Algal Research 11 (2015) 368–374

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Algal Research

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Phototrophic culture of Chlorella sp. using charcoal ash as an inorganicnutrient source

María A. Sandoval R. ⁎, María F. Flores E., Ricardo A. Narváez C., Jesús López-VilladaInstituto Nacional de Eficiencia Energética y Energías Renovables (INER), 6 de Diciembre N33-32, Quito, Ecuador

⁎ Corresponding author.E-mail address: msandovaluio@gmail.com (M.A. Sand

http://dx.doi.org/10.1016/j.algal.2015.07.0082211-9264/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 January 2015Received in revised form 7 June 2015Accepted 11 July 2015Available online xxxx

Keywords:Chlorella sp.AlgaeCultureBiomass ashLeachateTrace metalsNutrientsEcuador

Although several studies have recognized the suitability of employing ashes as a component of a culturemediumor fertilizer, the number of these studies remains limited. The use of biomass ash as a nutrient source for algalculture is an unexplored research topic that should be investigated to analyse the possibility of reducing thecosts associated with commercial microalgae culture.In this study, biomass ash from charcoal was used as a source of nutrients for the cultivation of Chlorella sp., andtwo alternative processes for nutrient supply were studied. First, various culture media containing the leachatestock solution from solid ash at different concentrations were prepared. Second, different amounts of solid ashwere added directly to the culture media. The results indicated that the direct use of biomass ash mixed inwater enables the formation of a more suitable medium compared with Guillard's f/2 medium because itpromotes faster cell growth and higher biomass productivity. The higher biomass productivities were reachedover the same period compared with those achieved with the culture media based on biomass ash leachates.Moreover, the nutrients in the media containing ash leachates are sufficient to maintain cell growth rates andbiomass productivities that are comparable to those achieve with Guillard's f/2 medium.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Oneof the primary objectives of research onmicroalgal biotechnologyis to produce basic commodities in large-scale and cost-effectiveways [1].One of the key issues of algal cultivation is the employment of abundantand low-cost nutrient sources to reduce the production costs. Regardlessof their form in the media, carbon, nitrogen, and phosphorus (macronu-trients) are the threemost important nutrients, and their uptake dependson the environmental conditions, species, nutrient ratios, and growthrates. It is also important to note that algae need other micronutrients,such as, K, Na, Fe, Mg, Ca, B, Cu, Mn, Zn, Mo, Co, V, and Se [1]. Many ofthese micronutrient elements are important in enzyme reactions andfor the biosynthesis of many compounds.

At the bench scale, ordinary culture media are composed of highlypurified and thus expensive chemical compounds. Nevertheless, theuse of these supplies for large-scale cultivation is non-profitable, andthus, available alternatives may represent lower production costs andlower biomass yields. Ashraf et al. [2] suggested the replacement ofpure and expensive nutrientmediawith low-cost commercial fertilizersused in current fish production and agriculture. Nevertheless, it is impor-tant to consider that the manufacturing of these fertilizers is based on anintense use of fossil fuels as a feedstock and energy source [3]. Hence, thisalternative may be adequate in the short term but not in the mid to long

oval R.).

term due to the scarcity and unstable prices of fossil fuels. As a result, therecycling of waste materials as a nutrient source for large-scale algalcultivation may be an interesting option that should be evaluated. Yanget al. [4] demonstrated that the recycling of harvest waste reduces thenutrient usage by 55% and that the use of sea/wastewater as the culturemedia eliminates the need of all of the nutrients with the exception ofphosphate. Fenton et al. [5] showed that the nutrient content of agricul-turally derived organic fertilizers, runoff and drainage waters has the po-tential to facilitate algal biomass growth. In particular, these researchersdemonstrated that the surplus of pig and poultry manures or otherbioproducts from anaerobic digestion are potentially viable sources ofnutrients for algal growth. Skorupskaite et al. [6] investigated several po-tential inexpensive waste materials for microalgae Chlorella sp. biomassproduction rate, including technical glycerol and the liquidwaste fractionof the digestate after biogas production. This research demonstrated thatthe microalgae growth was strongly dependent on the nitrogen concen-tration in the growth medium.

One alternative waste material that should be investigated as anutrient source for algal growth is biomass ash (BA). In one scenario,the increasing number of combustion facilities of natural biomass forenergy generation appears to be one of themain drivers for biofuel pro-motion in many countries worldwide in the near future [7,8,9], and oneof the key issues associated with natural biomass facilities is themanagement of BA. In contrast, BA is a complex inorganic–organic mix-ture with a poly-component, heterogeneous and variable compositionin the solid, liquid and gaseous phases [10]. According to Vasilev et al.

369M.A. Sandoval R. et al. / Algal Research 11 (2015) 368–374

[10], the chemical elements present in BA (in decreasing order of abun-dance) are commonly O N Ca N K N Si NMg N Al N Fe N P NNa N S NMn N Tias well as some Cl, C, H, N and other trace elements, such asB N Au N CdN (Cr,Mn) N Ag N Zn N (Be, Cu, Se) NNi N Rb. These researchersalso state that thewater-soluble elements leached fromBAs are common-ly Cl N S N K N Na N Sr N Ni NMn N Cd N Cr N Zn N Co N Si NMo N Li N (Mg,Pb) N Ca N Cu N Ba N P N Se N Sb N Al N Fe N (Br, Hg) N (As, B, Sn, Ti, V).

These facts indicate that BAmay be a nutrient source for algal cultiva-tion. However, very few studies have evaluated the effects of BA on algalgrowth, andmost of these have focused on the environmental impacts ofusing wood ash in aquatic systems [11,12,13]. Hence, it is clear that theuse of BAas anutrient source for algal cultivationneeds to be investigated,which may allow reducing the costs of algal products in commercialmicroalgal culture and indicate that BA may be a suitable solution to themanagement of ash waste that would otherwise be disposed in landfills.

In this study, we investigated the effects of using charcoal ash as aninorganic nutrient source for the cultivation of Chlorella sp. We firstanalysed the chemical composition of the ash and its leaching properties.We then studied the effects of two alternative nutrient supplies on thegrowth of Chlorella sp. The first alternative nutrient supply involved thepreparation of culture media with a leachate stock solution of solid ashat different concentrations, and the second involved the first addition ofdifferent amounts of solid ash to the culture media. We also comparedthe results obtained with those achieved with Guillard's f/2 as thereference medium.

