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Integrated biomass production and biodegradation of olive mill wastewater by cultivation of Scenedesmus sp.
Algal Research 9 (2015) 306–311
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Integrated biomass production and biodegradation of olive mill wastewater by cultivation of Scenedesmus sp. Fabrizio Di Caprio, Pietro Altimari ⁎, Francesca Pagnanelli Department of Chemistry, Sapienza University of Rome, Ple. Aldo Moro 5, 00185 Rome, Italy
a r t i c l e
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Article history: Received 30 December 2014 Received in revised form 20 March 2015 Accepted 3 April 2015 Available online xxxx Keywords: Microalgae Olive mill wastewater Biofuel Biodiesel Heterotrophic growth Scenedesmus sp.
a b s t r a c t Biomass production and bio-treatment of olive mill wastewater (OMW) were simultaneously achieved by cultivation of Scenedesmus sp. The influence of medium composition was addressed by analyzing the effect of the addition of unsterilized OMW (9% v·v−1) and of inorganic nutrients, and the use of tap water. Performance of implemented treatments was assessed by analysis of the following variables: biomass productivity, growth rate, content of target microalgal fractions (lipids and carbohydrates), and consumption of OMW organic carbon fractions (reducing sugars and phenols). The addition of unsterilized OMW (9% v·v−1) caused darkening of microalgal cultures inducing transition to heterotrophic growth. Growth rate values attained in heterotrophic cultures (with OMW) (0.0181 and 0.023 h−1) were comparable to those attained in autotrophic cultures (without OMW) (0.0272 and 0.0249 h−1). Nonetheless, significantly lower biomass productions were achieved by heterotrophic growth (0.22 and 0.35 g·L−1 vs. 0.56 and 0.51 g·L−1). Even though leading to lower biomass productions, heterotrophic growth ensured carbohydrate productions comparable to those attained by autotrophic growth (0.1 and 0.16 vs. 0.12 and 0.17 g·L−1). Biodegradation of OMW phenols was found to be significantly affected by the concentration of inorganic nutrients. The inhibition of microalgal growth induced by phenols, and the interaction between phenol biodegradation and biomass production were analyzed. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Microalgae can attain biomass productivity values per hectare larger than that of any terrestrial plant and do not compete with agricultural activities for arable lands. These characteristics, along with the ability of microalgae to accumulate large lipid fractions (even larger than 60% of dry weight), have motivated over the past decade an increasing interest towards the application of microalgae in the industrial production of biofuels [1,2]. Numerous studies have reported that biofuels generated from microalgae are close to competing with fossil fuels and oldergeneration biofuels [3,4]. Despite such claims, no economically feasible process for the production of biofuels from microalgae has been realized yet [5,6]. Major obstacles to the achievement of this latter objective are the capital cost of equipment and the operating costs associated with energy and nutrient consumption [4,6]. A promising approach towards the achievement of economic feasibility is represented by the use of wastes as nutrients. This approach can significantly reduce the costs associated with nutrient supply ensuring, at the same time, co-depuration of the employed waste [7,8]. In this framework, the application of agro-industrial wastes is particularly attractive. These wastes are characterized by elevated concentrations of ⁎ Corresponding author. E-mail address: [email protected] (P. Altimari).
http://dx.doi.org/10.1016/j.algal.2015.04.007 2211-9264/© 2015 Elsevier B.V. All rights reserved.
nitrogen and phosphorus (the most relevant microalgal nutrients), and organic carbon [9]. In microalgal cultivation, the presence of elevated organic carbon concentrations can induce transition from autotrophic to mixotrophic or heterotrophic metabolism [10], allowing for high biomass productivities even with scarce light intensity or even in the dark. This excludes the need for expensive reactors with elevated surface to volume ratio required in autotrophic growth. Cost savings can thus be attained by application of conventional reactors and achievement of larger productivity per hectare. Olive oil production is an agro-industrial activity particularly active in the Mediterranean basin, which is characterized by the production of elevated volumes of wastewaters (olive mill wastewaters (OMW)). This waste contains phosphorus, nitrogen, and organic carbon and can be used as fertilizer [11]. Nevertheless, this application is prevented by the presence of phenols, which are responsible for anti-microbial and phytotoxic effects [11,12]. Several studies investigated the application of OMW as a source of nutrients in the cultivation of microalgae. The influence of light intensity, temperature, OMW fraction and OMW pre-treatment on biomass productivity, growth rate and accumulation of target microalgal fractions (lipids, carbohydrates and proteins) was analyzed for the microalgal strain Scenedesmus obliquus by Hodaifa et al. [13–17]. These latter studies were characterized by the application of sterilized OMW.
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This can reduce the risk of contamination however results could hardly be replicated in a relevant environment. In industrial practice, sterilization of the employed medium may be economically unfeasible [8]. Further, considerable differences may be found between the cases of application of sterilized and unsterilized OMW. For example, fungi and bacteria can significantly reduce biomass production by consuming dissolved oxygen which is employed by microalgae to oxidize the organic fraction [18]. In Hodaifa et al. [13–17], the use of a medium realized by dissolution of sterilized OMW in distilled water determined biomass productivities significantly lower than those attained by autotrophic growth with a standard culture medium. Several factors including the presence of toxic compounds, the lack of nutrients and the darkening of culture preventing photosynthetic activity, were indicated as possible causes of the observed result. However, a detailed analysis of the mechanisms responsible for the observed variations in biomass productivity is far from being proposed. In particular, scarce attention has been paid to the influence of phenols on microalgal growth. Microbiological studies have evidenced that elevated concentrations of phenols can inhibit microalgal growth [11,19,20]. Further, microalgae can biodegrade phenols and thus offer a route towards the degradation of such toxic compounds in wastewaters [21,22]. Research in this field has mainly analyzed the mechanisms exploited by microalgae to degrade low molecular weight phenols while reduced efforts have been aimed at investigating biodegradation of large polyphenol compounds in microalgal cultures. Large polyphenols cover most of the OMW phenol fraction and require, unlike low molecular weight phenols, extracellular enzymes to be degraded [23,24]. Despite the practical relevance of these arguments, no study was reported analyzing the effect of OMW polyphenols on biomass production and accumulation of target microalgal fractions. In the present study, the microalgal strain Scenedesmus sp. was cultivated in media enriched with OMW. The influence of OMW on biomass production and the role played by mineral salts in OMW biodegradation were investigated. Unlike previously published studies [13–17], the employed culture medium was realized by the addition of unsterilized OMW to water. This allowed testing the influence of contamination and provided indications about the scale up of the proposed cultivation strategy. Following this latter perspective, the possibility to replace distilled water by tap water and to avoid the addition of micronutrients (mineral salts) in the preparation of the culture medium was also explored. A further element of novelty is the addition of inorganic nitrogen (nitrate) to OMW. This allowed excluding inhibition caused by the lack of inorganic nitrogen in OMW. 2. Materials and methods 2.1. Cultivation A strain of Scenedesmus sp. was selected in Siracusa (Sicily, Italy) and maintained in Petri dishes with BG11 solid medium (Table 1). Microalgae were successively transferred from Petri dishes to 1000 mL roux bottles by using the BG11 liquid medium and then were reinoculated in 1000 mL roux bottles by 1:10 dilution. In this latter dilution, BG11 or a tap water based medium (TWBM) was employed depending on the performed experiment. TWBM was obtained by adding to tap water NaNO3 and K2HPO4 concentrations identical to those found in BG11 medium. To evaluate the effects of employed medium (BG11 and TWBM) and addition of OMW on biomass production, a 22 factorial design was performed. This consisted of four different treatments performed by cultivating microalgae with the following media: BG11 medium, TWBM, BG11 medium with addition of 9% v·v−1 OMW, and TWBM with addition of 9% v·v−1 OMW. Compositions of BG11 medium and tap water are reported in Table 1. Composition of tap water was obtained by data published by ACEA SpA (municipality owned company responsible for water management in Rome, Italy).
