Bioresource Technology 135 (2013) 598–603

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Treatment of agro-industrial wastewater using microalgae–bacteria consortium

combined with anaerobic digestion of the produced biomass
D. Hernández a, B. Riaño a, M. Coca b, M.C. García-González a,⇑
a
Agricultural Technological Institute of Castilla y León, Ctra. Burgos, km. 119, 47071 Valladolid, Spain
b
Department of Chemical Engineering and Environmental Technology, University of Valladolid, C/Doctor Mergilina s/n, 47011 Valladolid, Spain

h i g h l i g h t s

" Two agroindustrial wastewaters are used as culture medium for microalgae production.
" Phosphorous limitation triggers lipid accumulation in produced biomass.
" Anaerobic batch experiments are carried out using the produced biomass.
" Lower TCOD/VS ratio (0.5) promotes higher methane yield.
" The higher lipid content (30%), the higher methane yield.

a r t i c l e i n f o a b s t r a c t

Article history: Two combined processes were studied in order to produce second generation biofuels: microalgae bio-
Available online 23 September 2012 mass production and its further use to produce biogas. Two 5 L photobioreactors for treating wastewater
from a potato processing industry (from now on RPP) and from a treated liquid fraction of pig manure
Keywords: (from now on RTE) were inoculated with Chlorella sorokiniana and aerobic bacteria at 24 ± 2.7 °C and
Biofuels 6000 lux for 12 h per day of light supply. The maximum biomass growth was obtained for RTE wastewa-
Agro-industrial wastewater ter, with 26.30 mg dry weight L1 d1. Regarding macromolecular composition of collected biomass, lipid
Algal-biomass
concentration reached 30.20% in RPP and 4.30% in RTE. Anaerobic digestion results showed that methane
Methane production
yield was highly influenced by substrate/inoculum ratio and by lipids concentration of the biomass, with
a maximum methane yield of 518 mL CH4 g COD1added using biomass with a lipid content of 30% and a
substrate/inoculum ratio of 0.5.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction as methane, is the optimal strategy from the energetic and eco-
nomic point of view (IEA, 2010).
The potential of microalgae as an alternative to biofuels is cur- The first study on energetic recovery from algal biomass was
rently subjected to strong research (Sialve et al., 2009). In fact, al- published by Golueke et al. (1957), in which the energetic recovery
gae have a high number of potential advantages compared to was carried out throughout anaerobic digestion of the biomass.
higher plants: (1) it is estimated that the production of algae is This research effort was reintroduced in the 1970s and 1980s
ten-fold higher than those of higher plants; (2) algae growth is due to the first global energy crisis, and nowadays there is a re-
independent from arable lands, attenuating food and feed compe- newed interest on anaerobic digestion motivated again by the ac-
tition (Rittman, 2008; Stephens et al., 2010); (3) algae biomass is tual fuel crisis and the ability to treat and to convert a wide range
rich in lipids, proteins and starch, which could be converted into of organic wastes into renewable energy, including microalgae
energy using thermochemical processes, biochemical processes biomass.
and esterification of fatty acids to produce biodiesel (Markou and However, microalgae production requires high amount of nutri-
Georgakakis, 2011). Products obtained through these processes ents, for which environmental and economic impact may not be
can be considered ‘‘first generation’’ biofuels. However, the use of suitable (Halleux et al., 2008; Sialve et al., 2009). One alternative
the resulting biomass to obtain ‘‘second generation’’ biofuels such to synthetic culture media is to use wastewaters, especially those
derived from agro-industrial facilities which usually present high
nutrient concentration (Markou and Georgakakis, 2011). In this
⇑ Corresponding author. Tel.: +34 983 317389.
sense, microalgal–bacterial systems for agro-industrial wastewater
E-mail addresses: [email protected] (D. Hernández), [email protected] (M.C.
García-González). treatment have been gaining special attention in last years. In