2. Materials and methods

2.1. Microalgal isolation and initial culture conditions

Chlorella sp. (Chlorophyceae) was isolated fromwastewater collectedfrom a river near Quito, Ecuador. The strain was propagated andmaintained under laboratory conditions in batch culture in Guillard'sf/2 medium [14] with constant air bubbling. The temperature rangewas 25 to 28 °C, the light intensity was 3000 lx with a photoperiodof 12:12 h, and the pH was controlled between 8 and 10 (see Fig. 1).

2.2. Culture medium under phototrophic conditions

2.2.1. Charcoal ash preparationFive hundred grammes of wood charcoal from a local Laurel tree

(Morella pubescens) were placed in a porcelain crucible and burnt for4 h in a Thermolyne FD1535Mmuffle furnace at 575 + 25 °C followingthe procedure described by Sluiter et al. [15]. The ash obtained wasground and sieved to 38 μm.

2.2.2. Charcoal ash thermogravimetric analysis (TGA)Amass of 14.183mg of charcoal ash was introduced into a Shimatzu

TG60Wthermogravimeter set to a constant heating rate of 35 °C/min. Theanalysis was initiated at ambient temperature and terminated at 900 °C.During the analysis, the sample was maintained in a nitrogen-inertatmosphere with a flow rate of 45 mL/min.

2.2.3. Leachate stock solution preparationThe leachate solution (LS) was prepared by adding 1 g of dried

charcoal ash to a litre of ASTM Type II water (ASTM D 1193) followingthe Standard DIN 38414–4 [16].

2.2.4. Culture medium conditionsCulture media from leachates and with solid ashes, Guillard's f/2

medium and a blank medium without nutrients were prepared bytriplicate in 500 mL flasks. Each flask was inoculated with Chlorella sp.strain to obtain a cell density of 1·106 cells mL−1. The inoculum ofnative Chlorella sp. was previously filtered using N° 4 filter paper(pore diameter of 20 to 25 μm) and then washed with Type II water.The culture conditions for all of the media were 25 °C with a constant

air flux of 2 L perminute, awhite light intensity of 3000 lx and a photope-riod of 12:12 h. All of the culturemedia lacked buffer solution, and the pHwas thus measured at the beginning and end of the runs.

2.2.4.1. Guillard's f/2 reference medium. Guillard's f/2 was chosen as thereference medium. Macronutrient, micronutrient and vitamin stocksolutions were prepared using the formulation described in [14,17].Table 1 shows the preparation of the Guillard's f/2 reference medium,including the volumes of inoculum and of the vitamin, macronutrientand micronutrient stock solutions.

2.2.4.2. Leachate medium. Different volumes of the stock leachatesolutionweremixedwith 145mL of the inoculum, 0.5ml of the vitaminstock solution and the necessary volumes of ASTM Type II water toachieve concentrations of 70%, 35% and 18% to prepare the TM1, TM2and TM3 culture media, respectively (see Table 2). A control blankculture was prepared by mixing 145 mL of the inoculum with 354 mLof ASTM Type II water and 0.5 mL of the vitamin solution.

2.2.4.3. Solid ash medium. To prepare the solid ash medium TM4 andTM5, 1 g and 0,5 g of charcoal ash weremixedwith 145ml of inoculum,354 ml of ASTM Type II water and 0,5 ml of vitamin stock solution (seeTable 2).

2.2.5. Charcoal ash, leachate stock solution and final TM4, TM5 culturemedia chemical analyses

The concentrations of several metals in the dried charcoal ash,leachate stock solution, Guillard's stock solutions and the final solutionsof the TM4 and TM5 media after the experiment were determinedthrough inductively coupled plasma mass spectrometry (ICP-MS) usingan Agilent ICP-MS equipment model 7700. The samples were preparedtrough microwave digestion before elemental analysis by ICP-MS [18].In addition, the concentrations of nitrates, sulphates and phosphates inthe leachate stock solutionwerequantified throughmethods ion chroma-tography technique (Waters, LC 10 Ai). The electrical conductivity and pHof the leachate stock solution and Guillard's f/2 medium were measuredwith a multiparameter Edge HI 20X0-20 instrument.

2.3. Evaluation of cell density and biomass productivity

The cell densitywas estimated by counting directly using aMarienfeldNeubauer chamber with a depth of 0.1 mm and an area of 0.0025 mm2.The cell density was estimated during 15 alternate days starting on thethird day.

The kinetics growth curve, generation time (Gt), and specific growthrate (μ) were calculated for all of the batch cultures. The specific growthrate μ (in d−1) and biomass productivity X (in g L−1 d−1)were estimatedusing Eqs. (1) and (2), respectively [19]:

μ ¼ Ln N2=N1ð Þt2−t1ð Þ ð1Þ

X ¼ N2−N1

t2−t1ð Þ ð2Þ

N2 and N1 describe the biomass concentration (in g L−1) at times t2and t1 (in d).

2.4. Statistical analyses

The biomass growth rate data were analysed by ANOVA with an αvalue of 0.05 to compare the results of the different leachate, solid ashand Guillard's f/2 culture stock solution. Tukey's test with a confidencelevel of 95% was applied to the experiments using the Microsoft Excel(2010) programme [20].

Table 1Details of the preparation of the Guillard's f/2 reference medium [26] as well as the initial pH. This reference media was prepared in triplicate.

Culture medium Duration (days) Volume (ml) pH

Inoculum Micronutrient Stock Solution Macronutrient Stock Solution Water Vitamin Stock Solution

Guillard's f/2 (RM) 15 145 ± 0.80 0.5 ± 0.005 0.5 ± 0.005 352 ± 1.13 0.5 ± 0.005 7.5

370 M.A. Sandoval R. et al. / Algal Research 11 (2015) 368–374

3. Results and Discussion

3.1. Ash, leachate and culture media analysis

The burning of 500 g of charcoal resulted in 12.78 g of biomassash with a yield of 2.56%, which is in accordance with the literature[16,21,22]. Table 3 shows the chemical analysis of the ash obtained.The high contents of Si, Al, Na and especially Fe suggest that the charcoalbelong to the S-type biomass with mostly glass, silicates andoxyhydroxides (SiO2 + Al2O3 + Fe2O3 + Na2O + TiO2), accordingto the classification proposed by Vassilev et al. [21]. This biomasstype is very unusual for biomass from wood trees in this classification.However, this chemical composition is very similar to the compositionof the wood ash analysed by Steenary et al. [23] with the exception ofMn. A reasonable explanation for this composition and particularly forthe high content of Fe is that the Morella Pubescens tree usually growsin places with meteorised volcanic soil, which is typical of the Andeanregion of Ecuador. These soils present Acrisols, Alisols and Ferralsolswith high contents of iron oxides, silicates and aluminium [24].Moreover, it is possible that the charcoal was contaminated duringthe manufacturing process.