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Table 1 Chemical and physical properties of tap water and BG11 medium. Parameter
Tap water
BG11
pH Electrical conductivity (μS·cm−1) −1 ) HCO− 3 (mg·L Hardness (F°) −1 Ca (mg·L ) Mg (mg·L−1) Cl2 (mg∙L−1) NH4+ (mg·L−1) −1 ) NO− 3 (mg·L −1 ) NO− 2 (mg·L Cl− (mg·L−1) − −1 F (mg·L ) K (mg·L−1) Na (mg·L−1) SO42− (mg·L−1) As (μg·L−1) Fe (mg·L−1) Mn (μg·L−1) (mg·L−1) PO3− 4 EDTA (mg·L−1) H3BO3 (mg·L−1) Zn (mg·L−1) Mo (mg·L−1) Cu (μg·L−1)
7.34 553 387 32.7 101 18.3 0.18 b0.10 3.05 b0.05 7.1 0.14 1.9 5.0 16.76 b1.0 b0.5 0.3 b0.07 – – – – –
7.1 – 14.5 – 9.8 7.4 – 0.4 143 – 17.4 – 13.7 58.5 29.6 – 1.3 0.2 16.6 1 0.06 0.065 0.007 0.64
BG11 medium was prepared without NaNO3 which was added during the successive dilutions to reach a NO− 3 concentration of 0.143 ± 0.003 g·L−1 for each test. Experimental tests were conducted at room temperature (299 ± 3 K) under magnetic agitation. Roux bottles were maintained under constant illumination of 80 ± 10 μE m−2 s−1 by application of cool-white fluorescent lamps. Microalgal cultures were supplied with 7.5 L·d−1 of pure CO2. All tests were performed in duplicate. 2.2. Determination of biomass concentration Cell (L−1) and biomass concentrations (g·L−1) were determined by direct counting and spectrophotometric analysis, respectively. Counting was performed by a Leitz Laborlux 12 optical microscope in a 10−4 mL Thoma chamber. For the spectrophotometric analysis of biomass concentration, the sample was washed three times (solid–liquid separation performed by centrifugation at 8000 rpm) and re-suspended in distilled water. The absorbance was then measured at 690 nm by a UV–Visible spectrophotometer (Varian Cary 50 Scan). Biomass dry weight concentration was determined from sample absorbance by application of a calibration line. Dry weight of samples (20 mL of suspensions) employed for calibration was measured by filtration with 0.45 μm acetate cellulose filter. The filters were dried at 105 °C and then weighed. All the measures were performed in triplicate. 2.3. Lipid determination Microalgae obtained at the end of cultivation were separated by centrifugation at 3000 rpm for 5 min and washed three times with distilled water. After drying at 60 °C for 12 h, biomass was hand milled in a mortar to reduce pellet size. Lipid extraction was performed by the method reported in Ref. [25]. 2.4. Carbohydrate determination Carbohydrates were extracted from 100 mg of residual biomass obtained after lipid extraction. For this purpose, the M1 saccharification method reported in Ref. [26] was implemented. The solid–liquid suspension obtained by saccharification was centrifuged at 8000 rpm and total carbohydrates dissolved in the supernatant were determined by the Dubois method [27].
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2.5. Characterization of OMW OMW was generated by a three phase traditional pressing process from an industrial plant in Velletri (Rome, Italy). It was stored in a freezer at −20 °C and thawed before use. The OMW concentration of total reducing sugars was determined by the Dubois method [27]. To prevent interference, measures with and without addition of phenol solution were carried out according to the method reported by Chow and Landhäusser [28]. Total phenols were determined by the method reported by Atanassova et al. [29] and calculated as tyrosol equivalents. COD was determined by AQUANAL™ professional tube test purchased from Sigma-Aldrich. Sedimentable solids were determined by 2 h of sedimentation in an Ihmoff cone. Total solids were gravimetrically determined after drying at 105 °C. Elemental composition was determined by a CHN elemental analysis on total solids. Phosphorus concentration was measured by colorimetric ascorbic acid method [30]. Fig. 1. Temporal evolution of biomass concentration (g·L−1) for the four conditions of the factorial design.
3. Results and discussion 3.1. Wastewater and tap water characterization The chemical characteristics of the OMW employed in this work are summarized in Table 2. The concentration of reducing sugars is equal to 7.6 g·L−1. Reducing sugars along with organic acids are the most easily degradable components of the OMW organic fraction [31]. COD and phenol contents (49 and 4.88 g·L−1, respectively) are comparable to those reported in previous studies [11,13]. Total sedimentable solids were 240 ± 20 mL·L−1 and could be removed after 1 h of sedimentation before cultivation. In Table 1, the chemical and physical properties of tap water and BG11 medium are compared. Such comparison reveals that the concentrations of inorganic nitrogen and phosphorus are much lower in tap water than in BG11. On the other hand, HCO− 3 concentration is one order of magnitude larger in tap water than in BG11. Also, greater Ca2 + and Mg2 + concentrations are found in tap water. NaNO3 and K2HPO4 were added to tap water in concentrations identical to those found in the BG11 medium in order to promote microalgal growth.
respectively. In autotrophic tests, a stationary phase was attained after about 7–8 days of cultivation. In this latter case, stationary growth was induced by nitrate depletion. This was confirmed by the analysis of the evolution of nitrate concentration (data not shown here). The effects of the two considered factors, namely employed autotrophic medium (BG11 or TWBM) and addition of OMW, and the interaction between factors were quantified by analysis of variance (ANOVA). The results of this analysis are reported in Fig. 2. The effects of TWBM and OMW are defined as the variations in biomass concentration determined by using TWBM in place of the BG11 medium, and by addition of OMW, respectively. The estimate of the effects for each day was compared with the 95% confidence interval due to random errors determined from two replicates: effects exceeding such intervals are significant. In particular, the significance interval is estimated as follows:
L ¼ t α;v
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi MSE ðt Þ n
ð1Þ
3.2. Biomass production Evolutions of biomass concentration recorded during experimental tests are displayed in Fig. 1. For any performed test, no lag phase was found probably due to the use of an inoculum in exponential phase. Light could penetrate the culture medium in tests performed without addition of OMW while completely dark cultures were obtained in cases of OMW addition. Accordingly, heterotrophic and autotrophic growth modes were experienced in tests with and without OMW, respectively. In the following, tests performed with and without OMW will therefore be referred to as heterotrophic and autotrophic tests,
where MSE(t) is the sample variance due to random errors calculated at time t for the two replicates, tα,ν is the tabulated value of the t of Student for α = 0.05 and ν degrees of freedom [32].
