Development of a Culture Medium for Microalgae Production Based on Minimal Processing of Oil Palm Biomass Ash

With the increasing participation of biomass in the world energy matrix, large amounts of ash are produced through combustion, resulting in the need to dispose of this waste to minimize the environmental impact. An alternative is to use ashes as phosphorus supplements in microalgae cultures. The present work describes the development and use of a balanced culture medium based on the minimal processing of oil palm biomass ash to cultivate Arthrospira platensis Paracas, Neochloris oleoabundans UTEX 1185, and Dunaliella salina SAG 184. The acid extraction process of phosphorus (P) was defined by evaluating the following parameters: temperature (20 to 70 °C), acid load (0.01 to 0.03 mols/g of ash) of HNO3, and liquid/solid ratio (50 to 150 mLg−1). The best efficiency of the extraction process was 97%. The use of HNO3 allowed for the production of an extract containing balanced amounts of N and P sources, the BAX medium (Biomass Ash Extract). This medium was efficient for cultivating the three microorganisms studied, reaching biomass concentrations of 2.03, 0.902, and 0.69 g/L or 84%, 82%, and 99% of the control concentrations for A. platensis, N. Oleoabundans, and D. salina, respectively. In a final scaling-up test, A. platensis showed productivity of 0.047 g L−1d−1 in a 120 L tank in a greenhouse. BAX can be an alternative nutrient medium for microalgae cultivation, especially in integration with biomass-fueled biorefineries.


Introduction
Bioenergy participation in the world energy matrix has been growing by 8% yearly since 2006 [1]. It is estimated that by 2050, up to 33-50% of the world's primary energy consumption could be provided by biomass [2,3]. Today, plant biomass used directly as an energy source contributes to 10% of the global energy supply. Part of this use is for cooking and heat, generating a scattered residue. However, much of the biomass is used in industries-16 to 18% in the United States of America, India, and Brazil in 2009 [4]. This industrial use generates large and localized volumes of ash, which must be disposed of. Biomass presents significant advantages mainly because of the reduction of CO 2 emissions and reduced dependence on the use of fossil fuels [5].
The increased use of biomass, mainly by the industrial sector, also increases the amount of residual ash generated. Those wishing to apply ash to soil must consider the composition, which is variable and depends on factors such as soil type and composition, crop and cultivation method, season, and combustion temperature [6][7][8][9]. A possible use for biomass ash, rich in inorganic compounds, is its reintegration into the soil as a fertilizer. However, the residue is nutritionally unbalanced-virtually all the nitrogen is volatilized upon burning, and high concentrations of silicon, calcium, and aluminum are often present. Proper pretreatment is essential: although less toxic than coal ash, biomass ash can cause environmental problems [9].  Recovered, phosphorus can be used as a mineral nutrient for cultivating microalgae and cyanobacteria, since this is one of the essential elements in the cell composition [34,35]. On-site recovery also gives an alternative to value addition and zero-waste development in biorefineries, including a microalgal cultivation step and producing algal biomass rich in proteins, pigments, and even energetic and dietary lipids. Thus, the study's objective is to explore the use of nutrients present in minimally processed palm fiber ash to cultivate the cyanobacteria Arthospira platensis and the microalgae Neochloris oleoabundans and Dunaliella salina, with a focus on phosphorus recovery through treatment with nitric acid, thus developing a nitrogen-to-phosphorus balanced culture media based on minimal processing of biomass ash from the palm oil industry.

