Studies on NOx removal using

Dunaliella salina

algae

in photobioreactors

Kethineni Chandrika

, S. F. Choragudi

, Krishna Kireeti Kakarla

, Kolluru Sumanth

and

Ch Devika

Associate Professor, Department of Biotechnology, Koneru Lakshmaiah Education Foundation,

Vaddeswaram, Guntur, AP, India–522502

2,3,4,5

Department of Biotechnology, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur, AP,

India–522502

ABSTRACT

The capability of an algal species to remove NO2 and NO in the simulated  ue gas was established using Dunaliella

salina in Photobioreactors under two variants of NOx sources. The concentrations studies were in the range between

25ppm to 150ppm. The diffusion of NOx and subsequent reaction with water resulted in NO3- and NO2- in the growth

medium. Algal growth by absorption of NO3- and NO2- created a nitrate gradient in the bulk medium resulting in

NOx uptake rates from the gas phase of up to 96%, leaving the unconsumed nitrogen of up to 7 mg-N/L in the growth

medium. Algal species having an initial cell density of 2.8x105 cells/mL grew to the cell density of 1.73x107 cells/mL

and dry weight of 262 mg/L. The Nitrogen content of cells varied from 3-6%. The treatment of NOx in Photobioreactors

was investigated with reference to the gas removal ef ciency, cell growth and total nitrogen content in the biomass

KEY WORDS:

DUNALIELLA SALINA

, PHOTOBIOREACTORS, ALGAL GROWTH

674

Biotechnological

Communication

Biosci. Biotech. Res. Comm. 11(4): 674-680 (2018)

INTRODUCTION

Disproportionate usage of fossil fuels has been consid-

ered as the source for manmade toxic emissions com-

prising carbon dioxide, sulfur dioxide, nitrogen oxides,

volatile organic compounds and heavy metals (Mulhol-

land, 2008; Attilo et al., 2009). The by-products of fossil

fuels have been identi ed as one of the major anthro-

pogenic sources of this gas, contributing to global warm-

ing by the greenhouse effect. Therefore, it has become

obligatory to reduce these toxic emissions before they

are disposed into the environment. Nitric oxide (NO) and

ARTICLE INFORMATION:

Corresponding Authors: kkchandrika@kluniversity.in

Received 19

Oct, 2018

Accepted after revision 24

Dec, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA

Mono of Clarivate Analytics and Crossref Indexed

Journal Mono of CR

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/11.4/18

Kethineni Chandrika et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS NOX REMOVAL USING DUNALIELLA SALINA ALGAE IN PHOTOBIOREACTORS 675

Nitrogen dioxide (NO2) are the two main components

that make up NOx. These components are toxic and have

various environmental hazards as per Environmental

Protection Agency (Ronda, 2014). The nitrogen removal

is 94.9 % in the ammonium form nitrogen group using

bio lm (Yuxuan Zhu 2018).

There are several methods for treatment of NOx.

Selective catalytic reduction (SCR) is used, however it

is more expensive when applied for large-scale power

plants (Miller et al., 2010). Another way to treat NOx

from stationary sources is to use scrubbers to trans-

fer the risk into an aqueous solution, which still must

be treated or disposed of (Raja et al., 2007). Hence, to

develop an economical and practical process to handle

NOx exists. Cultivation of an algae to take up dissolved

NOx from a scrubber as a nitrogen source, and oxygen

only released as a by-product. This concept has been

worked to estimate that, algal strains and conditions,

algae can take up nitrogen from dissolved NOx (Nagase

et al., 1997). Toxic compounds present in the  ue gas

inhibit the growth rate. NO2 has high solubility in water

and therefore, reacts with water to form aqueous nitrates

(NO3) and nitrites (NO2), some of the unaccounted-for

nitrogen was lost due to volatization of gaseous nitro-

gen species, (Kaitlyan 2018).

Aqueous nitrate and nitrites are used by the algae as

a source of nitrogen for cell synthesis, (Mulholland &

Lomas, 2008). The dissolved NO2 and NO react to form

dissolved nitrogen compounds which are available to

ingest, biological conditions for the uptake of nitrate

or nitrite by the algae, (Lee & Schwartz 1981). The

nitrate or nitrite uptake by the algae reduce the soluble

NO2, thus increases the concentration gradient of NO2

between liquid and the air. Thus, apparent solubility

of NO2 is proportional to the NO2 gradient in the bulk

medium, (Skalska, et al 2010). Nitrogen mono-oxide and

sulfur dioxide can be removed by simultaneous absorp-

tion into aqueous mixed solutions of sul te and [Fe II

(edta)]H2O)]2−, ferrous ion coordinated to an anion of

ethylene-diaminetetraacetic acid, (Tomasz et al 2016). A

sequential process for the recovery and puri cation of

multiple products was used on a mixture of algal bio-

mass comprised of Spirulina platensis and Dunaliella

salina (Kethineni 2017).