Table 2 Chemical properties determined in olive mill wastewater (OMW). OMW Parameter −1
Phenols (g·L ) Sugars (g·L−1) Sedimentable solids (mL·L−1) Total solids (g·L−1) H2O (%) COD (g·L−1) pH −1 ) PO− 4 (mg·L C (%)a a H (%) N (%)a S (%)a a
Percentage values refer to total solids.
Average
±Δ
4.88 7.6 240 51.1 94.89 49 5.0 1347 40 5 1 0.9
0.05 0.1 20 0.2 0.02 5 0.2 1 5 1 0.8 0.2
Fig. 2. Effect of TWBM and OMW, and their interaction during cultivation of Scenedesmus sp. (solid line denotes the least significant difference for every day).
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A positive effect of TWBM on biomass growth was invariably found (Fig. 2). This effect is however statistically significant only during the first 5 days and can be attributed to a concentration of bicarbonate in tap water larger than in BG11 medium (Table 1). In heterotrophic growth, stationary phase was achieved with both BG11 and TWBM after 4 days of cultivation and maximum biomass concentration values lower than those attained in autotrophic growth were found (Fig. 1). Accordingly, a negative effect of OMW was determined by ANOVA (Fig. 2). On the other hand, specific growth rate values determined during the initial four days of heterotrophic growth were comparable to those found in autotrophic growth. The effect of OMW becomes therefore negative after five days of cultivation (Fig. 2). As reported in Fig. 2, a negative interaction between OMW and TWBM was found. However, this effect became statistically not significant following the initial eight days of cultivation. Addition of OMW may favor contamination by fungi and bacteria. In case of the presence of these latter microorganisms, application of the method illustrated in Section 2.2 could lead to overestimating the biomass concentration. This latter method entails weighing the dried solid resulting from filtration of the cultivation suspension and, hence, cannot distinguish between microalgae and foreign microorganisms. To verify the absence of relevant contamination, the temporal evolution of cell concentration was monitored by direct counting (data not shown here). This allowed for distinguishing microalgae from foreign microorganisms and excluded interferences induced by contamination. The effect of any investigated factor and interaction between factors was determined by analysis of variance of cell concentration data. Results were qualitatively identical to those reported in Fig. 2 confirming the reliability of biomass concentration data reported in Fig. 1. Overall biomass production and maximum productivity values obtained for the performed experimental tests are reported in Table 3. Independently of the employed medium (BG11 or TWBM), larger biomass production was found when microalgae were cultivated without OMW. Analysis of variance was also conducted evidencing a statistically significant reduction of maximum productivity in response to the addition of OMW. 3.3. OMW biodegradation Reductions in the concentrations of phenols, reducing sugars and COD were found following microalgal harvesting in media enriched with OMW (Fig. 3). No statistically significant difference was determined between the removals of reducing sugars attained with BG11 and TWBM. With both these two latter media, reducing sugar concentration decreased by about 60%. It can be assumed that consumed reducing sugars were mainly constituted by glucose and fructose. These compounds can easily be metabolized by microalgae and cover most of OMW carbohydrates [12]. Besides glucose and fructose, additional carbohydrates are present in OMW, which can hardly be biodegraded as, for example, pentose [11]. Unlike what was found for reducing sugars, a statistically significant difference was determined between phenol removals attained
309
Fig. 3. Reducing sugars, phenols (as tyrosol equivalents) and COD removed by microalgae in OMW. Results were obtained by cultivation of microalgae in TWBM and BG11 with 9% OMW.
with TWBM and BG11. In particular, phenol removal values around 6% and 22% were found with the media (TWBM + OMW) and (BG11 + TWBM), respectively. COD reduction changed from 36.5 ± 0.5% to 28 ± 4% as TWBM was used in place of the BG11 medium. This difference is however not statistically significant. An independent estimate of the COD reduction was derived by assuming that consumed reducing sugars and phenols (Fig. 3) were exclusively composed of glucose and tyrosol, respectively. In accordance with this latter assumption, 16% and 13% COD reductions were determined for the tests conducted with BG11 and TWBM, respectively. These values are significantly lower than measured COD variations reported in Fig. 2, evidencing that additional reduced substances were removed during cultivation along with carbohydrates and phenols. These additional substances are probably organic acids, which can be biodegraded by microalgae and reach high concentrations in OMW [11,12]. The contribution of organic acids to COD removal was estimated by assuming complete removal of acetic acid, citric acid, lactic acid and succinic acid. OMW concentration values reported in Ref. [12] were employed for these acids. COD removal comparable to actual measured values was found by this approach confirming biodegradation of organic acids.
3.4. Lipid and carbohydrate accumulation No statistically significant difference was found among lipid fractions of microalgae cultivated with the four considered media: an average lipid fraction around 32% (w/w) was invariably found (Fig. 4). A statistically significant positive effect on carbohydrate accumulation was in contrast induced by the addition of OMW. When Scenedesmus sp. was grown in the presence of OMW, stored carbohydrates increased from 34 ± 1% to 43 ± 9% with BG11, and from 21.4 ± 0.8% to 44.0 ± 0.2% with TWBM.
Table 3 Overall biomass production, maximum productivity, kinetic parameters and final concentrations of sugars, phenols and COD reached by Scenedesmus sp. in TWBM and BG11 with and without OMW.
x–x0 (g·L−1) Carbohydrate production (g·L−1) Lipid production (g·L−1) Maximum biomass productivity (g·L−1·d−1) μ (h−1) Cf,phenols (g·L−1)a Cf,reducible sugars (g·L−1)a Cf,COD (g·L−1)a a
TWBM - OMW
TWBM
BG11 – OMW
BG11
0.22 ± 0.05 0.1 ± 0.02 0.09 ± 0.04 0.06 ± 0.01 0.023 ± 0.002 0.42 ± 0.02 0.26 ± 0.03 2.83 ± 0.03
0.559 ± 0.001 0.1196 ± 3·10−4 0.1790 ± 4·10−4 0.11 ± 0.01 0.0272 ± 4·10−4 – – –
0.35 ± 0.09 0.16 ± 0.04 0.11 ± 0.03 0.08 ± 0.01 0.0249 ± 4·10−4 0.347 ± 0.007 0.27 ± 0.01 3.2 ± 0.3
0.512 ± 0.007 0.174 ± 0.002 0.164 ± 0.003 0.120 ± 0.008 0.0181 ± 8·10−4 – – –
Reported concentrations of phenols, reducing sugars and COD are computed for OMW 9% v·v−1.
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in biomass productivity observed in response to the addition of OMW cannot be imputed to the presence of fat matter.
Fig. 4. Comparison of lipid and carbohydrate content in Scenedesmus sp. biomass obtained in the four conditions investigated.