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.09.029
 D. Hernández et al. / Bioresource Technology 135 (2013) 598–603 599

these systems, microalgae produce oxygen during photosynthesis mechanically and stored at 4 °C for further use. Chemical charac-
that is used by bacteria metabolism whereas bacteria release CO2 terization of TE and PP is shown in Table 1.
needed for microalgae growth. Microalgal–bacterial systems for
wastewater treatment avoid the external oxygen supplementation 2.1.3. Experimental set-up
compared to conventional processes, allow nutrients recovery into The experimental set-up consisted in two photobioreactors,
biomass and reduce CO2 emissions to the atmosphere by its micro- which are open to the atmosphere for a treated liquid fraction of
algae use contributing to CO2 mitigation (Molinuevo-Salces et al., pig manure (RTE) and for a wastewater from a potato processing
2010). Therefore, the re-use of this kind of substrates can improve industry (RPP), with a total working volume of 5 L (17 cm wide,
the feasibility to produce microalgae biomass for its further valori- 30 cm long, 10 cm high). Each photobioreactor was illuminated
zation, like anaerobic digestion (González-Fernández et al., 2011). using four fluorescent lamps at 6000 lux (Philips 50 W) for 12 h
The aim of the study herein was to evaluate the integrated sys- per day. The lighting of the photobioreactors also provided heating
tem of combining a microalgal–bacterial system for wastewater for the cultivation medium. The average temperature was
treatment with anaerobic digestion of the produced biomass. For 24.1 ± 2.7 °C. The cultures were gently agitated with magnetic stir-
this purpose, two agro-industrial wastewaters (treated liquid frac- rers at 60 rpm. The volume was daily checked and the water lost
tion of pig manure and potato processing wastewater) were se- due to evaporation was corrected by adding distilled water (lower
lected for feeding separately two photobioreactors. The selection than 4% of culture broth volume). Dissolved oxygen (DO), pH and
of these wastewaters was based on the different phosphorous con- temperature were monitored in situ.
centration. The performance of the photobioreactors was evaluated Both photobiorreactors were initially filled with distilled water
in terms of organic matter and nutrient removal efficiency together and inoculated with 25 and 12 mg volatile suspended solids (VSS)
with biomass production and biochemical composition. Addition- L1 of microalgae C. sorokiniana and aerobic sludge, respectively.
ally, anaerobic batch experiments were carried out using the pro- Right after inoculation, the photobiorreactors were fed with TE
duced biomass in both photobioreactors. Finally, the influence of (photobioreactor RTE) and PP (photobioreactor RPP). The hydraulic
the substrate/inoculum ratio was determined in terms of methane retention time (HRT) was 10 days, corresponding to an ammonium
yield. loading rate (ALR) of 1.2 mg NH4+–N L1 day1 for both
photobioreactors.
Culture broths were collected separately in two settlers for bio-
2. Methods mass sedimentation. Samples of the influent and effluent from the
top of the settlers were collected periodically in order to determine
2.1. Photobioreactors and culture conditions total chemical oxygen demand (TCOD), soluble chemical oxygen
demand (SCOD), total solids (TS), volatile solids (VS), total sus-
2.1.1. Microalgae–bacteria inoculum pended solids (TSS), VSS, total Kjeldahl nitrogen (TKN), soluble
Chlorella sorokiniana was obtained from the culture collection of phosphorus (SP), ammonia (NH4+–N), nitrites (NO2–N) and
the University of Goettingen (Goettingen, Germany). Microalgae nitrates (NO3–N). In addition, TS and VS were monitored period-
inoculum was prepared according to Guieysse et al. (2002). The ically in culture broths. Biomass was purged from the bottom of
average temperature was 24 ± 2.7 °C. Before inoculation, microal- the settlers at the end of the experimental time. These purges were
gae were centrifuged (4000 rpm; Centrifuge 5810R, Eppendorf) analysed for TS, VS and TKN determination. Moreover, a fraction of
for 20 min and resuspended in distilled water. The aerobic sludge collected biomass was lyophilized (Lyoquest 85 Plus Eco, Spain) for
was obtained from an activated sludge reactor of the municipal lipid content determination.
wastewater treatment plant of Valladolid (Spain).
2.2. Anaerobic biodegradability experimental set-up

