Professional Documents
Culture Documents
De Mattos, Bastos - 2016 - COD and Nitrogen Removal From Sugarcane Vinasse by Heterotrophic Green Algae Desmodesmus SP
De Mattos, Bastos - 2016 - COD and Nitrogen Removal From Sugarcane Vinasse by Heterotrophic Green Algae Desmodesmus SP
To cite this article: Luiz Felipe Arjonilla de Mattos & Reinaldo Gaspar Bastos (2015): COD and nitrogen removal
from sugarcane vinasse by heterotrophic green algae Desmodesmus sp., Desalination and Water Treatment, DOI:
10.1080/19443994.2015.1028454
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions
Desalination and Water Treatment (2015) 1–9
www.deswater.com
doi: 10.1080/19443994.2015.1028454
ABSTRACT
*Corresponding author.
obliquus, reporting a 70% removal of calcium after this biomass concentration. After that, biomass was
period. In autotrophic cultivations, most nitrogen centrifuged at 1,275 g and inoculated in vinasse.
removal is caused by pH alkalinization, which gives Heterotrophic cultivation of Desmodesmus sp. was
microalgae an important role in secondary effluent carried out in stirred batch bioreactor (covered to pre-
treatment [10]. Accordingly, Ji et al. [11] obtained 99% vent light incidence) with vinasse at pH 7.0, 25˚C, 1
removal of nutrients after 96 h autotrophic cultivation VVM, and 3 g of stirrer speed. Parameters such as pH,
of S. obliquus reporting a specific growth speed of dissolved oxygen, and carbon dioxide were monitored
0.047 h−1. Similar level was observed by Samorı̀ et al. and recorded every 30 min. Samples from the bioreac-
[12] using Desmodesmus communis and by Martı́nez tor were collected every 3 h for 30 h.
et al. [6] using S. obliquus.
Desmodesmus, a recently created genus from
Scenedesmus accordingly to An et al. [13] due to its 2.3. Analysis
spiny formations and genotypic differences, has mem- COD of vinasse was obtained by the dichromate
bers with close capacity of consuming wastewater method, according to APHA [17], using an acid
nutrient under heterotrophic conditions. Bastos et al. solution made of sulfuric acid and dichromate for the
[14], using vinasse as growth media for Desmodesmus samples digestion at 150˚C for 2 h in an HACH®
sp., reported specific growth rate of 0.096 h−1 and digestor block. Digested samples were cooled to ambi-
Downloaded by [Fudan University] at 14:00 17 May 2015
pointed that these results are promising for high ent temperature and the absorbance read at 600 nm in
added value biomass generation in agroindustrial HACH® spectrophotometer using 4 mL glass cuvettes
effluents. Biomass generated with microalgae produc- and calibration curve with biftalate solution.
tion using industrial effluents as substrate seems to be Total nitrogen (TN) of vinasse was made using TN
an alternative for expensive wastewater treatment, Test’N Tube™ HACH® vials kit. In the first step,
since it can be used in aquaculture as a valuable feed samples were digested in an alkaline media with
ingredient [15]. Thus, the aim of this research was to persulfate for 30 min at 105˚C in HACH® digestor
evaluate chemical oxygen demand (COD), nitrogen block, similar to COD, cooled at ambient temperature.
removal, and growth of Desmodesmus sp. in sugarcane A bisulfate reagent was poured into the vial and
vinasse. mixed followed by a reaction time of 3 min after
which an indicator was added to react for 2 min. For
the final step, samples of 2 mL from the digested
2. Materials and methods
tubes were added to acid reagent tubes for the color
2.1. Vinasse formation and absorbance reading in a HACH®
spectrophotometer at 410 nm.
Vinasse was collected from a sugarcane processing
Suspended solids for estimation of biomass was
industry in the city of Araras, São Paulo, Brazil,
performed by vacuum filtration using a Millipore®
directly after the ethanol distillation at 96˚C, fraction-
filtration apparatus with 0.22 μm Sartorius® cellulose
ated in 2 L bottles at approximately 80˚C in the labora-
acetate membranes previously dried at 105˚C for
tory, and frozen until use.