4. Discussion Growth rate values comparable or even larger than those attained in autotrophic cultures were found by cultivation of microalgae in media enriched with OMW (Figs. 1–2 and Table 3). This result is in contrast with data reported by Hodaifa et al. [16,33]. These authors evidenced the achievement of a growth rate with OMW significantly lower than the value attained by autotrophic cultivation in a standard medium. A culture medium realized by the addition of only sterilized OMW to distilled water was employed in the performed experiment. We attribute the revealed contradiction to the addition of nitrates to the culture media employed in the present study. This can be verified by noting that the medium (TWBM + OMW), which ensures growth rates close to those attained in autotrophic cultures, differs from a medium composed only of OMW and water (as the one employed by Hodaifa et al. [16,33]) because of the addition of nitrates and phosphates (used in the preparation of TWBM). On the other hand, phosphates added along with nitrates to TWBM are not expected to influence microalgal growth in (TWBM + OMW) medium because they are already abundant in OMW (Table 2). Therefore, nitrates can be considered responsible for the increased growth rate achieved in the present study by cultivation in media enriched with OMW. It must be remarked that nitrogen is not absent in OMW and can even reach values in such wastewater concentration close to the ones supplied in the present study by the addition of nitrates. OMW nitrogen is however mainly present in peptides and amino acids. Degradation of these compounds is required to generate ammonium which can be exploited to synthesize proteins. However, this imposes the production of extracellular enzymes which is uncommon in algae [34]. This can explain the observed positive effect of inorganic nitrogen on specific growth rate. Results reported in Fig. 1 indicate that the addition of OMW determines the early achievement of stationary phase. This leads to heterotrophic biomass productivity values lower than those attained by autotrophic growth. This result has been previously attributed to the absence of photosynthetic activity and to OMW fat matter adsorbing on microalgal surface and limiting nutrient availability [16,33]. We consider these two latter factors insufficient to explain the arrest of growth at lower biomass concentrations. To verify it, let us consider the following arguments: – Even in the absence of photosynthetic activity, microalgae grown with OMW accumulated a large amount of organic carbon (Fig. 4). This organic carbon could have been employed to derive the energy required for growth. – While evidencing an increase in growth rate, the application of defatted OMW led to biomass productivity values lower than those attained in autotrophic growth [16,33]. Accordingly, the reduction
A further element which may be responsible for the early arrest of microalgal growth is the depletion of nitrogen. However, this must also be discarded because, with the same initial nitrate concentration (0.143 g·L−1), autotrophic tests led to biomass concentration significantly higher than that attained in heterotrophic tests (Fig. 1). Further proof that nitrogen was not limiting growth in heterotrophic cultures is given by the analysis of carbohydrates and lipids (Fig. 4). About 75% dry weight of microalgae cultivated in heterotrophic conditions is composed of lipids and carbohydrates. By assuming that the remaining 25% is constituted by proteins alone (that is an overestimation), nitrogen fraction not larger than 4% would be found. This latter value corresponds, with the attained biomass concentration around 0.3 g·L− 1 (Fig. 1), to a consumption of about 40% of initial nitrogen. In this framework, the fundamental role appears to be played by OMW phenols. Although microalgae can degrade phenols, studies have evidenced that such compounds can induce toxic effects in microalgal cells. The hypothesis of early arrest of growth determined by phenols is supported in the present study by the apparent relationship between phenol consumption and biomass concentration. The consumption of phenols was with the medium (BG11 + OMW) significantly larger than with the medium (TWBM + OMW) (Fig. 3), the larger phenol consumption corresponding to the achievement of increased biomass concentration (Table 3). This suggests that microalgal growth is enhanced as mechanisms lowering phenol concentration are activated. While supporting the hypothesis of inhibition induced by phenols, the previous analysis raises the question of why phenol consumption could be attained in the medium (BG11 + OMW) (about 22% removal) and not with the (TWBM + OMW) medium (about 6% removal). This must necessarily be imputed to differences between the compositions of the BG11 and TWBM media. BG11 contains micronutrients and EDTA which were not added to TWBM, while a significantly larger concentration of Ca2 + ions can be found in TWBM because of the application of tap water. For what concerns micronutrients, iron concentration reached a value of 1.3 mg·L−1 in BG11 while it was not detectable in tap water (b 0.5 mg·L−1). Iron and other metal ions are employed by enzymes as co-factors in the degradation of phenols [35–37]. The absence of micronutrients may therefore explain the negligible consumption of phenols in (TWBM + OMW) medium. Further, micronutrients could be not assimilated by microalgae in the absence of EDTA. This compound can form complexes with metal ions and can thus improve the access of such metals to the cell. Ca2+ concentration in TWBM higher than in BG11 must also be taken into account as the interaction between calcium ions and phenols can influence phenol biodegradability [38]. A further important characteristic of inhibition occurring in heterotrophic cultivation is that it appears after five days of cultivation (Fig. 1). This latter result indicates that toxic effects induced by phenols require a latent time to appear. Even though not influencing the microalgal lipid fraction, the addition of OMW improved accumulation of carbohydrates (Fig. 4). This result allowed for achieving heterotrophic carbohydrate productivity close to those found in autotrophic growth (Table 3) and suggests a possible application of the employed cultivation strategy in bioethanol production. 5. Conclusions The study investigated the cultivation of the microalgal strain Scenedesmus sp. with unsterilized OMW. The main conclusions of the illustrated study can be stated as follows: – Elevated robustness of the selected strain towards the risk of contamination was found suggesting the possibility to successfully
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scale-up the described cultivation strategy to pilot and large industrial scale. – The addition of inorganic nitrogen can prevent reduction in the specific growth rate. From this perspective, media realized by mixing OMW and wastewaters rich in nitrogen should be explored. – Early achievement of stationary phase was observed in response to the addition of OMW. This phenomenon is responsible for a reduction of biomass productivity and is mainly determined by antimicrobial activity of phenols. – Significantly larger removal of phenols was found in the case of addition of OMW to BG11 medium rather than to TWBM. This difference highlights the fundamental role played by concentration of mineral salts in the degradation of phenols. The conclusions above encourage studies aimed at further characterizing the interaction between phenol degradation and microalgal growth in culture media enriched with OMW. References [1] S.S. Oncel, Microalgae for a macroenergy world, Renew. Sustain. Energy Rev. 26 (2013) 241–264. [2] T.M. Mata, A.A. Martins, N.S. Caetano, Microalgae for biodiesel production and other applications: a review, Renew. Sustain. Energy Rev. 14 (2010) 217–232. [3] N.H. Norsker, M.J. Barbosa, M.H. Vermuë, R.H. Wijffels, Microalgal production — a close look at the economics, Biotechnol. Adv. 29 (2011) 24–27. [4] F.G. Avién, J.M. Fernández, J.J. Magán, E. Molina, Production cost of a real microalgae production plant and strategies to reduce it, Biotechnol. Adv. 30 (2012) 1344–1353. [5] E. Waltz, Biotech's green gold? Nature 27 (2009) 15–18. [6] R. Slade, A. Bauen, Micro-algae cultivation for biofuels: cost, energy balance, environmental impacts and future prospects, Biomass Bioenergy 53 (2013) 29–38. [7] W. Zhou, P. Chen, M. Min, X. Ma, J. Wang, R. Griffith, F. Hussain, P. Peng, Q. Xie, Y. Li, J. Shi, J. Meng, R. Ruan, Environment-enhancing algal biofuel production using wastewaters, Renew. Sustain. Energy Rev. 36 (2014) 256–269. [8] Y.H. Wu, H.Y. Hu, Y. Yu, T.Y. Zhang, S.F. Zhu, L.L. Zhuang, X. Zhang, Y. Lu, Microalgal species for sustainable biomass/lipid production using wastewater as resource: a review, Renew. Sustain. Energy Rev. 33 (2014) 675–688. [9] G. Chen, L.Z.Y. Qi, Enhancing the productivity of microalgae cultivated in wastewater toward biofuel production: a critical review, Appl. Energy 137 (2015) 282–291. [10] F. Pagnanelli, P. Altimari, F. Trabucco, L. Toro, Mixotrophic growth of Chlorella vulgaris and Nannochloropsis oculata: interaction between glucose and nitrate, J. Chem. Technol. Biotechnol. 89 (2014) 652–661. [11] S. Dermeche, M. Nadour, C. Larroche, F. Moulti-Mati, P. Michaud, Olive mill wastes: biochemical characterizations and valorization strategies, Process Biochem. 48 (2013) 1532–1552. [12] C. Faraloni, A. Ena, C. Pintucci, G. Torzillo, Enhanced hydrogen production by means of sulfur-deprived Chlamydomonas reinhardtii cultures grown in pretreated olive mill wastewater, Int. J. Hydrog. Energy 36 (2011) 5920–5931. [13] G. Hodaifa, M.E. Martínez, S. Sánchez, Daily doses of light in relation to the growth of Scenedesmus obliquus in diluted three-phase olive mill wastewater, J. Chem. Technol. Biotechnol. 84 (2009) 1550–1558. [14] G. Hodaifa, M.E. Martínez, S. Sánchez, Influence of pH on the culture of Scenedesmus obliquus in olive-mill wastewater, Biotechnol. Bioprocess Eng. 14 (2009) 854–860. [15] G. Hodaifa, M.E. Martínez, S. Sánchez, Influence of temperature on growth of Scenedesmus obliquus in diluted olive mill wastewater as culture medium, Eng. Life Sci. 10 (2010) 257–264.