2.1.2. Substrate composition Anaerobic biodegradability assays were carried out at

Treated liquid fraction of pig manure (TE) was collected from a 37 ± 0.4 °C for 50 days in 0.57 L bottles. Quantities were calculated
pig manure treatment plant located in Cuellar (Segovia, Spain). to reach a final volume of 0.25 L, allowing a headspace of 0.32 L for
Treatment consisted of a solid-liquid separation (with addition of gas accumulation. The bottles were closed with a septum and the
coagulants and flocculants) and a treatment of the liquid fraction headspace flushed with N2 to remove the O2. The biogas produc-
by nitrification–denitrification. Potato processing wastewater tion was measured by the overpressure in the headspace with time
(PP) was obtained from a potato industry located in Medina del frequency (Colleran et al., 1992). Constant agitation was provided
Campo (Valladolid, Spain). Wastewaters were homogenized by a shaker at 200 rpm.
Anaerobic sludge inoculum was collected at the municipal
Table 1
wastewater treatment plant of Valladolid (Spain). Anaerobic sludge
Characterization of treated piggery effluents (TE) and potato processing wastewater presented a TS and VS concentration of 20.0 and 10.2 g L1, respec-
(PP). Standard deviation is shown in brackets. tively. For these assays, microalgal–bacterial biomass collected
TE PP
from the bottom of the two settlers described in Section 2.1.3 at
the end of the experimental period was used. Specifically, biomass
pH 7.5 (0.3) 5.8 (0.2)
TS (mg L1) 3319 (147.9) 1603 (388.2)
was concentrated by centrifugation (8000 rpm; Beckman Coulter,
VS (mg L1) 1031 (96.5) 903 (320.2) Avanti centrifuge J-30I) for 10 min. For the determination of
TCOD (mg L1) 616 (44.8) 1536 (529.1) endogenous methane production, blanks containing only anaero-
SCOD (mg L1) 465 (38.5) 745 (227.2) bic sludge were also tested.
BOD5 (mg L1) 63.0 (18.3) 917 (166.9)
The influence of the substrate/inoculum ratio (from now on
TKN (mg L1) 32.9 (8.0) 33.7 (10.1)
NH4+–N (mg L1) 12.3 (1.7) 12.1 (1.7) TCOD/VS ratio) was evaluated according to González-Fernández
NO3–N (mg L1) 53.8 (6.1) n.d. and García-Encina (2009). TCOD/VS ratios ranged between 0.5
NO2–N (mg L1) 131.7 (5.7) n.d. and 2.0, which were achieved by keeping a constant inoculum con-
TP (mg L1) 50.1 (9.0) 4.2 (0.01) centration of 2.2 g L1. Treatments T1 to T4 were performed using
SP (mg L1) 47.5 (4.4) 3.4 (0.6)
microalgal biomass produced in photobioreactor RTE, whereas
n.d.: not detected. treatments T5–T8 were performed using biomass produced in pho-
600 D. Hernández et al. / Bioresource Technology 135 (2013) 598–603