60 min and weighted. Filtration was performed with
5 mL samples and a blank from the suspended
2.2. Inoculum and cultivation solids of vinasse was made before the inoculation of
Desmodemsus sp. in the bioreactor. After filtration,
Green microalgae (Chlorophytae) Desmodesmus sp. was membranes were dried at 105˚C until constant mass
maintained in culture bank in modified BGN media [16] measurement and the dry weight of suspended
with the following composition: 30 mg L−1 K2HPO4, solids was obtained by weighting the difference
75 mg L−1 MgSO4·7H2O, 36 mg L−1 CaCl2·2H2O, between initial and after filtration weight of the
6 mg L−1 ferric ammonium citrate, 1 mg L−1 Na2 EDTA, membranes.
6 mg L−1 citric acid, 20 mg L−1 Na2CO3, 1.5 mg L−1
NaNO3, 72 mg L−1 NaCl, 2.86 mg L−1 H3BO3,
−1
1.81 mg L MnCl2·4H2O, 0.22 mg L−1 ZnSO4·7H2O,
2.3.1. Green algae and bacteria cell counting
0.39 mg L−1 NaMoO4·2H2O, 0.079 mg L−1 CuSO4·5H2O,
and 0.04 mg L−1 CoCl2·6H2O. Desmodesmus sp. cells were counted using
Desmodesmus sp. inoculum was grown autotrophi- improved Neubauer chamber, counting the corner
cally in the bioreactor using BGN media with pH 7.5, squares and the central one. Average of the counted
at 25˚C, aeration of 1VVM, 3 xg mechanic agitation, values was performed and multiplied by the dilution
and photo flux of 45 μmol m−2 s−1 until 1 g L−1 factor and 105 for the cells per mL concentration.
L.F.A. de Mattos and R.G. Bastos / Desalination and Water Treatment 3
Bacterial colony-forming units (CFU) were mea- for adjustment of pH for the cultivation of green algae
sured using Petrifilm Aqua™ from 3 M® applying since pH of propagation medium BGN is 7.6. Another
1 mL of the samples per plate and incubated for 48 h important point is the C/N ratio of vinasse sample
at 37˚C. In this method, entire plate area is counted, around 20. This value calculated from the ratio in
the value is multiplied by the dilution factor and the COD and TN is in the optimum range for growing
result is expressed in CFU. these micro-organisms [18].
Fig. 1 presents dissolved oxygen and carbon diox-
ide profiles in vinasse during the growth of Desmod-
2.3.2. Kinetic analysis esmus. sp. As shown, oxygen consumption increased
Percentage efficiency of substrate removal from until the 15th hour of cultivation, stabilizing at 20% of
COD or TN was determined by following equation: the oxygen pumped into reactor until the end of the
experiment. In this period, oxygen consumption
s0 s occurred at a constant rate (non-limiting) of
ES¼ 100 (1) 0.186 mg L−1 h−1, following zero-order kinetics. On the
s0
other hand, the production of carbon dioxide
remained constant from 6 to 21 h at a rate of
From the biomass concentration, COD and TN
2,608 mg L−1 h−1.
were calculated by the specific growth rate and con-
Downloaded by [Fudan University] at 14:00 17 May 2015
Table 1
Vinasse composition
pH COD (mg L−1) TN (mg L−1) TP (mg L−1) Total solids (mg L−1)
3.5 27,100 1,420 2.61 26,220
Notes: COD: chemical oxygen demand; TN: total nitrogen; TP: total phosphorus.
Downloaded by [Fudan University] at 14:00 17 May 2015
Fig. 1. Oxygen consumption and CO2 production during the growth of Desmodesmus sp. in sugarcane vinasse.
shown in the biomass production topic, nitrogen indicating that its sequestration from the media did
concentration in the media started to decrease when not happen by physical–chemical ways but by
the log phase of biomass growth was reached, biological consumption.
L.F.A. de Mattos and R.G. Bastos / Desalination and Water Treatment 5
Downloaded by [Fudan University] at 14:00 17 May 2015
Fig. 3. Nitrogen and COD consumption by Desmodesmus sp. in sugarcane vinasse at 30 h experiment.