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Algal Research journal homepage: www.elsevier.com/locate/algal
Integrated biomass production and biodegradation of olive mill wastewater by cultivation of Scenedesmus sp. Fabrizio Di Caprio, Pietro Altimari ⁎, Francesca Pagnanelli Department of Chemistry, Sapienza University of Rome, Ple. Aldo Moro 5, 00185 Rome, Italy
a r t i c l e
i n f o
Article history: Received 30 December 2014 Received in revised form 20 March 2015 Accepted 3 April 2015 Available online xxxx Keywords: Microalgae Olive mill wastewater Biofuel Biodiesel Heterotrophic growth Scenedesmus sp.
a b s t r a c t Biomass production and bio-treatment of olive mill wastewater (OMW) were simultaneously achieved by cultivation of Scenedesmus sp. The influence of medium composition was addressed by analyzing the effect of the addition of unsterilized OMW (9% v·v−1) and of inorganic nutrients, and the use of tap water. Performance of implemented treatments was assessed by analysis of the following variables: biomass productivity, growth rate, content of target microalgal fractions (lipids and carbohydrates), and consumption of OMW organic carbon fractions (reducing sugars and phenols). The addition of unsterilized OMW (9% v·v−1) caused darkening of microalgal cultures inducing transition to heterotrophic growth. Growth rate values attained in heterotrophic cultures (with OMW) (0.0181 and 0.023 h−1) were comparable to those attained in autotrophic cultures (without OMW) (0.0272 and 0.0249 h−1). Nonetheless, significantly lower biomass productions were achieved by heterotrophic growth (0.22 and 0.35 g·L−1 vs. 0.56 and 0.51 g·L−1). Even though leading to lower biomass productions, heterotrophic growth ensured carbohydrate productions comparable to those attained by autotrophic growth (0.1 and 0.16 vs. 0.12 and 0.17 g·L−1). Biodegradation of OMW phenols was found to be significantly affected by the concentration of inorganic nutrients. The inhibition of microalgal growth induced by phenols, and the interaction between phenol biodegradation and biomass production were analyzed. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Microalgae can attain biomass productivity values per hectare larger than that of any terrestrial plant and do not compete with agricultural activities for arable lands. These characteristics, along with the ability of microalgae to accumulate large lipid fractions (even larger than 60% of dry weight), have motivated over the past decade an increasing interest towards the application of microalgae in the industrial production of biofuels [1,2]. Numerous studies have reported that biofuels generated from microalgae are close to competing with fossil fuels and oldergeneration biofuels [3,4]. Despite such claims, no economically feasible process for the production of biofuels from microalgae has been realized yet [5,6]. Major obstacles to the achievement of this latter objective are the capital cost of equipment and the operating costs associated with energy and nutrient consumption [4,6]. A promising approach towards the achievement of economic feasibility is represented by the use of wastes as nutrients. This approach can significantly reduce the costs associated with nutrient supply ensuring, at the same time, co-depuration of the employed waste [7,8]. In this framework, the application of agro-industrial wastes is particularly attractive. These wastes are characterized by elevated concentrations of ⁎ Corresponding author. E-mail address: [email protected] (P. Altimari).
http://dx.doi.org/10.1016/j.algal.2015.04.007 2211-9264/© 2015 Elsevier B.V. All rights reserved.
nitrogen and phosphorus (the most relevant microalgal nutrients), and organic carbon [9]. In microalgal cultivation, the presence of elevated organic carbon concentrations can induce transition from autotrophic to mixotrophic or heterotrophic metabolism [10], allowing for high biomass productivities even with scarce light intensity or even in the dark. This excludes the need for expensive reactors with elevated surface to volume ratio required in autotrophic growth. Cost savings can thus be attained by application of conventional reactors and achievement of larger productivity per hectare. Olive oil production is an agro-industrial activity particularly active in the Mediterranean basin, which is characterized by the production of elevated volumes of wastewaters (olive mill wastewaters (OMW)). This waste contains phosphorus, nitrogen, and organic carbon and can be used as fertilizer [11]. Nevertheless, this application is prevented by the presence of phenols, which are responsible for anti-microbial and phytotoxic effects [11,12]. Several studies investigated the application of OMW as a source of nutrients in the cultivation of microalgae. The influence of light intensity, temperature, OMW fraction and OMW pre-treatment on biomass productivity, growth rate and accumulation of target microalgal fractions (lipids, carbohydrates and proteins) was analyzed for the microalgal strain Scenedesmus obliquus by Hodaifa et al. [13–17]. These latter studies were characterized by the application of sterilized OMW.