Dunaliella salina is a green algae known to with

stand high salinity and accumulate carotenes. The

nitrate requirement for algae is more for accumulation

of biomass than for accumulation of carotene. Nitrates

in the range of 1- 10mM is suitable for algal growth

(Tafreshi et al., 2009). Harter et al, (2012) performed a

mass balance for nitrogen from NOx for Dunaliella cul-

tures in a column reactor. In a lab scale trail under simu-

lated  ue gas the results indicate that with an in ow of

150 μg N/L day NOX along with CO2 the alga could able

to maintain a net in ux of 0.52.73 μg N L-1 d-1 which

amounts to 35% NOx removal. Nagase et al, (1997) stud-

ied the removal rate of nitric oxide by Dunaliella tertio-

lacta supplied in the range between 25-500ppm. At an

inlet concentration of 500ppm NO in addition to CO2, it

was shown to remove 110 μmoles per hour at a  ow rate

of 150mL/min. Also, it was shown that within a range of

100 to 400mL/min gas  ow rate, a maximum of 60% of

the NO was removed. These results suggest that D. salina

is a potential algal species for NOx removal.

the ability

of thegreen algae, Chlorellato acclimate to high level

of NOx and the potential usage of Chlorella strains in

biological NOx removal (DeNOx) from industrial  ue

gases,

(Tianpei and Gang Xu 2016).

To understand the

NOX removal process and to increase its range of appli-

cability. The use of microalgae for simultaneous removal

of CO

, SO

and NO

from  ue gas is an environmen-

tally benign process, (Hong‐Wei Yen et al, 2016), 75%

decrease of the nitrogen concentration in the medium,

with respect to the optimal values for growth, increased

the lipid fractions of algal species, ( Attilio Converti et

al 2009).

It is very important to undertake biological NO

 xa-

tion. Therefore, in this work, two individual experiments

were conducted to productively remove the NOx from

simulated  ue gas with varying NOx loading rates by

estimating the optimal growth parameters. Different

NOx concentrations were supplied to each photobiore-

actor inoculated with Dunaliella salina. NOx removal

ef ciency and algal growth were determined in each

experiment.

MATERIAL AND METHODS

DUNALIELLA CULTURE

All three reactors were inoculated with 600 mL of pure

Dunaliella salina (SAG:42.88) grown in Dunaliella

medium (=Dun) at 250C and a pH of 7.0. The inoculum

was grown to a 1 x107 cells/mL, with an initial cell den-

sity of 2.8x105 cells/mL

GROWTH MEDIUM

The modi ed Dunaliella growth medium was used for

inoculum and algae growth experimentation. All the

nitrogen uptake by the algal cells was provided through

inlet simulated gas. Dunaliella salina was grown in

modi ed Dunaliella medium. A nitrogen free stock solu-

tion was prepared with K2HPO4, 0.1 g/ 100 mL. 20 mL

of this nutrient was mixed with 30 mL of the soil extract

and 930 mL arti cial seawater to make a liter solution.

The growth medium was given in Table 1.

Kethineni Chandrika et al.

676 NOX REMOVAL USING DUNALIELLA SALINA ALGAE IN PHOTOBIOREACTORS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Operating conditions of the reactor

Case1: pure NO2 feed source:

NO2 gas diluted with ambient air was used as the simu-

lated  ue gas for the  rst run. As the boiling point of

is approximately 20

C at atmospheric pressure; NO

was initially released as a liquid in the tubing. NO2 was

blended with 3Lpm of air to get NOx concentrations of

100ppm, 200ppm,350 ppm in photobioreactors 1a, 1b,

and 1c respectively. Experimental conditions were given

in Table 2.

ing 5000 ppm and 9000 ppm NO2 concentration. Thus,

in this case, the need for the liquid NO2 trap was ignored

as the gas mixture was already in a vapor state. Thus,

pumping calibration gas achieved steady inlet concen-

trations. Experimental conditions were given in table3.

The calibrated NO2 is blended using air for the required

NOX concentrations. The calibrated gases were supplied

to the reactor at 3 Lpm, having NO2 concentrations of

25 ppm, 50 ppm, 120 ppm respectively. The concentra-

tion range was chosen to resemble real-time power plant

NOx concentrations. Two aquarium stones of 12cm were

used to diffuse the gas into the reactor. CO2 was sup-

plied in the system, at a concentration of 400ppm until

the pH remains 7 However when the pH is below 7, the

pH 7 was maintained using 1.0 g/L solution of sodium

bicarbonate (NaHCO3). The system was run for six days

after loading of NOx. In uent and ef uent concentra-

tions of NO and NO2 in gas samples were measured

using an analyser, (Testo 350-S/-XL, USA).