As expected, the contents of the alkaline metals Na, K, Ca and Mg inthe ash are relatively high. In addition, it should be noted that importantinorganic macronutrients, such as phosphhorous and nitrogen, arepresent at concentrations of 6000 and 622 mg kg−1, respectively.Regarding micronutrient elements, it is important to note that the ashis very rich in biologically active heavy metals that are necessaryfor the growth of microalgae, such as Zn, Mn, Co and Cu, as well as themetals such as Pb and Cr, which may cause inhibition phenomena.

The leachate stock solution presented a conductivity of 1950 μS cm−1

and a pH of 9.3, whereas the conductivity and pH of Guillard's mediumwere 633 μS cm−1 and 7.5. These values indicate that the stock leachingsolution contains more salts than the Guillard's f/2 medium. For thatreason, it would be advisable to complement this experiment with atestmediumwhere the amount of ash ismuch lower due to the represen-tative quantities of metal in ashes, but with nitrate supply with thepurpose of obtaining similar concentrations to Guillard f/2 medium.Table 3 provides the concentrations of some components of the stockleachate solution, Guillard's stock solution (RM), test media (TM1, TM2,and TM3) and Guillard's f/2 reference medium (RM). The most abundantelements in the leachate solution in decreasing order of abundance arethe following: K≈ Ca N Cl N Mg N Na. This finding may be partly causedby the basic pH resulting from the loss of organic acids during biomassburning and the production of soluble Ca, Mg, K and Na oxides, hydrox-ides, carbonates and bicarbonates in BA [6]. The presence of carbonateswas confirmed by TGA analysis (Fig. 2).

Table 2Details of the preparation of the test culture media from charcoal ash leachate and solid charcoprepared in triplicate.

Culture media Duration (days) Solid Ash Weight (g) Volume (m

Inoculum

Test media 1 (TM1): 70% 15 - 145 ± 0.80Test media 2 (TM2): 35% 15 - 145 ± 0.80Test media 3 (TM3): 18% 15 - 145 ± 0.80Test media 4 (TM4) 15 1 ± 0.021 145 ± 0.80Test media 5 (TM5) 15 0.5 ± 0.021 145 ± 0.80Blank (B) 15 - 145 ± 0.80

The TGA profile shows an initial smallmass loss at low temperaturesand a significant mass loss of 1.4 mg at temperatures between 640 °Cand 745 °C. The initial mass loss observed at temperatures lower than200 °C is due to the evaporation of water adsorbed by the ash when itis stored for a period of time after the preparation. Themass loss observedat temperatures greater than 600 °C is caused by the decomposition ofcarbonates of both calcium and potassium into the corresponding oxidesand CO2 [16,22].

Moreover, the leachate solution was found to contain very lowamounts of nitrates (0.40 mg/L). This result is consistent with a studyof leachability of nutrients from biochar [26] which showed that N(in the form of NH4

+ and NO3−) was the least water extractable element

by several orders of magnitude compared to other elements leached(Ca, K,Mg, Mn, P, S, Na). The phosphates contentmay be also importantbut difficult to infer due to the complexity of the sample.

The comparison of the compositions of the leachate and Guillard's f/2stock solutions revealed that the former has higher concentrations of K,Ca, Cl, Mg, Al, Cu and SO4

2−, but amuch lower content of Fe and Co.More-over, the content of nitrates in the leachate stock solution is around a 200times lower than the Guillard's stock solution. Regarding toxic heavymetals, both stock solutions have low contents of Hg, Pb, Cd, and Cr.With respect to Zn and Mo, the analyses indicate concentrations of thesame order. The comparison of the compositions of the Guillard's f/2and the TM1, TM2 and TM3 test media revealed that the former hasmuch lower concentrations of all the metals and some anions such asSO4

2− and Cl−. However, the nitrate content of TM1, TM2 and TM3media is approximately only 3, 1.5 and 0.8 times more than the corre-sponding Guillard's f/2 reference medium. That means that the nitratederived fromcharcoal ash acts as the limiting nutrient and, for that reasonit is necessary to add relatively important amounts of ash to obtain similarconcentrations than those of the Guillard's f/2 medium.

Table 4 provides the results of the analyses of some metals in thefinal solutions of the TM4 and TM5 media. Although the TM4 and TM5solutionswere prepared by diluting 1 and 0.5 g of ash in 500mL, respec-tively, TM4 only presents a slightly higher content of metals in the finalsolution. The comparison of these results with the leachate media TM1,TM2 and TM3 reveals that the contents of Al, Ca, Cd, Cr, Fe, Pb, K, Na andCl in the TM4 and TM5 media are markedly higher.

3.2. Cell density and biomass productivity of chlorella sp.

The average results of Chlorella sp. growth in the different culturemedia described in the methodology are presented in this section.Figs. 3 and 4 and Table 5 show the kinetics behaviour and growthparameters of the different media. As expected, the cell density in theblank (B) decreased after the second day because of the total absence

al ashes. The column on the right indicates the initial pH of the culture. Each culture was

l) pH

Leachate Stock Solution Water Vitamin Stock Solution

354 ± 1.13 0 0.5 ± 0.005 9.2177 ± 1.13 177 ± 1.13 0.5 ± 0.005 8.588.5 ± 1.13 265.5 ± 1.13 0.5 ± 0.005 8.1- 354 ± 1.13 0.5 ± 0.005 8.5- 354 ± 1.13 0.5 ± 0.005 8.0- 354 ± 1.13 0.5 ± 0.005 7.5

Table3

Chem

ical

analyses

ofthesolid

dryash,

ashleacha

testocksolution

andGuilla

rd'sStoc

kSo

lution

s.Te

stmed

ia(TM1,TM

2,an

dTM

3)an

dreferenc

emed

ia(R

M)co

ncen

trations

werecalculated

from

thean

alyses

data

oftheresp

ective

stocksolution

s.