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This can reduce the risk of contamination however results could hardly be replicated in a relevant environment. In industrial practice, sterilization of the employed medium may be economically unfeasible [8]. Further, considerable differences may be found between the cases of application of sterilized and unsterilized OMW. For example, fungi and bacteria can significantly reduce biomass production by consuming dissolved oxygen which is employed by microalgae to oxidize the organic fraction [18]. In Hodaifa et al. [13–17], the use of a medium realized by dissolution of sterilized OMW in distilled water determined biomass productivities significantly lower than those attained by autotrophic growth with a standard culture medium. Several factors including the presence of toxic compounds, the lack of nutrients and the darkening of culture preventing photosynthetic activity, were indicated as possible causes of the observed result. However, a detailed analysis of the mechanisms responsible for the observed variations in biomass productivity is far from being proposed. In particular, scarce attention has been paid to the influence of phenols on microalgal growth. Microbiological studies have evidenced that elevated concentrations of phenols can inhibit microalgal growth [11,19,20]. Further, microalgae can biodegrade phenols and thus offer a route towards the degradation of such toxic compounds in wastewaters [21,22]. Research in this field has mainly analyzed the mechanisms exploited by microalgae to degrade low molecular weight phenols while reduced efforts have been aimed at investigating biodegradation of large polyphenol compounds in microalgal cultures. Large polyphenols cover most of the OMW phenol fraction and require, unlike low molecular weight phenols, extracellular enzymes to be degraded [23,24]. Despite the practical relevance of these arguments, no study was reported analyzing the effect of OMW polyphenols on biomass production and accumulation of target microalgal fractions. In the present study, the microalgal strain Scenedesmus sp. was cultivated in media enriched with OMW. The influence of OMW on biomass production and the role played by mineral salts in OMW biodegradation were investigated. Unlike previously published studies [13–17], the employed culture medium was realized by the addition of unsterilized OMW to water. This allowed testing the influence of contamination and provided indications about the scale up of the proposed cultivation strategy. Following this latter perspective, the possibility to replace distilled water by tap water and to avoid the addition of micronutrients (mineral salts) in the preparation of the culture medium was also explored. A further element of novelty is the addition of inorganic nitrogen (nitrate) to OMW. This allowed excluding inhibition caused by the lack of inorganic nitrogen in OMW. 2. Materials and methods 2.1. Cultivation A strain of Scenedesmus sp. was selected in Siracusa (Sicily, Italy) and maintained in Petri dishes with BG11 solid medium (Table 1). Microalgae were successively transferred from Petri dishes to 1000 mL roux bottles by using the BG11 liquid medium and then were reinoculated in 1000 mL roux bottles by 1:10 dilution. In this latter dilution, BG11 or a tap water based medium (TWBM) was employed depending on the performed experiment. TWBM was obtained by adding to tap water NaNO3 and K2HPO4 concentrations identical to those found in BG11 medium. To evaluate the effects of employed medium (BG11 and TWBM) and addition of OMW on biomass production, a 22 factorial design was performed. This consisted of four different treatments performed by cultivating microalgae with the following media: BG11 medium, TWBM, BG11 medium with addition of 9% v·v−1 OMW, and TWBM with addition of 9% v·v−1 OMW. Compositions of BG11 medium and tap water are reported in Table 1. Composition of tap water was obtained by data published by ACEA SpA (municipality owned company responsible for water management in Rome, Italy).
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Table 1 Chemical and physical properties of tap water and BG11 medium. Parameter
Tap water
BG11
pH Electrical conductivity (μS·cm−1) −1 ) HCO− 3 (mg·L Hardness (F°) −1 Ca (mg·L ) Mg (mg·L−1) Cl2 (mg∙L−1) NH4+ (mg·L−1) −1 ) NO− 3 (mg·L −1 ) NO− 2 (mg·L Cl− (mg·L−1) − −1 F (mg·L ) K (mg·L−1) Na (mg·L−1) SO42− (mg·L−1) As (μg·L−1) Fe (mg·L−1) Mn (μg·L−1) (mg·L−1) PO3− 4 EDTA (mg·L−1) H3BO3 (mg·L−1) Zn (mg·L−1) Mo (mg·L−1) Cu (μg·L−1)
7.34 553 387 32.7 101 18.3 0.18 b0.10 3.05 b0.05 7.1 0.14 1.9 5.0 16.76 b1.0 b0.5 0.3 b0.07 – – – – –
7.1 – 14.5 – 9.8 7.4 – 0.4 143 – 17.4 – 13.7 58.5 29.6 – 1.3 0.2 16.6 1 0.06 0.065 0.007 0.64
BG11 medium was prepared without NaNO3 which was added during the successive dilutions to reach a NO− 3 concentration of 0.143 ± 0.003 g·L−1 for each test. Experimental tests were conducted at room temperature (299 ± 3 K) under magnetic agitation. Roux bottles were maintained under constant illumination of 80 ± 10 μE m−2 s−1 by application of cool-white fluorescent lamps. Microalgal cultures were supplied with 7.5 L·d−1 of pure CO2. All tests were performed in duplicate. 2.2. Determination of biomass concentration Cell (L−1) and biomass concentrations (g·L−1) were determined by direct counting and spectrophotometric analysis, respectively. Counting was performed by a Leitz Laborlux 12 optical microscope in a 10−4 mL Thoma chamber. For the spectrophotometric analysis of biomass concentration, the sample was washed three times (solid–liquid separation performed by centrifugation at 8000 rpm) and re-suspended in distilled water. The absorbance was then measured at 690 nm by a UV–Visible spectrophotometer (Varian Cary 50 Scan). Biomass dry weight concentration was determined from sample absorbance by application of a calibration line. Dry weight of samples (20 mL of suspensions) employed for calibration was measured by filtration with 0.45 μm acetate cellulose filter. The filters were dried at 105 °C and then weighed. All the measures were performed in triplicate. 2.3. Lipid determination Microalgae obtained at the end of cultivation were separated by centrifugation at 3000 rpm for 5 min and washed three times with distilled water. After drying at 60 °C for 12 h, biomass was hand milled in a mortar to reduce pellet size. Lipid extraction was performed by the method reported in Ref. [25]. 2.4. Carbohydrate determination Carbohydrates were extracted from 100 mg of residual biomass obtained after lipid extraction. For this purpose, the M1 saccharification method reported in Ref. [26] was implemented. The solid–liquid suspension obtained by saccharification was centrifuged at 8000 rpm and total carbohydrates dissolved in the supernatant were determined by the Dubois method [27].
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2.5. Characterization of OMW OMW was generated by a three phase traditional pressing process from an industrial plant in Velletri (Rome, Italy). It was stored in a freezer at −20 °C and thawed before use. The OMW concentration of total reducing sugars was determined by the Dubois method [27]. To prevent interference, measures with and without addition of phenol solution were carried out according to the method reported by Chow and Landhäusser [28]. Total phenols were determined by the method reported by Atanassova et al. [29] and calculated as tyrosol equivalents. COD was determined by AQUANAL™ professional tube test purchased from Sigma-Aldrich. Sedimentable solids were determined by 2 h of sedimentation in an Ihmoff cone. Total solids were gravimetrically determined after drying at 105 °C. Elemental composition was determined by a CHN elemental analysis on total solids. Phosphorus concentration was measured by colorimetric ascorbic acid method [30]. Fig. 1. Temporal evolution of biomass concentration (g·L−1) for the four conditions of the factorial design.