Oil Palm Ash
Ash from oil palm was obtained from BioPalma S.A., located in Moju, PA, Northern region of Brazil. Oil palm ash was collected and sieved to remove non-calcined materials, such as fibers. The granulometry distribution ( Figure 1) was determined using a 6-screens lab sifter (Bertel VP-01, Lazanjeiras, SP, Brazil). Ashes used for extractions were homogenized lightly in a mortar. To remove the moisture eventually adsorbed, the ash was dried in a laboratory oven (Fanem Orion 502, Guarulhos, SP, Brazil) at 85 • C for 15 h.
The residue was analyzed by X-ray fluorescence spectrometry (FRX) ( Table 2). The equipment used was an Axios Max (Malvern PANalytical, Almelo, Netherlands). The results were interpreted using the software SuperQ 5l ® , from the same manufacturer.  The residue was analyzed by X-ray fluorescence spectrometry (FRX) ( Table 2). The equipment used was an Axios Max (Malvern PANalytical, Almelo, Netherlands). The results were interpreted using the software SuperQ 5l ® , from the same manufacturer.

Nutrient Extraction from Ash
Ashes were leached to extract phosphate. A 3 3 experimental design was used, based on conditions described in the literature for of acid extraction of phosphate from diverse

Nutrient Extraction from Ash
Ashes were leached to extract phosphate. A 3 3 experimental design was used, based on conditions described in the literature for of acid extraction of phosphate from diverse ash materials [36][37][38][39][40][41][42]. The independent variables were temperature, mols of acid (as nitric acid), and Liquid/Solid Ratio (L/S). In all experiments, 1 g of ash was mixed with the acid and an adequate amount of Milli-Q water to reach the final intended volume. The levels of the variables are shown in Table 3. The contact time was defined as 24 h, previously defined as enough time for reaching quasi-equilibrium. After extraction, the mixtures were filtered through nitrocellulose membranes with 0.45 µm pore size to separate the remaining ash from the extract. The extracts were analyzed for the quantification of phosphate.

Kinetics of Phosphate Extraction
The extraction kinetics of phosphate was evaluated to determine the minimum contact time necessary for extraction. The extraction was performed for 30 h, using the mildest conditions of the initial factorial design (lower temperature and acid concentration, i.e., 20 • C; 0.01 moles of HNO 3 ; L/S 150 mL g −1 , periodic stirring). Samples were periodically collected in 2 mL Eppendorf tubes, centrifuged at 10,000 rpm for 5 min, and the supernatant was evaluated for phosphate concentration.

Microorganisms and Culture Media
Three microorganisms suitable for cultivation on a large scale were used as models for studying ash extracts as culture media: the cyanobacterium Arthrospira platensis strain Paracas and the microalgae Neochloris oleoabundans UTEX 1185 and Dunaliella salina SAG 184.8.
Inoculum and control culture media: The inoculum of Arthrospira platensis was routinely maintained by successive culturing in Zarrouk medium [43], with initial pH 9.5 and at 30 • C. The inoculum of Neochloris oleoabundans was routinely maintained by successive cultures in BG11 medium [44], with initial pH 7.5 and at 25 • C. The inoculum of Dunaliella salina was routinely maintained by successive culturing in Modified Johnson's Medium (MJM) [45], adjusted to pH 7.1 and at 25 • C. The three cultures were maintained in 3 L Erlenmeyer flasks aerated with 1 vvm of air and irradiation of 25 µmol photons m −2 s −1 . Control cultures for comparison with extracts had the same compositions and conditions, except for aeration-which was by diffusion, with daily manual homogenization.
BAX (Biomass Ash Extract) media: To evaluate the suitability of oil palm ash extracts as a nutrient source for microalgae growth, the extracts were diluted to match the control media concentration. The conditions for extraction were selected among the experiments that showed the highest phosphate solubilization efficiency in the factorial design. Contact time was 24 h. The supernatant (extract) was filtered through a nitrocellulose membrane with 0.45 µm pore size. The extract was separated into three fractions and diluted to match the theoretical usable concentration of P in each Control medium (Zarrouk, MJM, and BG11), respecting the Redfield ratio of 16 mols of nitrogen for each mol of phosphorus. The pH was adjusted with 10 M NaOH to match the initial pH in control cultures. Additionally, 87.7 g L −1 NaCl was added for Dunaliella salina, specifically to maintain adequate salinity. Culture volume was 500 mL (450 mL culture medium + 50 mL inoculum) in 1 L Erlenmeyer flasks, with aeration by diffusion and gentle manual shaking every 24 h. Initial concentrations were 0.09 g L −1 for both A. platensis and N. oleoabundans and 0.13 g L −1 for D. salina. Light intensity was 25 µmol photons m −2 s −1 , with continuous light, and constant temperature of 25 • C for Neochloris oleoabundans, Dunaliella salina, and 30 • C (±5 • C) for Arthrospira platensis. Each control and treatment were conducted in triplicate.