Compo

nent

Charco

alDry

Ash

Leacha

teStoc

kSo

lution

Guilla

rd'sStoc

kSo

lution

sTe

stCu

ltureMed

iaGuilla

rd'sf/2med

ia

(mg/kg

)(m

g/L)

(mg/L)

TM1(m

g/L)

TM2(m

g/L)

TM3(m

g/L)

(mg/L)

Aluminium

7744

±23

230.28

±0.08

b0.02

0.20

±0.06

0.10

±0.03

0.05

±0.01

5b2.0·

10−

5

Cadm

ium

0.60

±0.18

b2.0·

10−

47.0·

10−

4b1.4·

10−

4b7.1·

10−

5b3.5·

10−

57.0·

10−

7±

2·10

−7

Calcium

190,00

0±

57,000

18±

5b0.1

13±

46.4±

1.8

3.2±

0.9

b1.0·

10−

4

Chloride

s-

10±

21.22

±0.3

7.1±

1.4

3.5±

0.7

1.8±

0.4

1.2·

10−

3±

3·10

−4

Chromium

42±

133.4·

10−

3±

1.0·

10−

37·

10−

4±

2·10

−4

2.4·

10−

3±

7·10

−4

1.2·

10−

3±

4·10

−4

6.0·

10−

4±

2·10

−4

7.0·

10−

7±

2.1·

10−

7

Coba

lt11

±3

6·10

−4±

2·10

−4

4.1·

10−

3±

1.2·

10−

34.2·

10−

4±

1.4·

10−

42.1·

10−

4±

7·10

−5

1.1·

10−

4±

4·10

−5

4.1·

10−

6±

1.2·

10−

6

Copp

er14

0±

420.05

±0.02

2.5·

10−

3±

8.0·

10−

40.03

5±

0.01

40.01

8±

7·10

−3

8.9·

10−

3±

4·10

−3

3.6·

10−

6±

8·10

−7

Iron

12,000

±36

00b0.04

1.0±

0.3

b0.02

8b0.01

4b7·

10−

31.0·

10−

3±

3·10

−4

Lead

198±

59b1·

10−

32·

10−

3±

6·10

−4

b7·

10−

4b4·

10−

4b2·

10−

42.0·

10−

6±

6·10

−7

Mag

nesium

15,000

±45

002.6±

0.8

b0.04

1.8±

0.6

0.92

±0.29

0.46

±0.15

b4.0·

10−

5

Man

gane

se52

3±

157

b1·

10−

38.2·

10−

3±

2.5·

10−

3b7.1·

10−

4b3.5·

10−

5b1.8·

10−

48.2·

10−

6±

2.6·

10−

6

Mercu

ryb0.1

b2·

10−

4b2.0·

10−

4b1.4·

10−

47.1·

10−

53.5·

10−

5b2.0·

10−

7

Molyb

denu

m5.9±

1.8

0.04

7±

0.01

40.02

3±

0.00

70.03

3±

0.01

00.01

7±

5·10

−3

8.3·

10−

3±

3·10

−3

2.3·

10−

5±

7·10

−6

Nitrates(total

Nforsolid

ash)

622±

187

0.40

±0.09

90±

200.28

±0.04

0.14

±0.02

0.07

±0.01

0.09

±0.02

Phosph

ates

(total

Pforsolid

ash)

6000

±18

00b5

2.0±

0.7

b3.5

b1.8

b0.89

2.0·

10−

3±

2·10

−4

Potassium

56,000

±16

,800

50±

155.5±

1.7

35±

1118

±5

8.9±

2.7

5.5·

10−

3±

1.8·

10−

3

Silic

on17

50±

524

4.3±

1.3

4.0±

1.2

3.0±

0.9

1.5±

0.5

0.76

±0.24

4.0·

10−

3±

1.2·

10−

3

Sodium

12,000

±36

0011

±3

4.3±

1.3

7.8±

2.1

3.9±

1.1

1.9±

0.5

0.04

3±

2·10

−3

Sulpha

tes(total

Sforsolid

ash)

2906

±87

23.3±

0.7

0.01

8±

0.00

42.3±

0.5

1.2±

0.3

0.58

±0.13

1.8·

10−

5±

4·10

−6

Van

adium

25±

84·

10–3

±1·

10–3

b4·

10−

42.8·

10−

3±

7·10

−4

1.4·

10−

3±

4·10

−4

7.1·

10−

4±

1.8·

10−

4b4.0·

10−

7

Zinc

625±

187

b0.01

8.6·

10–3

±2.6·

10–3

b7.1·

10−

3b3.5·

10−

3b1.8·

10−

38.6·

10−

6±

2.7·

10−

6

371M.A. Sandoval R. et al. / Algal Research 11 (2015) 368–374

of nutrients in this culture medium. According to Fig. 3, it can be affirmedthat the TM1 and TM3 media showed a similar pattern throughout theexperimental period. The test medium TM2 showed a lower cell densityduring the first days compared with TM1 and TM3. Nevertheless, itreached a slightly higher peak at day 13 with a density of 3.85·106 cellsmL−1. Chlorella sp. started to grow in all of thesemedia on day 3, whereasthe growth began earlier in the RM (Guillard's f/2), which achieved a celldensity of approximately 1·106 cells mL−1 greater than the densitiesobtained with the TM1, TM2 and TM3 media up to day 9, when a peakin the cell density of 3.75·106 cells mL−1 was obtained. Afterward, theRM cell density decreased markedly because of the scarcity of nutrients.

As shown in Fig. 4, the TM4 medium prepared with solid ashpresented the highest cell density (13.82·106 cells mL−1) at day 15.The growth curve obtained in the TM5 medium showed that the celldensity obtained with TM5 was equal to almost 40% of that obtainedwith TM4 throughout the experimental period (5.24·106 cells mL−1).The growth in both of these media presented an extended exponentialphase until day 15 but did not reach a peak compared with the growthrates obtained with the other media studied (Figs. 3 and 4). Thecomparison of the TM4 and TM5 media with RM Guillard's f/2 mediumrevealed that RM resulted in a cell density that was 20–30% higher thanthose obtained with TM4 and TM5 up to day 9. A cell density of approx-imately 3.8·106 cells mL−1 was reached with all of the media on day 9,and afterward, the cell density obtained with TM4 increased sharply,the cell density obtained with the TM5 medium showed a smoothgrowth, and the density obtained with RM decreased markedly.

