to the major environmental stressors such as salinity, temperature and light intensity, depending on their indigenous environment to facilitate carbon uptake mechanisms and provide undisturbed CO2
availability. Such metabolic plasticity enables enhanced photosynthesis and biomass productivity by additional carbon supply. Air pumping with addition of 2–5% of CO2
is one of the commonly used ways to increase carbon fixation, as well as the provision of organic carbon substrate for support of the mixotrophic growth [41
]. As seen from the experiments in PBR2 and PBR4 effects of organic P and CO2
addition, respectively, have both positively impacted biomass productivity of Synechococcus
sp. MK568070. This is in accordance with previous studies elucidating the sufficient carbon supply as a prerequisite for lipid biosynthesis under nitrogen stress conditions [37
]. Moreover, Concas et al. [43
] provided a mathematical model for metabolic adaptation of Chlorella vulgaris
to 100% (v
) of CO2
Unicellular microalgae and cyanobacteria are quite often richer in lipids than filamentous species, known to produce large quantities of polysaccharides. Prokaryotes due to their small size, metabolic plasticity and resilience to rough environmental conditions are widely considered as good candidates for open-pond lipid production [40
]. In addition to lipid total yield, the structure of the synthesized triglycerides is very important for biodiesel production. Most of the cyanobacteria have a fatty acid profile dominated by C14, C16 and C18 saturated and monounsaturated fatty acids. However, quite often they contain significant quantities of some essential polyunsaturated fatty acids such as C18 linoleic (18:2ω6) and α-linolenic (18:3ω3) acids whose production in some cases can reach up to 20% of the cellular dry weight [18
]. The FAME profiles of Synechococcus
sp. MK568070 contained mostly saturated and monounsaturated FAME. The PUFA (18:2 ω6) was present only in the inoculum. Higher proportion of shorter acyl chain, high degree of saturation in the stationary phase, UND as low as 0.45 and 66% of C16 and an absence of polyunsaturated FA provide good biofuel feedstock characteristics. In gaseous CO2
supplied systems, if the CO2
flux is too high, biosynthesis can be directed towards production of high amounts of carbohydrate, sometimes exceeding 60% of the dry biomass. Although the carbohydrate dynamics during Synechococcus
sp. MK568070 growth in PBR4 shows high production, the maximum proportion does not exceed 34%. Moreover, the FTIR spectral analysis and (C=O/Amid I) ratio show continuously increasing proportion of carboxylic group in respect to proteins, confirming the prevalence of the lipid biosynthesis within the metabolic pathways during photosynthesis.
One of the advantages of using microalgae in the biofuel production is the possibility of wastewater treatment. In this study for the first time an insight is given on all energetically important groups of molecules synthesized by Synechococcus
sp. MK568070 during its cultivation on the oil refinery wastewater. These kind of wastewaters are very demanding regarding their high content of pollutants, and the capability of any microalgae for remediation of such waters while producing a notable amount of biomass is of high interest from the perspective of the lowering risks of coastal water eutrophication and improvements in cost-efficiency of the blue economy. Therefore the nutrient removal efficiency is an important condition for both, the biomass productivity, and for the wastewater bioremediation. The NH4+
removal >99% in six days makes Synechococcus
sp. MK568070 a very efficient species in bioremediation of wastewaters enriched with nitrogen. Efficient N-sequestration, metabolic plasticity and a high tolerance to a wide range of ammonium/ammonia concentrations open up the considerations for the genetic remodeling of its biochemical aspects [44
]. In order to evaluate the suitability of Synechococcus
sp. (MK568070) for biofuel production on the industrial scale, the metabolic mechanisms of lipid synthesis and growth kinetics should be explored in more detail. The potential of lipid accumulation, due to the functional photosynthesis even at the minimal cell quota of nutrients, is critical for the usage of excess industrial CO2
and its more cost-effective industrial transformation to biodiesel. To conclude, this study of biomass and lipid production by the Synechococcus
sp. MK568070 when cultivated on oil refinery wastewater with excess CO2
, provides useful data for further work in order to bring this species onto the industrial scale of biomass production and contributes to the findings for future prospects of wastewater bioremediation through algae cultivation.