tobioreactor RPP. All experiments were carried out in duplicate and shown in Fig. 1a, DO was never limited in reactor RTE. Therefore,
the results were expressed as means. the remaining COD was acting as recalcitrant for this system.
COD removal efficiencies observed in reactor RTE were slightly
2.3. Analytical procedures lower than those reported by De Godos et al. (2009) when treating
piggery wastewaters (76%). These better COD removal behaviour
TS, VS, TSS, VSS, TCOD, SCOD, biological oxygen demand (BOD5) could be probably promoted by higher pig manure biodegradabil-
TKN and SP were analysed in duplicates according to APHA Stan- ity. In the case of photobioreactor RPP, DO decreased during the
dard Methods (2005). NH4+–N, NO2–N and NO3–N concentra- experimental time up to values below 1 mg O2 L1 at day 23
tions were determined using electrodes, Orion 900/200 (Thermo (Fig. 1a), which indicated that microalgae limited the system and
Electron Corporation, Beverly, USA). DO, pH and temperature in higher COD removal efficiencies could be achieved with a proper
the reactors were determined using a multi-probe system model consortia development.
YSI 556 MPS (YSI incorporated, USA).
Lipids were extracted from the lyophilized biomass with chlo- 3.1.2. Nutrient removal
roform–methanol following the method proposed by Kochert ALR was similar in both photobioreactors (1.2 mg NH4+–N L1 d1).
(1978). Once the extraction was done, the lipids were quantified Ammonium was removed up to 82.7% in RTE and it was almost
by gravimetric analysis. Proteins were calculated by multiplying exhausted in RPP (Table 2). These high removal efficiencies were
the TKN by 5.95 (González-López et al., 2010). Carbohydrates were expected since ALR was low in comparison with those applied in
estimated by subtracting the percentage of ashes, lipids and pro- other studies (Sialve et al., 2009; Xin et al., 2010; Wang et al.,
teins out of 100% (Wilkie and Mulbry, 2002). 2012). In order to quantify NH4+–N stripping, the free ammonia
Microalgae identification was carried out by microscopical concentration was calculated according to Hansen et al. (1998).
examination (OLYMPUS IX70, USA) of culture broth samples fixed The results indicated that ammonia volatilization was not the main
with formaldehyde at 0.5% and stored at 4 °C prior to analysis, mechanism for ammonium removal since ammonia stripping
according to Phytoplankton Manual (Sournia, 1978). accounted for 25% in RTE and for 3% in RPP (Table 2). This differ-
Biogas composition was analyzed using a gas chromatograph ence was attributed to the higher pH achieved in reactor RTE
(Bruker 430-Gc) with a thermal conductivity detector, provided (8.8) compared to RPP (8.0), as a consequence of the higher SCOD
by a CP-Molvsieve5A column (15 m  0.53 mm  15 lm) followed removals in photobioreactor RPP (Fig. 1b), and therefore to higher
by a CP-Porabond Q column (25 m  0.53 mm  10 lm). Hydrogen bacterial activity in this reactor. Therefore, biomass nitrogen
(13.6 mL min1) was used as carrier gas. The injection port temper- assimilation was determined by the daily TKN increase, being
ature was set at 150 °C and the detector temperature was 175 °C. higher in RPP (0.25 mg TKN g1 TS d1) than in RTE (0.08 mg
Total volatile fatty acids (VFA) were analyzed at the end of the TKN g1 TS d1).
assays using a gas chromatograph (Agilent 7820A) equipped with a Photobioreactor RTE showed a nitrite removal efficiency higher
Teknokroma 10% SP1000 capillary column and a flame ionization than 75%. Even when ammonia stripping and assimilation were