Table 5 shows the specific growth rates (μ), biomass productivities(X) and final pH of the culture media studied. The results clearly showthat the pH of all of the culture media decreased as a result of the acid-ification effect of CO2 dilution caused by air bubbling [14]. ANOVA (α=0.05) of the biomass productivities resulted in a p-value lower than0.0001, indicating the existence of significant differences in the biomassproductivities between the culture media tested. The Tukey's testresults suggest that the best media by far is TM4, followed by TM5,RM and then TM3, TM2 and TM1. The biomass productivities obtainedwith TM1 and TM2 were not significantly different. Both of theseshowed a similar behaviour and achieved a lower cell density of Chlorellasp. comparedwith that obtainedwith the referencemedia RM. Addition-ally, the different biomass productivities obtained with the TM1, TM2and TM3media reveal the low effects on cell growth obtained by chang-ing the metal and macronutrient contents in the test media preparedwith the leachate stock solution. This is due to the excess amount ofmetals available in the solution media in the period of the experiment.As the nitrate acts a limiting nutrient, to observe this effect on TM1,TM2 and TM3 media it would be necessary to extend the experimentfor more days.

One remarkable comparison can be made between the TM1 and TM5culture media. First, a metal concentration of 70% in the leachate stocksolution was found after the addition of 1 g of charcoal ash to 1 L. Thisvalue is equivalent to the leachate obtained by adding 0.35 g of ash to0.5 L of solution. Second, 0.5 g of charcoal ash was added to 0.5 L ofsolution medium. Tukey's test revealed that the biomass productivityobtainedwith the TM5medium is significantly higher than that achievedwith TM1. As shown in Figs. 3 and 4, both culture media showed verysimilar growth until day 7. From day 7 to day 15, TM5 showed a largergrowth, achieving a cell density of 5.24·106 cells mL−1, whereas the celldensity obtainedwith TM1 only reached 3.30·106 cells mL−1. One expla-nation for these results is the larger amount of charcoal ash added in theTM5mediumwith respect to the equivalent ashmass used to prepare theleachate in the TM1 medium. This behaviour indicates that microalgaehad sufficient nutrients in both media up to day 7, but afterward, somenutrients became scarce in TM1 medium, thereby slowing growth. Thisfindingwas not observed with TM5 because the solid ash in this mediumcontinuously supplied the necessary nutrients for microalgal growth.Thus, this finding obtained with the use of ash to supply nutrients formicroalgal development may be an important advantage.

372 M.A. Sandoval R. et al. / Algal Research 11 (2015) 368–374

The major growth rate and biomass productivity of TM4 show thepositive effects of the larger amount of solid ash on Chlorella sp. growth,which leads to our hypothesis that the use of the dilution of 1 g of ash in0.5 L of water with vitamin solution as a nutrient source supplies aricher media for promoting faster cell growth compared with the RM.This suggests that dry ash is able to continuously supply the necessarynutrients during the growth phase and that their direct addition towater is amore efficient nutrient source than the preparation of leachatesolutions. Additionally, it appears that the relatively low levels ofnutrients in the ash leachate-containing media TM1, TM2, and TM3are sufficient to maintain cell growth rates and biomass productivitiescomparable to those achieved with RM.

In principle, the results indicate that to obtain obtained similargrowth rates than Guillard's f/2 medium when using charcoal as nutri-ent source, it is necessary to use relatively larger quantities of charcoalash. One of the more obvious explanations is the low leachability ofnitrates from charcoal ash [26] because of the N is bound in a sparselywater soluble form.Moreover, it is important to consider themulticom-ponent feature of the leachate solutions, which could exert importantsynergistic effects on algae growth. Franklin et al. [25] demonstratedsynergistic and antagonistic toxic effects of Zn, Cu and Cd mixtures onChlorella vulgaris growth depending on the concentrations levels ofeach element. According to Pietrokswa et al. [27], the biosorption ofheavy metals from nutrient media results in reductions in Chlorellavulgaris growth and metabolite level. In addition, Huang et al. [28]demonstrated that cadmium and zinc can stimulate the growth ofboth autotrophic and heterotrophic Chlorella vulgaris. Nevertheless,excessive concentrations of Cd or Zn in the culture media markedlyinhibit algal growth. Ouyang HuiLing et al. [29] showed that there wasno significant inhibition effect under the 0.05 μmol L−1 treatments ofPb, Cr and Cd metals. At 0.5 μmol L−1 the inhibition effect was 25%during the first 24 h for Cd and Pb and 15% with Cr. After that period,the inhibition effectwith this concentration for the threemetals becameweaker and practically was reduced to zero 96 h later. Only whenexposure concentration increased to 5 μmol L−1 biomass were lowerthan the blank. Then we can assume that the presence of Cr, Pb andCd in our cultures will have a weak effect on the growth of the Chlorellasp. in the long term. Furthermore, the solid ash present in the culturemay supply these metals slowly giving algae the opportunity to beadapted to this environment but also may supply some necessarymacronutrients and micronutrients for the development of Chlorellasp. The effect of toxic metals is evident during the first 5 days. Afterthat period, algae begin to growth, showing a strong effect from day10 due to the possible abundant of macronutrients such as nitrates,phosphates and other essential metallic micronutrients. Another studyof Zhang et al. [30] showed that the growth rates of Chlorella vulgaris

Fig. 1. General outline of the units used to perform the experiments. All of the experiments wephotoperiod of 12:12 h.

exposed to Pb(II) for long and short-term (7–24 days and 24–120 h)generally declined with increasing Pb(II) concentration. Compared tothe controls, 50 and 80mg L−1 of Pb(II) exposure significantly inhibitedthe growth of C. vulgaris, while lower level of Pb(II) exposure (1 and5 mg L−1) only resulted in weak influence in all the treated groups.Similar results were found by Xiong et al. [31], where concentrationsof 1 mg L−1 of Pb only lead to less than 5% of inhibition. Although theconcentration of Pb may seem high in the wood ash of our study,the content of Pb in the leachates and our cultures is much lowerthan 1 mg/L, as shown in Tables 3 and 4, which indicates that Pbpresent a weak effect on the Chlorella sp. growth.