Analytical Methods
Phosphate was determined using a colorimetric assay with Malachite Green (Millipore Sigma, St. Louis, MI, USA) [46]. All tests were performed using genuine triplicates.

Statistical Analysis
Experimental design planning and analysis were performed using the software Statistica ® V. 7.0. Significant effects at the level of 5% confidence were considered through analysis of variance (ANOVA). All figures were prepared using MS-Excel ® V. 16.0.

Results and Discussion
Preliminary tests using raw ash added with nitrogen sources showed poor microalgal growth, presumably because of self-shading caused by insoluble, suspended particulate material and poor nutrient leaching at culture pH. To evaluate the suitability of culturing microalgae using nutrients from ash, we first evaluated and optimized the extraction, then tested extracts as the base for culture media, and finally chose conditions for scaling up.

Phosphate Extraction
It is well known that lower pH promotes more significant dissolution of P from ashes [36,38,41,51,52]. Typically, strong acids such as H 2 SO 4 and HCl are used to dissolve phosphorus due to their low cost [36,38,41,52,53]. In this investigation, we used nitric acid: it is a strong acid, forms only soluble salts, and it is suitable as a nitrogen source for microalgal cultures, which would have to be added downstream if another acid were used as extractant. The temperature and liquid/solid ratio used were based on the literature, while the amount of acid was calculated based on the amount of phosphate theoretically extractable to give extracts respecting the Redfield ratio [54,55]. Table 4 shows the experimental conditions and results for a 3 3 experimental design. The amount of HNO 3 chosen was defined according to the foreseen formulation of culture media. Absolute amounts from 0.01 to 0.03 mols acid g −1 ash were estimated to reach the ideal N:P ratio. The acid concentrations ranged from 0.067 to 0.6 mol L −1 . This reasoning made it possible to evaluate the L/S ratio without any conflict between variables simultaneously. An 8% efficiency gain in dissolution between 0.01 and 0.02 mol was observed, but a slight increase of 2% from 0.02 to 0.03 mols of HNO 3 . With the L/S studied (50/1, 100/1, and 150/1), 0.02 and 0.03 mols of HNO 3 presented better results. An analysis of the concentration showed that the highest initial acid concentrations did not necessarily give the best dissolution efficiencies: stoichiometry and temperature are more important.
Analyzing temperature, acid addition, and L/S ratio as variables influencing phosphorus extraction, the temperature was the variable that showed the most significant influence on the dissolution, followed by the number of mols, while L/S was not significant at p = 5%. The increase in temperature from 20 to 45 • C promoted an increase of only 2% in the extraction, which may not justify the energy expenditure of a process where average temperatures can be maintained around 20 • C ( Figure 2). However, from 20 to 70 • C, there is an increase of 10% in the dissolution efficiency of P. The increase in temperature and acidity increases solubility of phosphorus from palm biomass ash, similarly to what was is described in the literature for sludge and biomass ashes [36,51].  The dissolution of P 2 O 5 varied by 40%. The lowest P dissolution occurred under pH 1.58, while the highest efficiency, 97% (Table 4), was at pH 1.12. Extraction yields near to 100% were reported at pH 1.0 using H 2 SO 4 to treat sewage sludge ash [38], with a far lower recovery (40%) using 0.6M HNO 3 , however, with an L/S ratio of 5.0, which is 10 to 30 times lower than what was used in this present study. In another study with sewage sludge ash, 0.0075 mol of H 2 SO 4 in 150 mL g −1 and 0.015 mol of HCl in 150 mL g −1 gave satisfactory results, close to 100% phosphate dissolution [36]. A similar result was only obtained in the present study at 70 • C with 0.02 mol of HNO 3 . This difference may have been due to the intermittent agitation: ash sedimentation may have reduced contact with the liquid. The extraction efficiency also likely varies for different materials because of different compositions [41] and acid neutralization by alkali oxides in the ashes.
The L/S ratio showed no significant influence (at P = 5%) on the dissolution of P at the levels tested, presenting a slightly negative variation of 1.8% from 50 to 150 mL g −1 .
That could be explained not because the volume does not matter, but because all chemical species formed are soluble at the level tested, and that acid-to-ash stoichiometry and the consequent final pH is critical for this dissolution.