detector. Carrier gases were nitrogen, hydrogen and air and the indicated as the main mechanisms for nitrogen removal in open
temperature of the injector was 375 °C. The temperature of the reactors (Molinuevo-Salces et al., 2010), the present study demon-
oven was set at 160 °C. strates that denitrification also occurs when pH ranges between
8.0 and 8.8. In this manner, denitrification accounted for 53.8% in
RTE. It should be noted that denitrification requires low DO in
3. Results and discussion the medium and RTE presented DO concentration up to 4 mg
O2 L1 (Fig. 1a). Nevertheless, DO could be much lower in the
3.1. Photobioreactor performance flocks formed during the treatment (De Kreuk et al., 2005;
Molinuevo-Salces et al., 2010). Additionally, even when nitrate is
3.1.1. Organic matter removal not the preferred nitrogen form for microalgae uptake (Travieso
The organic loading rates (OLR) applied to photobioreactors et al., 2006), this process could also have contributed to the
during the whole experimental time were 0.06 and 0.15 g TCOD nitrogen removal from the culture broth. In photobioreactor RPP,
L1 day1 for RTE and RPP, respectively. The highest COD removal NO2–N or NO3–N was not detected during the whole experimen-
efficiencies were observed when treating potato processing waste- tal time, which indicated that nitrification process did not occur.
water. More specifically, TCOD removal was 62.3% for RTE and As shown in Table 2, SP removal efficiency was 58.0% and 80.7%
84.8% for RPP (Table 2). SCOD removal efficiencies accounted for in RTE and RPP, respectively. These results were similar to those
58.1% and 86.1% for RTE and RPP, respectively. As stated by other from Wang et al. (2012) who obtained a SP removal efficiency of
authors, higher COD removal efficiency observed in photobioreac- 60.6% when treating diluted piggery wastewater with similar
tor RPP could be attributed to the higher influent strength COD concentration using the microalgae Chlorella pyrenoidosa.
(Molinuevo-Salces et al., 2010; Wang et al., 2012). Additionally, These differences in phosphorous removal could be attributed to
BOD5/TCOD ratio in PP wastewater (0.59) was higher than in TE the higher SP loading rate applied in RTE (4.75 mg L1 d1) than
(0.10) and therefore, organic matter was more degradable. As in RPP (0.34 mg L1 d1). SP removals achieved in present study
were high compared with those reported by De Godos et al.
Table 2
COD removal, ammonium removal, ammonium removed by stripping, nitrification,
(2009), who obtained efficiencies of 10% working with high rate al-
denitrification and soluble phosphorous removal in the two photobioreactors. gal ponds. In this sense, it should be noticed that high pH achieved
Standard deviation is shown in brackets. in both reactors may be involved in PO43 precipitation (Nurdogan
RTE% RPP%
and Oswald, 1995).
Removed TCOD 62.3 (2.0) 84.8 (3.2)
Removed SCOD 58.1 (4.5) 86.1 (2.6)
3.1.3. Biomass productivity and biochemical profile
Removed NH4+–N 82.7 (3.0) >95 Biomass growth was measured as the dry weight (total solids)
Removed by stripping 25.4 (8.0) 2.9 (1.3) of produced biomass per day and litre of the photobioreactor.
Removed by nitrification 75.7 (11.8) – RTE produced 26.3 mg dry weight (DW) L1 d1 while RPP pro-
Removed by denitrification 53.8 (10.1) –
duced 18.8 mg DW L1 d1. Hence, the higher phosphorous avail-
Removed SP 58.0 (7.5) 80.7 (12.3)
ability in RTE resulted in higher biomass growth. These results
 D. Hernández et al. / Bioresource Technology 135 (2013) 598–603 601