Moreover, it is well known that Al may be toxic to the growth ofChlorella sp. in certain conditions. The study of Al toxicity to algae iscomplicated by the chemical properties of Al itself, its low solubilityand the interactions with counter ions such as phosphates, sulphates,chlorides and carbonates [32]. Aluminium forms different species inthe media depending strongly on the pH, temperature and concentra-tions of other ions [32,33]. Although according to some authors theseAl concentrations found in our treatments are high enough to inhibitChlorella growth [32,34,35] others show high tolerance to Al [32,36,37,38,39]. Toxicity data often ranged 2 orders of magnitude at any givenpH, which is likely a result of the widely different methods and taxaused in different studies. Generally, Al ismost toxic to algae under slightlyacidic conditions (pH ~ 6.0) and almost all the studies of the effects of Alon Chlorella growth found in the literature were performed at acidic pH[32]. However, in our study the initial and final pH of the cultures is inthe range 9.0–7.7, conditions in which predominates the aluminateanion [Al(OH)4]−, Al(OH)3 as very insoluble gibbsite and possible smallconcentrations of [Al(OH)5]2− [32,33]. Until now the toxic effects ofaluminate anion on green algae and specifically on Chlorella sp. havenot been studied. One possible explanation of its apparent less toxicityis that the proteins which are part of the cell membranes possesshydroxyl and carboxyl- groups that act as partners for a coordinatebinding of metal cations. With increasing pH the amount of negativecharges increases and the amount of positive charges decreases,what may explain the binding metal cations in the pH range 3–6and the difficulties for the adsorption of [Al(OH)4]− at basic pH. More-over, rapidly exchangeable cations (Na, K, Mg, and especially Ca), whichare relatively abundant in our ash wood cultures, may reduce the toxiceffects of Al [32] through cation competition for surface binding sites onalgae. Also complexation by other dissolved anions as SO4

2− decreasesAl toxicity by reducing the available Al [32].

The chemical analysis shown in Table 4 reveals that charcoal ash andthe leaching stock solution are important sources of magnesium, calcium,potassium, sulphates and chlorides in culture media. These componentsmay be present at markedly lower concentrations or even at trace levels

re conducted at 25 °C with an air flux of 2 L per minute, a light intensity of 3000 lx and a

13.8 mg

640 oC

12.4 mg

745 Co

Fig. 2. TGA profile of the charcoal ash. The red line indicates theweight progress over time,and the green line presents the temperature evolution.

0,0

1,0

2,0

3,0

4,0

5,0

0 2 4 6 8 10 12 14 16

Cel

l Den

sity

(10

6 m

l-1)

Days

TM1 TM2 TM3 RM B

Fig. 3. Kinetic growth curves in the test media TM1, TM2 and TM3, Guillard's ReferenceMedia (RM) and Blank (B).

373M.A. Sandoval R. et al. / Algal Research 11 (2015) 368–374

in the RMmedia. It has beendemonstrated that Ca andMgplay an impor-tant role in the biochemistry of Chlorella sp. growth [14,40,41,42]. Accord-ing to [14], artificial media that contain large quantities of calcium andmagnesiummay have “protective effects” on cells, neutralizing the effectsof some injurious compounds, such as toxins, that can inhibit microalgalgrowth. Finkle and Appleman [40] reported that the cell population inmagnesium cultures of Chlorella vulgaris increased rapidly and that theirreproduction slows down considerably when magnesium becomes un-available. Furthermore, these researchers stated that Ca appears to bean important metal in subcultures of Chlorella vulgaris and that a lack ofdiluted calcium in media causes a reduction in the growth of thesemicroalgae. Moreover, Mandalam et al. [41] demonstrated that Mg is animportant element that should be added to the culturemedia for Chlorellasp. A more recent study [42] revealed that the presence of Mg has a pos-itive effect on algal biomass productivity. High doses of Mg induce a highbiomass productivity because chlorophyll requires one magnesium ionfor each molecule. Moreover, Ca appears to not have significant effectsin the biomass yield. Nevertheless, both of these metals appear to playan important role in the accumulation of lipids in Chlorella vulgaris.

Considering all of these results, it can be affirmed thatwood charcoalash may be a suitable solution for providing these elements to Chlorellasp. cultures, which means that further multidisciplinary investigations,including studies of the chemical and physical characterization of BAs,BA treatment, biomass combustion conditions, synergistic effects ofdifferent elements, optimization of the composition of algae cultures,and environmental impacts, are needed to evaluate whether this prom-ising option may play an important role in the cultivation of algae.

12,5

15,0

l-1)

4. Conclusions

Chemical analyses of the charcoal ash and leachates indicated thatthey contain a high variety of chemical elements. The contents of K,Ca, Mg and Na in the ash and leachate solutions were relatively high.

Table 4Chemical analyses of some metals in the final solutions of the test media TM4 and TM5.

Component Final solution Test culture media

TM4 (mg/L) TM5 (mg/L)

Aluminium 1.20 ± 0.36 0.90 ± 0.27Cadmium 0.01 ± 3·13−3 0.01 ± 3·10−3

Calcium 32.51 ± 9.75 24.38 ± 7.31Chlorides 136.06 ± 40.82 120.84 ± 36.25Chromium 0.01294 ± 3.9·10−3 7.35·10−3 ± 2.2·10−3

Iron 0.14 ± 0.04 0.11 ± 0.03Lead 0.12097 ± 0.04 0.12528 ± 0.04Potassium 96.94 ± 29.08 88.68 ± 26.60Sodium 42.91 ± 12.87 42.88 ± 12.86

Moreover, important inorganic macronutrients, such as phosphatesand nitrates, were found to be present in the leachate solution. However,the nitrate concentration of the leachate stock solution was 200 timeslower than those than those of the Guillard's f/2 stock solution, whichmeans that this important macronutrient acts as a limiting factor, andalso, some metals with possible toxic effects such as Al, Pb, Cr, Cd arepresent in the culture media derived from wood ash. Additionally, therelatively low levels of nutrients in the ash leachate-containing mediaare sufficient to maintain cell growth rates and biomass productivitiescomparable to those obtained with Guillard's f/2 medium. The directaddition of solid ash to the culture media (1 g of solid ash in 500 mL)presented marked increases in algae growth and biomass productivitycompared with those obtained with the leachate-containing culturemedia and Guillard's f/2 medium. Hence, it can be concluded thatwood charcoal ash may be a suitable solution for providing macronutri-ents and micronutrients to Chlorella sp. culture media, which may allowreductions in the costs of algae products used in commercial microalgalculture.