Kinetics of Extraction
The extraction efficiency was plotted against contact time for 30 h to evaluate the dissolution kinetics. The mildest extraction conditions were chosen: 20 • C; 0.01 mols of HNO 3 ; 150 mL g −1 . P dissolution is highly dependent on the contact time [36,52] and increases with time as an asymptotic curve (Figure 3). Only 15 min was enough to dissolve 45% of the phosphate, but 8 h were needed to reach the equilibrium concentration (an efficiency of 60%). The relatively fast extraction of phosphorus may be due to its presence as soluble salts in the ashes [56] and the small particle size [41]. The L/S ratio showed no significant influence (at P = 5%) on the dissolution of P at the levels tested, presenting a slightly negative variation of 1.8% from 50 to 150 mLg −1 . That could be explained not because the volume does not matter, but because all chemical species formed are soluble at the level tested, and that acid-to-ash stoichiometry and the consequent final pH is critical for this dissolution.

Kinetics of Extraction
The extraction efficiency was plotted against contact time for 30 h to evaluate the dissolution kinetics. The mildest extraction conditions were chosen: 20 °C; 0.01 mols of HNO3; 150 mLg −1 . P dissolution is highly dependent on the contact time [36,52] and increases with time as an asymptotic curve (Figure 3). Only 15 min was enough to dissolve 45% of the phosphate, but 8 h were needed to reach the equilibrium concentration (an efficiency of 60%). The relatively fast extraction of phosphorus may be due to its presence as soluble salts in the ashes [56] and the small particle size [41].

Cultivation of A. platensis, N. oleoabundans, and D. salina on BAX Medium
Both nitrogen and phosphorus must be present in microalgae culture media. Although H2SO4 and HCl are more common for phosphate dissolution, the use of HNO3 as extractant acid allowed the supplementation of adequate amounts of nitrogen to cultivate cyanobacteria and microalgae. In balanced growth conditions, microalgae biomass has an average molar proportion of 106:16:1 (C:N:P) on its composition [34], although deviations exist [55,57]. After the extraction of P, it was examined which conditions have the N:P ratio closest to 16 (Figure 4).
Values above 16 mean nitrogen excess and below 16 mean phosphorus excess. Besides presenting the best extraction of P, Condition 4 presented the best ratio for cultivation: 16.9 is near the Redfield ratio with a slight excess of nitrogen, recovering 97% of the P extracted from oil palm ash.