16
(a)
14

12

10
DO (mg L )
-1

0
0 5 10 15 20 25 30 35
Time (d)

10
(b)

9
pH

7
0 5 10 15 20 25 30 35
Time (d)

Fig. 1. (a) Dissolved oxygen and (b) pH measured in situ in photobioreactor RTE (discontinuous line) and RPP (continuous line).

were in agreement with those reported by Riaño et al. (2011), who Nitrogen is directly implicated in aminoacid synthesis and
observed an increase in biomass productivity concomitantly with hence in protein formation (Sialve et al., 2009). Percentage of pro-
increasing nitrogen and phosphorous loading rate when treating teins was 8.6% for photobioreactor RTE and 26.6% for RPP. These
fish processing wastewater in open ponds. differences could be attributed to higher ammonia volatilization
The carbon and nutrients contained in wastewater were in photobioreactor RTE and, therefore lower ammonia assimilation
assimilated by the biomass and converted to organic macro- by biomass in this reactor. These results were in agreement with
molecules. those reported by Piorreck et al. (1984), who obtained that increas-
There is a great interest in microalgal biomass as a renewable ing ammonia concentrations from 30 to 1000 mg L1 NH4Cl, led to
energy source due to their availability to accumulate substantial
quantities of lipids. In the present study, significant differences
were found in lipids content of biomass produced in photobioreac- Table 3
Methane yields and total solid removal efficiency obtained after anaerobic process of
tor RTE (4.3%) and RPP (30.2%). Nutrient limitation is an efficient
biomass produced in RTE (T1–T4) and RPP (T5–T8).
trigger to increase lipid content per algal biomass (Xin et al.,
2010; Wang et al., 2012). In this context, it should be stressed that Substrate/inoculum % TS removal Methane yield
ratio (g TCOD/g VS) (mL CH4 g COD1added)
although this experiment was conducted under similar ALR in both
reactors, SP concentration was almost 14-folds higher in TE waste- T1 0.5 12.24 (0.37) 329.8
T2 1.0 18.36 (2.92) 207.2
water than in PP wastewater (Table 1). This fact may cause higher
T3 1.5 17.17 (2.08) 172.0
lipid accumulation in biomass produced in RPP. The results herein T4 2.0 21.33 (1.80) 200.5
were in agreement with those reported by Goldberg and Cohen T5 0.5 15.81 (5.09) 517.5
(2006), who observed that under the condition of phosphorous T6 1.0 23.32 (1.48) 408.2
limitation, the total cellular lipid content increased from 6.5% up T7 1.5 24.14 (1.34) 460.1
T8 2.0 24.97 (1.82) 404.6
to 39.3%.
602 D. Hernández et al. / Bioresource Technology 135 (2013) 598–603

600

500

400
added
-1
mL CH4 g COD

300

200

100

0
0 5 10 15 20 25 30 35 40 45 50 55
Time (d)

Fig. 2. Net methane yields in the evaluated treatments for RTE (discontinuous line) and for RPP (continuous line) at substrate/inoculum ratio of 0.5 (d), 1.0 (N), 1.5 (}) and
2.0 (j) g TCOD/g VS.

a significant protein content reduction (from 8% up to 54% of Biochemical composition of microalgal-biomass also deter-
proteins) in different microalgae species. mined methane yield obtained. Specifically, methane yield in-
In this study, carbohydrates were the main cellular component creased between 157% and 268% in the case of the biomass from
obtained for both photobioreactors. Percentage of carbohydrates RPP in comparison to biomass obtained from RTE (Fig. 2). This fact
was found to be 2-fold higher in biomass produced in RTE than could be explained by the lower lipid content of biomass from RTE
in RPP (data not shown), as a consequence of the lower content than from RPP as stated before. Regarding this, several authors
in lipids and proteins. Therefore, it was detected that the character- reported that lipids showed a higher biogas production potential
istics of wastewater presented a high influence on macromolecular compared with proteins and carbohydrates (Cirne et al., 2007; Li
composition of microalgal–bacterial biomass produced. Specifi- et al., 2002). The results herein demonstrated that an increase in
cally, the use of a substrate with low phosphorous concentration lipid content of digested, also increases the potential methane
was elucidated as an important factor affecting percentage of lipids yield. However, an excess of the percentage of lipids in biomass
in biomass, and therefore, it could determine the further valoration could lead to VFA accumulation, causing the inhibition of the
of this added-value product. anaerobic process (Park and Li, 2012).
The percentage of methane in biogas varied between 71.9% and
3.2. Overall anaerobic biodegradability performance 76.1% for biomass from RTE and between 76.5% and 77.0% for bio-
mass from RPP (data not shown). These results revealed a good
3.2.1. Biogas production and methane yields conversion of the microalgal–bacterial biomass into methane. High
Anaerobic experiments lasted for 50 days. Table 3 shows the methane content in anaerobic digester implies a steady balance of
accumulated biogas production at substrate/inoculum ratio from methane and carbon dioxide, which are products of methanogene-
0.5 to 2.0 g TCOD/g VS when using biomass produced in photobior- sis and acetogenesis, respectively (Park and Li, 2012). The values
eactor RTE (T1–T4) and RPP (T5–T8). The methane volumes were obtained in present study were similar to those reported by Sialve
corrected by subtracting the mean methane volume of the blanks et al. (2009).
(endogenous production) and were converted to standard temper-
ature and pressure (STP, 0 °C and 760 mm Hg). Methane yields 3.2.2. Solid removal efficiency
were calculated by dividing the corrected methane volume by TS removal efficiency improved with the increase of substrate/
TCOD added to each digester. As seen in Table 3 and Fig. 2, the inoculum ratio (Table 3). For biomass produced in RTE, TS removal
highest methane yields were achieved by the treatments T1 and
Table 4
T5 corresponding to a substrate/inoculum ratio of 0.5 g TCOD/g pH and NH4+–N during anaerobic process of biomass produced in RTE (T1–T4) and
VS ratio. The rest of treatments showed a similar methane produc- RPP (T5–T8).
tion, varying between 172.0 and 207.2 mL CH4 g COD1added in the
pH NH4+–N (mg L1)
case of the digestion of the biomass produced in photobioreactor
RTE and between 404.6 and 460.1 mL CH4 g COD1added for Initial Final Initial Final