3. Results and discussion 3.1. Wastewater and tap water characterization The chemical characteristics of the OMW employed in this work are summarized in Table 2. The concentration of reducing sugars is equal to 7.6 g·L−1. Reducing sugars along with organic acids are the most easily degradable components of the OMW organic fraction [31]. COD and phenol contents (49 and 4.88 g·L−1, respectively) are comparable to those reported in previous studies [11,13]. Total sedimentable solids were 240 ± 20 mL·L−1 and could be removed after 1 h of sedimentation before cultivation. In Table 1, the chemical and physical properties of tap water and BG11 medium are compared. Such comparison reveals that the concentrations of inorganic nitrogen and phosphorus are much lower in tap water than in BG11. On the other hand, HCO− 3 concentration is one order of magnitude larger in tap water than in BG11. Also, greater Ca2 + and Mg2 + concentrations are found in tap water. NaNO3 and K2HPO4 were added to tap water in concentrations identical to those found in the BG11 medium in order to promote microalgal growth.
respectively. In autotrophic tests, a stationary phase was attained after about 7–8 days of cultivation. In this latter case, stationary growth was induced by nitrate depletion. This was confirmed by the analysis of the evolution of nitrate concentration (data not shown here). The effects of the two considered factors, namely employed autotrophic medium (BG11 or TWBM) and addition of OMW, and the interaction between factors were quantified by analysis of variance (ANOVA). The results of this analysis are reported in Fig. 2. The effects of TWBM and OMW are defined as the variations in biomass concentration determined by using TWBM in place of the BG11 medium, and by addition of OMW, respectively. The estimate of the effects for each day was compared with the 95% confidence interval due to random errors determined from two replicates: effects exceeding such intervals are significant. In particular, the significance interval is estimated as follows:
L ¼ t α;v
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi MSE ðt Þ n
ð1Þ
3.2. Biomass production Evolutions of biomass concentration recorded during experimental tests are displayed in Fig. 1. For any performed test, no lag phase was found probably due to the use of an inoculum in exponential phase. Light could penetrate the culture medium in tests performed without addition of OMW while completely dark cultures were obtained in cases of OMW addition. Accordingly, heterotrophic and autotrophic growth modes were experienced in tests with and without OMW, respectively. In the following, tests performed with and without OMW will therefore be referred to as heterotrophic and autotrophic tests,
where MSE(t) is the sample variance due to random errors calculated at time t for the two replicates, tα,ν is the tabulated value of the t of Student for α = 0.05 and ν degrees of freedom [32].
Table 2 Chemical properties determined in olive mill wastewater (OMW). OMW Parameter −1
Phenols (g·L ) Sugars (g·L−1) Sedimentable solids (mL·L−1) Total solids (g·L−1) H2O (%) COD (g·L−1) pH −1 ) PO− 4 (mg·L C (%)a a H (%) N (%)a S (%)a a
Percentage values refer to total solids.
Average
±Δ
4.88 7.6 240 51.1 94.89 49 5.0 1347 40 5 1 0.9
0.05 0.1 20 0.2 0.02 5 0.2 1 5 1 0.8 0.2
Fig. 2. Effect of TWBM and OMW, and their interaction during cultivation of Scenedesmus sp. (solid line denotes the least significant difference for every day).
F. Di Caprio et al. / Algal Research 9 (2015) 306–311
A positive effect of TWBM on biomass growth was invariably found (Fig. 2). This effect is however statistically significant only during the first 5 days and can be attributed to a concentration of bicarbonate in tap water larger than in BG11 medium (Table 1). In heterotrophic growth, stationary phase was achieved with both BG11 and TWBM after 4 days of cultivation and maximum biomass concentration values lower than those attained in autotrophic growth were found (Fig. 1). Accordingly, a negative effect of OMW was determined by ANOVA (Fig. 2). On the other hand, specific growth rate values determined during the initial four days of heterotrophic growth were comparable to those found in autotrophic growth. The effect of OMW becomes therefore negative after five days of cultivation (Fig. 2). As reported in Fig. 2, a negative interaction between OMW and TWBM was found. However, this effect became statistically not significant following the initial eight days of cultivation. Addition of OMW may favor contamination by fungi and bacteria. In case of the presence of these latter microorganisms, application of the method illustrated in Section 2.2 could lead to overestimating the biomass concentration. This latter method entails weighing the dried solid resulting from filtration of the cultivation suspension and, hence, cannot distinguish between microalgae and foreign microorganisms. To verify the absence of relevant contamination, the temporal evolution of cell concentration was monitored by direct counting (data not shown here). This allowed for distinguishing microalgae from foreign microorganisms and excluded interferences induced by contamination. The effect of any investigated factor and interaction between factors was determined by analysis of variance of cell concentration data. Results were qualitatively identical to those reported in Fig. 2 confirming the reliability of biomass concentration data reported in Fig. 1. Overall biomass production and maximum productivity values obtained for the performed experimental tests are reported in Table 3. Independently of the employed medium (BG11 or TWBM), larger biomass production was found when microalgae were cultivated without OMW. Analysis of variance was also conducted evidencing a statistically significant reduction of maximum productivity in response to the addition of OMW. 3.3. OMW biodegradation Reductions in the concentrations of phenols, reducing sugars and COD were found following microalgal harvesting in media enriched with OMW (Fig. 3). No statistically significant difference was determined between the removals of reducing sugars attained with BG11 and TWBM. With both these two latter media, reducing sugar concentration decreased by about 60%. It can be assumed that consumed reducing sugars were mainly constituted by glucose and fructose. These compounds can easily be metabolized by microalgae and cover most of OMW carbohydrates [12]. Besides glucose and fructose, additional carbohydrates are present in OMW, which can hardly be biodegraded as, for example, pentose [11]. Unlike what was found for reducing sugars, a statistically significant difference was determined between phenol removals attained
309
Fig. 3. Reducing sugars, phenols (as tyrosol equivalents) and COD removed by microalgae in OMW. Results were obtained by cultivation of microalgae in TWBM and BG11 with 9% OMW.
with TWBM and BG11. In particular, phenol removal values around 6% and 22% were found with the media (TWBM + OMW) and (BG11 + TWBM), respectively. COD reduction changed from 36.5 ± 0.5% to 28 ± 4% as TWBM was used in place of the BG11 medium. This difference is however not statistically significant. An independent estimate of the COD reduction was derived by assuming that consumed reducing sugars and phenols (Fig. 3) were exclusively composed of glucose and tyrosol, respectively. In accordance with this latter assumption, 16% and 13% COD reductions were determined for the tests conducted with BG11 and TWBM, respectively. These values are significantly lower than measured COD variations reported in Fig. 2, evidencing that additional reduced substances were removed during cultivation along with carbohydrates and phenols. These additional substances are probably organic acids, which can be biodegraded by microalgae and reach high concentrations in OMW [11,12]. The contribution of organic acids to COD removal was estimated by assuming complete removal of acetic acid, citric acid, lactic acid and succinic acid. OMW concentration values reported in Ref. [12] were employed for these acids. COD removal comparable to actual measured values was found by this approach confirming biodegradation of organic acids.
3.4. Lipid and carbohydrate accumulation No statistically significant difference was found among lipid fractions of microalgae cultivated with the four considered media: an average lipid fraction around 32% (w/w) was invariably found (Fig. 4). A statistically significant positive effect on carbohydrate accumulation was in contrast induced by the addition of OMW. When Scenedesmus sp. was grown in the presence of OMW, stored carbohydrates increased from 34 ± 1% to 43 ± 9% with BG11, and from 21.4 ± 0.8% to 44.0 ± 0.2% with TWBM.