Cultivation of A. platensis, N. oleoabundans, and D. salina on BAX Medium
Both nitrogen and phosphorus must be present in microalgae culture media. Although H 2 SO 4 and HCl are more common for phosphate dissolution, the use of HNO 3 as extractant acid allowed the supplementation of adequate amounts of nitrogen to cultivate cyanobacteria and microalgae. In balanced growth conditions, microalgae biomass has an average molar proportion of 106:16:1 (C:N:P) on its composition [34], although deviations exist [55,57]. After the extraction of P, it was examined which conditions have the N:P ratio closest to 16 (Figure 4). After adjusting the pH, BAX was diluted 4.3, 34.3, and 30.6 times to match the theoretical usable concentration of P in each control medium (Zarrouk, BG11, and MJM, respectively), respecting the Redfield ratio. (Figure 5) shows the biomass production of A. platensis, N. oleoabundans, and D. salina in the BAX medium.
A. platensis: At the beginning of the cultivation ( Figure 5A), both control (Zarrouk medium) and BAX were similar. The cyanobacteria initially had a low growth rate, probably due to the low initial cell concentration, of 0.09 gL −1 . After 18 days, BAX cultivation growth started to slow down. The difference between control and BAX at this point is noticeable. On day 31, BAX cultivation reached 82% of the control biomass final concentration. Growth of Arthrospira sp. using POME (palm oil mill effluent) was observed in N:P ratios of 2.7:1 and 2.6:1 [58], while Zarrouk had an N:P ratio of 10:1. It is possible that A. platensis does not need an N:P ratio of 16:1, and the excess of nitrate in the BAX medium may have influenced the growth from day 18, promoting a slight reduction in biomass [59]. However, a hypothesis has been raised that the main reason for better growth in the Zarrouk medium is its initially large concentration of CO2 as sodium bicarbonate.
N. oleoabundans: From day 4 to 8, the BAX culture exhibited a slightly reduced growth compared to the control (BG11 medium), probably due to the process of acclimatizing the cells to the new medium. After day 12, N. oleoabundans elevated the growth rate and finished the 31 days of culture with 0.9 gL −1 , while the control had 1 gL −1 ( Figure 5B). Kinetics of cyanobacterial and microalgal growth was examined with poultry litter ash and showed better results when the N source was only ammonium [60]. However, the authors reported that 2.5 gL −1 NaNO3 was added to prevent nitrogen limitation, 100 mgL −1 Na2-EDTA was added as a chelating agent, and cultures had aeration of 2 L of air.min −1 and luminous intensity of 121.5 μmol photons m −2 s −1 , far superior than the diffusion aeration and 27 μmol photons.m −2 S −1 used in the present study.
D. salina: ( Figure 5C) shows that this microalga had the best growth in the BAX medium compared to the control (MJM medium). The cultures had similar growth since day 0, reaching final concentrations of 0.69 and 0.70 gL −1 , respectively.
For all microorganisms, 27 to 31 days of cultivation, without agitation or forced aeration, was necessary to evaluate the behavior of long-term cultures. All considered, the biomass production and growth rate were comparable in BAX and control media, and to the authors' best knowledge, there are no published data of oil palm Ash used to grow cyanobacteria and microalgae. The results were satisfactory and proved that phosphorus in the ashes could be used to grow cyanobacteria and microalgae at rates close to or equivalent to widely popular media. Values above 16 mean nitrogen excess and below 16 mean phosphorus excess. Besides presenting the best extraction of P, Condition 4 presented the best ratio for cultivation: 16.9 is near the Redfield ratio with a slight excess of nitrogen, recovering 97% of the P extracted from oil palm ash.
After adjusting the pH, BAX was diluted 4.3, 34.3, and 30.6 times to match the theoretical usable concentration of P in each control medium (Zarrouk, BG11, and MJM, respectively), respecting the Redfield ratio. (Figure 5) shows the biomass production of A. platensis, N. oleoabundans, and D. salina in the BAX medium.
A. platensis: At the beginning of the cultivation ( Figure 5A), both control (Zarrouk medium) and BAX were similar. The cyanobacteria initially had a low growth rate, probably due to the low initial cell concentration, of 0.09 g L −1 . After 18 days, BAX cultivation growth started to slow down. The difference between control and BAX at this point is noticeable. On day 31, BAX cultivation reached 82% of the control biomass final concentration. Growth of Arthrospira sp. using POME (palm oil mill effluent) was observed in N:P ratios of 2.7:1 and 2.6:1 [58], while Zarrouk had an N:P ratio of 10:1. It is possible that A. platensis does not need an N:P ratio of 16:1, and the excess of nitrate in the BAX medium may have influenced the growth from day 18, promoting a slight reduction in biomass [59]. However, a hypothesis has been raised that the main reason for better growth in the Zarrouk medium is its initially large concentration of CO 2 as sodium bicarbonate.
N. oleoabundans: From day 4 to 8, the BAX culture exhibited a slightly reduced growth compared to the control (BG11 medium), probably due to the process of acclimatizing the cells to the new medium. After day 12, N. oleoabundans elevated the growth rate and finished the 31 days of culture with 0.9 g L −1 , while the control had 1 g L −1 ( Figure 5B). Kinetics of cyanobacterial and microalgal growth was examined with poultry litter ash and showed better results when the N source was only ammonium [60]. However, the authors reported that 2.5 g L −1 NaNO 3 was added to prevent nitrogen limitation, 100 mg L −1 Na 2 -EDTA was added as a chelating agent, and cultures had aeration of 2 L of air min −1 and luminous intensity of 121.5 µmol photons m −2 s −1 , far superior than the diffusion aeration and 27 µmol photons m −2 S −1 used in the present study.
D. salina: ( Figure 5C) shows that this microalga had the best growth in the BAX medium compared to the control (MJM medium). The cultures had similar growth since day 0, reaching final concentrations of 0.69 and 0.70 g L −1 , respectively.  For all microorganisms, 27 to 31 days of cultivation, without agitation or forced aeration, was necessary to evaluate the behavior of long-term cultures. All considered, the biomass production and growth rate were comparable in BAX and control media, and to the authors' best knowledge, there are no published data of oil palm Ash used to grow cyanobacteria and microalgae. The results were satisfactory and proved that phosphorus in the ashes could be used to grow cyanobacteria and microalgae at rates close to or equivalent to widely popular media.