biomass from RPP. According to González-Fernández and T0 8.0 7.7 153.0 (1.4) 162.5 (2.9)
García-Encina (2009), high COD/VS ratios were responsible for T1 7.9 7.8 168.0 (1.4) 190.0 (1.5)
T2 8.0 7.7 161.5 (0.7) 198.0 (2.6)
methane production delay due to the accumulation of VFA. T3 8.0 7.8 175.0 (0.0) 236.0 (0.5)
Therefore, the substrate/inoculum ratio can be an essential T4 8.1 7.6 166.5 (0.7) 241.0 (3.0)
parameter to influence the methane yield in the batch anaerobic T5 7.6 7.6 155.0 (0.0) 167.0 (1.6)
digestion of microalgal–bacterial biomass. The same conclusion T6 7.6 7.4 165.0 (0.0) 181.5 (1.0)
T7 7.5 7.3 160.5 (0.7) 210.8 (5.1)
was obtained by previous research using different substrates
T8 7.4 7.4 172.5 (0.7) 229.8 (1.7)
(Raposo et al., 2006, 2008; Foster-Carneiro et al., 2008).
 D. Hernández et al. / Bioresource Technology 135 (2013) 598–603 603

efficiency increased from 12.2% to 21.3% when substrate/inoculum Goldberg, I.K., Cohen, Z., 2006. The effect of phosphate starvation on the lipid and
fatty acid composition of the fresh water eustigmatophyte Monodus
ratio increased from 0.5 to 2.0. In the case of biomass produced in
subterraneus. Phytochemistry 67, 696–701.
reactor RPP, TS removal efficiency increased from 15.8% to 25.0% Golueke, C.G., Oswald, W.J., Gotaas, H.B., 1957. Anaerobic digestion of algae. Appl.
when substrate/inoculum ratio increased from 0.5 to 2.0. These Microbiol. 5, 47–55.
findings were in accordance with previous results obtained by González-Fernández, C., García-Encina, P.A., 2009. Impact of substrate to inoculum
ratio in anaerobic digestion of swine slurry. Biomass Bioenerg. 33, 1065–1069.
González-Fernández et al. (2011), who accomplished TS removal González-Fernández, C., Molinuevo-Salces, B., García-González, M.C., 2011.
efficiencies of 14.7–32.9% by co-digesting algal biomass with swine Evaluation of anaerobic codigestion of microalgal biomass and swine manure
manure. via response surface methodology. Appl. Energ. 88 (10), 3448–3453.
González-López, C.V., Cerón-García, M.C., Acién-Fernández, F.G., Segovia-Bustos, C.,
Chisti, Y., Fernández-Sevilla, J.M., 2010. Protein measurements of microalgal
3.2.3. Process stability and cyanobacterial biomasa. Bioresour. Technol. 101, 7587–7591.
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