Acknowledgements

The authors would like to thank the research team of Biomass andGeothermal departments from National Institute of Energy Efficiencyand Renewable Energy (INER) for the support during the course ofthis study. In Addition, wewould like to dedicate this work to themem-ory of researcher and friend Dr. Jerko Labus, a personwho participated indiscussions and shared many personal and technical experiences whichinfluenced on the development of our research.

0,0

2,5

5,0

7,5

10,0

0 2 4 6 8 10 12 14 16Days

TM4 TM5 RM B

Cel

l Den

sity

(10

6 m

Fig. 4. Kinetic growth curves in the test media TM4 and TM5, Guillard's Reference Media(RM) and Blank (B).

Table 5Growth rate (μ), biomass productivity (X) and final pH of each test culture medium. The growth rate and biomass productivity were estimated from days 3 to 11.

Culture Media Growth Rate μ (d−1) Biomass Productivity X (g L−1 d−1) Maximum Cell Density (106 cells/ml) Final pH

Test media 1 (TM1) 0.14 (±0.01) 2.96 (±0.15) 3.32 (±0.18) 8.8Test media 2 (TM2) 0.14 (±0.01) 2.84 (±0.04) 3.32 (±0.07) 8.2Test media 3 (TM3) 0.16(±0.01) 3.15(±0.03) 3.53 (±0.18) 7.7Test media 4 (TM4) 0.25 (±0.02) 8.33 (±0.03) 13.93 (±0.11) 8Test media 5 (TM5) 0.17 (±0.02) 4.24 (±0.01) 5.61 (±0.20) 7.7Guillard's Reference Media (RM) 0.09 (±0.01) 3.51 (±0.04) 3.75 (±0.10) 7.2

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References

[1] A. Richmond, Q. Hu (Eds.), Handbook of Microalgal Culture: Biotechnology andApplied Phycology, 2nd editionJohn Wiley & Sons, Ltd., 2013 (ISBN 978-0-470-67389-8).

[2] M. Ashraf, M. Javaid, T. Rashid, M. Ayub, A. Zafar, S. Ali, M. Naeem, Replacement ofExpensive Pure NutritiveMedia with Low Cost Commercial Fertilizers forMass Cultureof Freshwater Algae, Chlorella vulgaris, Int. J. Agric. Biol. 13 (4) (2011) 484–490.

[3] M.C. Johnson, I. Palou-Rivera, E.D. Frank, Energy consumption during the manufactureof nutrients for algae cultivation, Algal Res. 2 (4) (2013) 426–436.

[4] J. Yang, M. Xu, X. Zhang, Q. Hu, M. Sommerfeld, Y. Chen, Life-cycle analysis on biodieselproduction frommicroalgae,Water footprint andnutrients balance, Bioresour. Technol.102 (2011) 159–165.

[5] O. Fenton,D.Ó. Huallacháin, Agricultural nutrient surpluses as potential input sources togrow third generation biomass (microalgae): A review, Algal Res. 1 (2012) 49–56.

[6] V. Skorupskaite, V. Makareviciene, D. Levisauska, Optimization of mixotrophic culti-vation of microalgae Chlorella sp. for biofuel production using response surfacemethodology, Algal Res. 7 (2015) 45–50.

[7] S.V. Vassilev, D. Baxter, L.K. Andersen, C.G. Vassileva, An overview of the compositionand application of biomass ash, Part 2, Potential utilisation, technological and ecologicaladvantages and challenges, Fuel 105 (2013) 19–39.

[8] A.K. James, R.W. Thring, S. Helle, H.S. Ghuman, Ash Management Review- Applicationsof Biomass Bottom Ash, Energy 5 (2012)http://dx.doi.org/10.3390/en5103856 3856-3873.

[9] B.A. Knapp, H. Insam, Recycling of Biomass Ashes – current technologies and futureresearch needs, Springer, 2011 (ISBN 978-3-642-19354-5).

[10] S.V. Vassilev, D. Baxter, L.K. Andersen, C.G. Vassileva, An overviewof the compositionand application of biomass ash. Part 1, Phase–mineral and chemical composition andclassification, Fuel 105 (2013) 40–76.

[11] T. Tulonen, L. Arvola, S. Ollila, Limnological effects of wood ash application to thesubcatchments of boreal, humic lakes, J. Environ. Qual. 31 (2002) 946–953.

[12] N.G.A. Ekelund, K.A. Aronsson, Changes in chlorophyll fluorescence in Euglenagracilis and Chlamydomonas reinhardtii after exposure to wood-ash, Environ. Exp.Bot. 59 (2007) 92–98.

[13] K.A. Aronsson, N.G.A. Ekelund, Effects on motile factors and cell growth of Euglenagracilis after exposure towood ash solution; assessment of toxicity, nutrient availabilityand pH-dependency, Water, Air, and Soil Pollution, 162, Springer 2005, pp. 353–368.

[14] R.A. Andersen, in: R.A. Andersen (Ed.), Algal Culturing Techniques, Academic Press,Burlington, 2005.

[15] A.D. Sluiter, B.R. Hames, R.O. Ruiz, C.J. Scarlata, J.B. Sluiter, D.W. Templeton,Determination of Ash in Biomass: Laboratory Analytical Procedure (LAP), NationalRenewable Energy Laboratory, Golden, CO, 2008 (NREL/TP-510–42622).

[16] DIN, German standard methods for the examination of water, waste andsludge— sludge and sediments, determination of leachability by water, DIN 38414-S4, Deutsches Institut für Normung, Berlin, 1984.

[17] J.A. Berges, D.J. Franklin, P.J. Harrison, Evolution of an artificial seawater media:Improvements in enriched seawater, artificial water over the last two decades, J.Phycol. 37 (2001) 1138–1145.