Scaling Up
The scaleup (120 L, open tank, greenhouse) was carried out to verify growth replicability in large scale, and also two hypotheses: (1) to verify if the aeration would compensate the limitation of the growth of the microalga cultivated in BAX medium when compared with the Zarrouk medium (limitation observed in the laboratory), and (2) because of the ability of Arthorspira to basify the medium to higher values (pH 11), to verify the possibility of reduced growth due to reduced availability of phosphate because of alkaline precipitation as Ca or Mg salts.
In growing on a larger scale (Figure 6), the growth pattern was similar to that observed in the laboratory, but the initial cell concentration was higher (0.22 g L −1 ). After 25 days of cultivation, the concentration obtained was 1.40 g L −1 and the productivity 0.0472 g L −1 d −1 , both similar to the laboratory scale culture in the same period. At 31 days of culture, the final cellular concentration observed was 1.6 g L −1 , corresponding to 80% of the value obtained in the culture on the laboratory scale. The difference of 20% in the cellular concentration between the scales evaluated can be attributed to natural temperature variability from 22 to 36.7 • C, with an average of 36.7 • C, which may have caused a decrease in culture growth-Arthorspira requires temperatures between 30 and 38 • C for optimal growth [61]. Another critical factor is that the cultivation on an enlarged scale was conditioned to a natural photoperiod of 13 h/11 h, unlike laboratory cultures where irradiation was constant during the 31 days.

Scaling Up
The scaleup (120 L, open tank, greenhouse) was carried out to verify growth replicability in large scale, and also two hypotheses: (1) to verify if the aeration would compensate the limitation of the growth of the microalga cultivated in BAX medium when compared with the Zarrouk medium (limitation observed in the laboratory), and (2) because of the ability of Arthorspira to basify the medium to higher values (pH 11), to verify the possibility of reduced growth due to reduced availability of phosphate because of alkaline precipitation as Ca or Mg salts.
In growing on a larger scale (Figure 6), the growth pattern was similar to that observed in the laboratory, but the initial cell concentration was higher (0.22 gL −1 ). After 25 days of cultivation, the concentration obtained was 1.40 gL −1 and the productivity 0.0472 gL −1 d −1 , both similar to the laboratory scale culture in the same period. At 31 days of culture, the final cellular concentration observed was 1.6 gL −1 , corresponding to 80% of the value obtained in the culture on the laboratory scale. The difference of 20% in the cellular concentration between the scales evaluated can be attributed to natural temperature variability from 22 to 36.7 °C, with an average of 36.7 °C, which may have caused a decrease in culture growth-Arthorspira requires temperatures between 30 and 38 °C for optimal growth [61]. Another critical factor is that the cultivation on an enlarged scale was conditioned to a natural photoperiod of 13 h/11 h, unlike laboratory cultures where irradiation was constant during the 31 days.