Table 3 Overall biomass production, maximum productivity, kinetic parameters and final concentrations of sugars, phenols and COD reached by Scenedesmus sp. in TWBM and BG11 with and without OMW.
x–x0 (g·L−1) Carbohydrate production (g·L−1) Lipid production (g·L−1) Maximum biomass productivity (g·L−1·d−1) μ (h−1) Cf,phenols (g·L−1)a Cf,reducible sugars (g·L−1)a Cf,COD (g·L−1)a a
TWBM - OMW
TWBM
BG11 – OMW
BG11
0.22 ± 0.05 0.1 ± 0.02 0.09 ± 0.04 0.06 ± 0.01 0.023 ± 0.002 0.42 ± 0.02 0.26 ± 0.03 2.83 ± 0.03
0.559 ± 0.001 0.1196 ± 3·10−4 0.1790 ± 4·10−4 0.11 ± 0.01 0.0272 ± 4·10−4 – – –
0.35 ± 0.09 0.16 ± 0.04 0.11 ± 0.03 0.08 ± 0.01 0.0249 ± 4·10−4 0.347 ± 0.007 0.27 ± 0.01 3.2 ± 0.3
0.512 ± 0.007 0.174 ± 0.002 0.164 ± 0.003 0.120 ± 0.008 0.0181 ± 8·10−4 – – –
Reported concentrations of phenols, reducing sugars and COD are computed for OMW 9% v·v−1.
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in biomass productivity observed in response to the addition of OMW cannot be imputed to the presence of fat matter.
Fig. 4. Comparison of lipid and carbohydrate content in Scenedesmus sp. biomass obtained in the four conditions investigated.
4. Discussion Growth rate values comparable or even larger than those attained in autotrophic cultures were found by cultivation of microalgae in media enriched with OMW (Figs. 1–2 and Table 3). This result is in contrast with data reported by Hodaifa et al. [16,33]. These authors evidenced the achievement of a growth rate with OMW significantly lower than the value attained by autotrophic cultivation in a standard medium. A culture medium realized by the addition of only sterilized OMW to distilled water was employed in the performed experiment. We attribute the revealed contradiction to the addition of nitrates to the culture media employed in the present study. This can be verified by noting that the medium (TWBM + OMW), which ensures growth rates close to those attained in autotrophic cultures, differs from a medium composed only of OMW and water (as the one employed by Hodaifa et al. [16,33]) because of the addition of nitrates and phosphates (used in the preparation of TWBM). On the other hand, phosphates added along with nitrates to TWBM are not expected to influence microalgal growth in (TWBM + OMW) medium because they are already abundant in OMW (Table 2). Therefore, nitrates can be considered responsible for the increased growth rate achieved in the present study by cultivation in media enriched with OMW. It must be remarked that nitrogen is not absent in OMW and can even reach values in such wastewater concentration close to the ones supplied in the present study by the addition of nitrates. OMW nitrogen is however mainly present in peptides and amino acids. Degradation of these compounds is required to generate ammonium which can be exploited to synthesize proteins. However, this imposes the production of extracellular enzymes which is uncommon in algae [34]. This can explain the observed positive effect of inorganic nitrogen on specific growth rate. Results reported in Fig. 1 indicate that the addition of OMW determines the early achievement of stationary phase. This leads to heterotrophic biomass productivity values lower than those attained by autotrophic growth. This result has been previously attributed to the absence of photosynthetic activity and to OMW fat matter adsorbing on microalgal surface and limiting nutrient availability [16,33]. We consider these two latter factors insufficient to explain the arrest of growth at lower biomass concentrations. To verify it, let us consider the following arguments: – Even in the absence of photosynthetic activity, microalgae grown with OMW accumulated a large amount of organic carbon (Fig. 4). This organic carbon could have been employed to derive the energy required for growth. – While evidencing an increase in growth rate, the application of defatted OMW led to biomass productivity values lower than those attained in autotrophic growth [16,33]. Accordingly, the reduction
A further element which may be responsible for the early arrest of microalgal growth is the depletion of nitrogen. However, this must also be discarded because, with the same initial nitrate concentration (0.143 g·L−1), autotrophic tests led to biomass concentration significantly higher than that attained in heterotrophic tests (Fig. 1). Further proof that nitrogen was not limiting growth in heterotrophic cultures is given by the analysis of carbohydrates and lipids (Fig. 4). About 75% dry weight of microalgae cultivated in heterotrophic conditions is composed of lipids and carbohydrates. By assuming that the remaining 25% is constituted by proteins alone (that is an overestimation), nitrogen fraction not larger than 4% would be found. This latter value corresponds, with the attained biomass concentration around 0.3 g·L− 1 (Fig. 1), to a consumption of about 40% of initial nitrogen. In this framework, the fundamental role appears to be played by OMW phenols. Although microalgae can degrade phenols, studies have evidenced that such compounds can induce toxic effects in microalgal cells. The hypothesis of early arrest of growth determined by phenols is supported in the present study by the apparent relationship between phenol consumption and biomass concentration. The consumption of phenols was with the medium (BG11 + OMW) significantly larger than with the medium (TWBM + OMW) (Fig. 3), the larger phenol consumption corresponding to the achievement of increased biomass concentration (Table 3). This suggests that microalgal growth is enhanced as mechanisms lowering phenol concentration are activated. While supporting the hypothesis of inhibition induced by phenols, the previous analysis raises the question of why phenol consumption could be attained in the medium (BG11 + OMW) (about 22% removal) and not with the (TWBM + OMW) medium (about 6% removal). This must necessarily be imputed to differences between the compositions of the BG11 and TWBM media. BG11 contains micronutrients and EDTA which were not added to TWBM, while a significantly larger concentration of Ca2 + ions can be found in TWBM because of the application of tap water. For what concerns micronutrients, iron concentration reached a value of 1.3 mg·L−1 in BG11 while it was not detectable in tap water (b 0.5 mg·L−1). Iron and other metal ions are employed by enzymes as co-factors in the degradation of phenols [35–37]. The absence of micronutrients may therefore explain the negligible consumption of phenols in (TWBM + OMW) medium. Further, micronutrients could be not assimilated by microalgae in the absence of EDTA. This compound can form complexes with metal ions and can thus improve the access of such metals to the cell. Ca2+ concentration in TWBM higher than in BG11 must also be taken into account as the interaction between calcium ions and phenols can influence phenol biodegradability [38]. A further important characteristic of inhibition occurring in heterotrophic cultivation is that it appears after five days of cultivation (Fig. 1). This latter result indicates that toxic effects induced by phenols require a latent time to appear. Even though not influencing the microalgal lipid fraction, the addition of OMW improved accumulation of carbohydrates (Fig. 4). This result allowed for achieving heterotrophic carbohydrate productivity close to those found in autotrophic growth (Table 3) and suggests a possible application of the employed cultivation strategy in bioethanol production. 5. Conclusions The study investigated the cultivation of the microalgal strain Scenedesmus sp. with unsterilized OMW. The main conclusions of the illustrated study can be stated as follows: – Elevated robustness of the selected strain towards the risk of contamination was found suggesting the possibility to successfully
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