Table 2
Kinetic parameters
Parameter Value
μmáx (h−1) 0.15 (R2 = 0.87)
qCOD (mg L−1 h−1) 342.8 (R2 = 0.95)
qTN (mg L−1 h−1) 32.88 (R2 = 0.95)
E—COD (%) 36.2 (30 h)
E—TN (%) 52.1 (30 h)
Notes: μmáx: maximum specific growth rate; qCOD: COD consumption specific rate; qTN: TN consumption specific rate; E—COD: maxi-
mum COD removal; E—TN: maximum TN removal; R2: coefficient of correlation for linear fit of growth, COD, and TN curves.
According to Table 2, which presents the kinetic slowed down, accompanied by a shock in the biomass
parameters as maximum specific growth rate of that stabilized the growth after that.
Desmodesmus sp. in vinasse, consumption rates, and The log phase of biomass growth started at the
maximum removals of COD and TN, nitrogen hour 9 lasting until hour 18 and biomass implementa-
consumption in bioreactor was 32.88 mg L−1 h−1, with tion in the bioreactor after 30 h cultivation with maxi-
maximum removal of 52% at 30 h, which agrees with mum specific growth rate of 0.15 h−1 (Fig. 4).
the results of nitrogen consumption by S. obliquus in The carbon dioxide production, oxygen consump-
autotrophic cultivation using artificial wastewater tion, COD, and TN concentration presented higher
shown by Nuñes et al. COD in the media decreased in variations after the log phase of biomass growth was
an exponential trend, reaching 36.2% in the 30 h reached, confirming that biomass was not only surviv-
cultivation, as shown in Fig. 3, with consumption rate ing the stressing media but growing in it, meaning
of 342.8 mg L−1 h−1. that the C/N ratio near to 20 for sugarcane vinasse is
The faster consumption period was in the first 20 h suitable for the growth of such micro-organisms even
and stabilized after that. This may be due to the qual- under dark conditions [18]. In fact, Xing et al. [21]
ity of the rest of organic matter present after that. A found that variations in the C/N ratio in cultivation
possible explanation is that in the first 20 h, low medium resulted in different rates of incorporation of
molecular weight organic compounds supported bio- carbon and nitrogen in the cells.
mass growth, and after its consumption, only high Thus, a possible explanation for the shortness of
molecular weight organic compounds were left. As the log phase is the misbalance of C/N ratio as
this is a hard consumption substrate, COD depletion nitrogen was consumed faster than carbon, which
6 L.F.A. de Mattos and R.G. Bastos / Desalination and Water Treatment
Downloaded by [Fudan University] at 14:00 17 May 2015
Fig. 4. Biomass concentration profile during the growth of Desmodesmus sp. in sugarcane vinasse.
may cause stressing situation in organic systems. to biomass in the first 15hours, agreeing with the
Nevertheless, other possible explanation is that the profiles of biomass and oxygen. These results suggest
high consumption of low-weight organic matter and that it is possible to obtain high yield of this green
the difficulty in consumption of larger molecules by algae in vinasse as byproduct of sugarcane processing,
the micro-organisms. which is an industrial activity with large-scale
Biomass productivity in this experiment was operations in Brazil.
101.1 mg L−1 h−1 and specific growth rate was Results of Desmodesmus sp. counting showed that
0.022 h−1, higher than that found by other authors for green algae was growing slowly (Fig. 6). Cell produc-
autotrophic growth and similar to heterotrophic sys- tivity calculated for Desmodesmus sp. in this experi-
tems [7,12,22]. ment was 13.105 cells L−1 h−1 and points the usual
Fig. 5 presents yield COD to biomass profile problem of contamination in heterotrophic cultiva-
suggesting considerable growth with high conversion tions, which differed from the conventional total solids
Fig. 5. Yield COD to biomass during the growth of Desmodesmus sp. in sugarcane vinasse.
L.F.A. de Mattos and R.G. Bastos / Desalination and Water Treatment 7
Downloaded by [Fudan University] at 14:00 17 May 2015
analysis that suggest a higher yield COD to biomass responsible for part of the nitrogen and COD
(Fig. 5). consumption. This situation, although very clear when
However, according to Fig. 7, results of bacteria it is heterotrophs crops green algae or cyanobacteria,
counting showed that bacteria grew fast in vinasse in is not addressed more consistently in the literature.
an exponential fit, with a lag phase of 9 h. At Additionally, these findings also would prevent
10 h-cultivation, the bacterial contamination was still the application of these micro-organisms since
small, which indicated that, at this period, the biomass research pointed enhancing in algae systems with
yield was due to growth of green algae. bacteria [23].