Practical Applications and Future Research
Microalgae are intensively researched as potential food, feed and bioenergy sources. However, one of the drawbacks of large-scale cultures of microalgae is the requirement for nutrients, which increases costs and makes only a few biomass products commercially viable at the moment. The successful cultivation of three important microalgae in ashextract based culture media (BAX) shows that commercial fertilizers can be substituted as nutrient sources for microalgae, and that the judicious use of nitric acid is adequate to

Practical Applications and Future Research
Microalgae are intensively researched as potential food, feed and bioenergy sources. However, one of the drawbacks of large-scale cultures of microalgae is the requirement for nutrients, which increases costs and makes only a few biomass products commercially viable at the moment. The successful cultivation of three important microalgae in ashextract based culture media (BAX) shows that commercial fertilizers can be substituted as nutrient sources for microalgae, and that the judicious use of nitric acid is adequate to enhance extraction and provide a nitrogen source. BAX could be prepared using other acids, such as sulfuric acid; however, a nitrogen source would have to be added tom complete a culture medium. The use of ash increases the circularity of processes, recovering phosphorus; the high yield shows that a similar process could also be used for recovery of phosphorus for other uses.
The BAX medium is simple to prepare, requiring only pH correction and, for marine microalgae, salt addition. It recycles phosphorus and other mineralized nutrients otherwise lost in biomass ash wastes. However, there are several aspects than can be improved; the three most important are (i) Scale-up was similar to laboratory cultures, but with modest productivity. It is possible that warmer temperatures lead to better growth. Repeated batches in a long running experiment will also show if some nutrient lacking, e.g., sodium for Spirulina, or in excess such as iron (usually present in low concentrations in synthetic media) limit the growth or affect the composition. (ii) Oil palm ash has a high concentration of potassium, a most needed element in plant fertilizers, but not important enough for microalgae growth. Fractionation of palm ash, e.g., through selective or sequential extraction could make even better use of this residue and maybe integrate it to palm production, reducing the need for fertilizer. (iii) Large-scale processing of palm oil ash would require reactors, pumps, and filters.
Even if the equipment is simple compared to the large-scale equipment used in palm processing, it involves a capital cost that would have to be compensated by the income from biomass products. This requires a process simulation and sensitivity analysis to indicate if microalgae production, although technically feasible, can be profitable in its biorefinery integration with palm processing.

Conclusions
This proof-of-concept study proved that minimally processed palm ash could be used in microalgae production. Because liquid media and nitrate as nitrogen source are required, it is sufficient to treat the ash with nitric acid. Phosphorus dissolution by HNO 3 presented satisfactory results at a temperature of 70 • C and an HNO 3 concentration of 0.2 M (a mass proportion of 1.26:1 HNO 3 :ash). In these conditions, up to 97% of the phosphorus was dissolved after 24 h. Lower temperatures (20 • C) are enough to extract about 72% of the phosphate, and 8 h was shown to be enough to reach equilibrium in the mildest conditions. The use of the BAX medium was shown to be efficient for the three microorganisms studied, giving biomass production and productivity similar to control cultures performed in synthetic media, albeit with room for improvement. A scaled-up, 120 L cultivation of A. platensis conducted in a greenhouse confirmed the feasibility of this residue use approach.