[18] M. Bettinelli, G. Beone, S. Spezia, C. Baffi, Determination of heavy metals in soils andsediments by microwave-assisted digestion and inductively coupled plasma opticalemission spectrometry analysis, Anal. Chim. Acta 424 (2000) 289.

[19] L. Barsanti, P. Gualtieri, Algae: Anatomy, Biochemistry and Biotechnology, 2nd edi-tion CRC Press, 2014 (ISBN 978–1–439–86732–7).

[20] D.G. Altman, J.M. Bland, Statistics Notes: Comparing several groups using analysis ofvariance, Br. Med. J. 312 (1996) 1472–1473.

[21] S.V. Vassilev, D. Baxter, L.K. Andersen, C.G. Vassileva, An overview of the chemicalcomposition of biomass, Fuel 89 (2010) 913–933.

[22] L. Etitgni, A.G. Campbell, Physical and Chemical Characteristics of Wood Ash,Bioresour. Technol. 37 (1991) 173–178.

[23] B.M. Steenari, K. Karlfeldt Fedje, Addition of kaolin as potassium sorbent in thecombustion of wood fuel – Effects on fly ash properties, Fuel 89 (2010) 2026–2032.

[24] C. Gardi, M. Angelini, S. Barceló, G. Comerma, C.E. Cruz, A. Rojas, A. Jones, P.Krasilnikov, S. Mendonça, L. Brefin, M. Montanarella, O. Ugarte, P. Schad, M.I. VaraRodríguez, R. Vargas (Eds.), Atlas de suelos de América Latina y el Caribe,Comisión Europea - Oficina de Publicaciones de la Unión Europea, Luxembourg,2014 (176 L-2995).

[25] N.M. Franklin, J. Stauber, R.P. Lim, P. Petocz, Toxicity of metal mixtures to a tropicalfreshwater alga (chlorella sp.): the effect of interactions between copper, cadmium,and zinc on metal cell binding and uptake, Environ. Toxicol. Chem. 21 (11) (2002)2412–2422.

[26] C.C. Hollister, J.J. Bisogni, J. Lehmann, Ammonium, Nitrate, and Phosphate Sorptionto and Solute Leaching from Biochars Prepared from Corn Stover (Zea mays L.)and Oak Wood (Quercus spp.), J. Environ. Qual. 1 (2012) 137–144.

[27] A. Piotrowska-Niczyporuk, A. Bajguz, E. Zambrzycka, B. Godlewska-Żyłkiewicz,Phytohormones as regulators of heavy metal biosorption and toxicity in green algaChorella vulgaris (Chlorophyceae), Plant Physiol. Biochem. 52 (2012) 52–65,http://dx.doi.org/10.1016/j.plaphy.2011.11.009.

[28] Z. Huang, L. Li, G. Huang, Q. Yan, B. Shi, X. Xu, Growth-inhibitory and metal-bindingproteins in Chlorella vulgaris exposed to cadmium or zinc, Aquat. Toxicol. 91 (2009)54–61.

[29] H.L. Ouyang, X.Z. Kong, W. He, N. Qin, Q.S. He, Y. Wang, R. Wang, F.L. Xu, Effects offive heavy metals at sub-lethal concentrations on the growth and photosynthesisof Chlorella vulgaris, Chin. Sci. Bull. 57 (25) (2012) 3363–3370.

[30] W. Zhang, B. Xiong, C. Lin, K. Lin, X. Cui, H. Bi, M. Guo,W.Wang, Toxicity assessmentof Chlorella vulgaris and Chlorella protothecoides following exposure to Pb(II),Environ. Toxicol. Pharmacol. 36 (2013) 51–57.

[31] B. Xiong, W. Zhang, C. Lin, K.-F. Lin, M.-J. Guo, W.-L. Wang, X.-H. Cui, H.-S. Bi, B.Wang, Effects of Pb(II) Exposure on Chlorella protothecoides and Chlorella vulgarisGrowth, Malondialdehyde, and Photosynthesis-Related Gene Transcription, Environ.Toxicol. 29 (2013) 1346–1354.

[32] R.W. Gensemer, R.C. Playle, The Bioavailability and Toxicity of Aluminum in AquaticEnvironments, Crit. Rev. Environ. Sci. Technol. 29 (4) (1999) 315–450.

[33] J. Saukkoriipi, Theoretical study of hydrolysis of Aluminum Complexes, Acta Univ.Oulu A554 (2010).

[34] S. Helliwell, G.E. Batley, T.M. Florence, B.G. Lumsden, Speciation and toxicity ofaluminum in a model freshwater, Environ. Technol. Lett. 4 (1983) 141–144.

[35] L. Parent, P.G.C. Campbell, Aluminum bioavailability to the Green Alga ChorellaPyrenoidosa in acidified synthetic soft water, Environ. Toxicol. Chem. 13 (4) (1994)587–598.

[36] C.D. Foy, G.C. Gerloff, Response of Chlorella pyrenoidosa to aluminum and low pH, J.Phycol. 8 (1972) 268–271.

[37] B.R. Folsom, N.A. Popescue, J.M. Wood, Comparative study of aluminum and coppertransport and toxicity in an acid-tolerant freshwater green alga, Environ. Sci.Technol. 20 (1986) 616–620.

[38] L.C. Rai, Y. Husaini, N. Mallick, pH-altered interaction of aluminium and fluoride onnutrient uptake, photosynthesis and other variables of Chlorella vulgaris, Aquat.Toxicol. 42 (1998) 67–84.

[39] J. Lindemann, E. Holtkamp, R. Hermann, The Impact of Aluminium on Green AlgaeIsolated from Two Hydrochemically Different Headwater Streams, Bavaria, Germany,Environ. Pollut. 67 (1990) 61–77.

[40] B.J. Finkle, I.D. Appleman, Theeffect ofmagnesiumconcentrationongrowthof chlorella,Plant Physiol. 28 (1953) 664–673.

[41] R.K. Mandalam, B. Palsson, Elemental balancing of biomass and media compositionenhances growth capacity in high-density Chlorella vulgaris cultures, Biotechnol.Bioeng. 59 (5) (1998) 605–611.

[42] P. Chandra, S.K. Bagchi, N. Mallick, Effects of calcium, magnesium and sodium chloridein enhancing lipid accumulation in two greenmicroalgae, Environ. Technol. 34 (13–14)(2013).