Moreover, these results confirm that biomass Bacterial biomass production in this study was
growth measured by total solids was masked by the of almost 103 UFC h−1, which indicates high affinity
bacterial contamination, and that bacteria were of bacteria for vinasse’s nutrients and a risk of
8 L.F.A. de Mattos and R.G. Bastos / Desalination and Water Treatment
wastewater supplemented with CO2 for nutrient [18] M.I. Queiroz, E.J. Lopes, L.Q. Zepka, R.G. Bastos, R.
removal and biomass production, Ecol. Eng. 58 (2013) Goldbeck, The kinetics of the removal of nitrogen and
142–148. organic matter from parboiled rice effluent by
[12] G. Samorı̀, C. Samorı̀, F. Guerrini, R. Pistocchi, cyanobacteria in a stirred batch reactor, Bioresour.
Growth and nitrogen removal capacity of Desmod- Technol. 98 (2007) 2163–2169.
esmus communis and of a natural microalgae consor- [19] B. Raboy, E. Padan, Active transport of glucose and
tium in a batch culture system in view of urban α-methylglucoside in the cyanobacterium Plectonema
wastewater treatment: Part I, Water Res. 47 (2013) boryanum, J. Biol. Chem. 253 (1978) 3287–3291.
791–801. [20] S.J. Hong, C.G. Lee, Evaluation of central metabolism
[13] S.S. An, T. Friedl, E. Hegewald, Phylogenetic relation- based on a genomic database of Synechocystis
ships of Scenedesmus and Scenedesmus-like coccoid PCC6803, Biotechnol. Bioprocess Eng. 12 (2007)
green algae as inferred from ITS-2 rDNA sequence 165–173.
comparisons, Plant Biol. 1 (1999) 418–428. [21] X.H. Xing, B.M. Jun, M. Yanagida, Y. Tanji, H. Unno,
[14] R.G. Bastos, L.F.A. Mattos, V.C. Dessia, S. Goldemberg, Effect of C/N values on microbial simultaneous
D. Da Silva, Heterotrophic growth of Desmodesmus sp. removal of carbonaceous and nitrogenous substances
using sugarcane vinasse in batch stirred reactor, in: in wastewater by single continuous-flow fluidized-bed
Second International Conference on Algal Biomass, bioreactor containing porous carrier particles, Biochem.
Biofuels and Bioproducts, Elsevier, San Diego, CA, Eng. J. 5 (2000) 29–37.
2012. [22] M. Prathima Devi, G. Venkata Subhash, S. Venkata
[15] P.M.P. Ferreira, Manual de cultivo e bioencapsulação Mohan, Heterotrophic cultivation of mixed microalgae
Downloaded by [Fudan University] at 14:00 17 May 2015
da cadeia alimentar para a larvicultura de peixes for lipid accumulation and wastewater treatment
marinhos (Guidebook to cultivation and bioencapsula- during sequential growth and starvation phases: Effect
tion of the food chain for larviculture of marine fishs), of nutrient supplementation, Renew. Energy 43 (2012)
INRB—IPIMAR, Lisboa, Portugal, 2009. 276–283.
[16] R. Rippka, J. Deruelles, J.B. Waterbury, M. Herdman, [23] H.S. Kim, Algal sphere bacteria enhance the growth of
R.Y. Stanier, Generic assignments strain histories and eukaryotic microalga Chlorella vulgaris, World Con-
properties of pure cultures of cyanobacteria, J. Gen. gress on Biotechnology and Bioprocessing, Orlando,
Microbiol. 111 (1979) 1–61. FL, 2012.
[17] APHA, AWWA, WEF, Standard Methods for the [24] J. Lee, D.H. Cho, R. Ramanan, B.H Kim, H.M. Oh,
Examination of Water and Wastewater, twenty-first H.S. Kim, Microalgae-associated bacteria play a key
ed., American Public Health Association, Washington, role in the flocculation of Chlorella vulgaris, Bioresour.
DC, 2005. Technol. 131 (2013) 195–201.