RESULTS AND DISCUSSION

NOX REMOVAL EFFICIENCY

Case 1: pure NO2 feed source

In this case, the reactors were fed with pure NO2 which

is delivered as a liquid at room temperature and then the

collected vapors were diluted with air. Using pure NO2

gas, actual average inlet concentrations for reactors 1a,

1b, and 1c were 108 ppm, 35ppm, 70 ppm respectively

(Table 4). Fig. 1 shows the Dunaliella growth curve in

reactors 1a, 1b, and 1c during 90-hour run.

Table1. Modi ed Dunaliella growth medium

Component

Stock sol.

(g/100mL)

Nutrient concentrate

ion (mL)

KOH 0.1 20

Soil extract 30 -

Arti cial seawater 930 -

Table 2. Experimental conditions for case 1

Setting Reactor 1a Reactor 1b Reactor 1c

Inlet NO

(ppm) 100 200 350

Inlet Gas Flow

Rate (Lpm)

333

pH 7-8 7-8 7-8

Temperature (

C) 20 20 20

The simulated gas entered each photobioreactor

through a sparger, pH was maintained in the range

of 7.0 to 8.0 using CO2. The carbon dioxide feed was

monitored by separate valves to each reactor which was

controlled based on the pH in the reactor. NOx removal

rates were monitored for four days. The reactors were

illuminated with three 1 m long  uorescent white lights

emitting, a total of 2700 Klux.

Case 2: Gas feed source calibration:

For the case 2, the reactors were inoculated before to

the start of NOx loading and left for two days during

which, only ambient air was supplied to the system. NOx

was given from NO2 calibration gas cylinders compris-

Table 3. Experimental conditions for case 2

Setting

Reactor

Inlet NO

(ppm) 25 50 120

Inlet Gas Flow Rate (Lpm) 3 3 3

In uent CO

(g) (ppm) 400 400 400

pH 7-8 7-8 7-8

Temperature (

C) 20 20 20

Table 4. In uent and ef uent NO

concentrations in reactors

Reactor

Ratio

(NO:NO2)

Inlet NOx (g)

Conc (ppm)

Outlet NO

(g)

Conc (ppm)

Average removal

Conc (%)

1a 0.47 108±55 47±33 49

1b 0.15 35±16 15±12 51

1c 0.35 70±32 8±7 81

2a 0.038 27±6 11±4 59

2b 0.035 57±9 2±8 96

2c 0.043 126±12 7±11 95

Kethineni Chandrika et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS NOX REMOVAL USING DUNALIELLA SALINA ALGAE IN PHOTOBIOREACTORS 677

FIGURE 1. Growth curves in Photobioreactors for case 1

FIGURE 2. Growth curves in Photobioreactors for case 2

All three reactors began with 2.8x10

cells/ml. As

shown in Fig. 1, the culture in reactor 1a began exhaus-

tive during the  rst 24 hours, therefore, the cells never

attained a density greater than the initial. The maximum

cell densities, for 1b, 1c were 1.46x106 cells/mL and

1.48x106 cells/mL respectively. Table 3 presents the NOx

removal data for the reactors under case 1. Inlet and out-

let NOx in Table 3 is the summation of measured NO and

NO2 concentrations in the inlet and outlet streams. The

ef ciency of NOx removal by the algal system is based

on total nitrogen through the reactor system and not

on any particular NOx component. Therefore, removal

percent of total NOx is only considered in the analysis.

Reactor 1a obtained an average NOx removal of 49%,

Kethineni Chandrika et al.

678 NOX REMOVAL USING DUNALIELLA SALINA ALGAE IN PHOTOBIOREACTORS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Table 5. Total suspended solids of Photobioreactors in

Case 2

Photo-bioreactor TSS Initial (mg/L) TSS Final (mg/L)

2a 6.0 210

2b 5.7 243

2c 6.3 222

FIGURE 3. I) & II) Concentrations of NO3 & NO2 in Reactors 2a, b, c

Table 6. Nitrogen mass balance data

Reactor

Input 890 1740 3450

Output 450 60 90

N Consumed from NO

gas

phase

440 1680 3360

Initial N in growth medium 23 22 23

Final NO

-271448

Final NO

- 74 13 163

Net Accumulation of NO

+NO

78 5 188

Initial Organic N 0 0 0

Final Organic N 310 750 1140

N Accumulated in Algal

Cells

310 750 1140

Total N Accumulated 388 755 1228

Mass Balance (% NO

uptake accounted for)

88% 45% 36%

Reactor 1b achieved an average 51% NOx removal, and

Reactor 1c has removed an average of 81% of inlet NOx.

The average NOx removal ef ciency for the  rst 60 h

was 39%, but the average removal for the last 25 hours

was 52%. As the algal culture was declining, removal

of NOx could be associated with the dissolution of NO2

into the medium.

Reactors 1b and 1c presented moderate growth fol-

lowing an extended lag phase, as shown in the  gure1.

Algae in reactors 1b, 1c has not truly experienced log

phase, and the run was ended after 90 hours.

Case 2: Calibration gas as a feed source

As shown in Fig. 2 all three reactors began with a cell

concentration of 2.8x105 cells/mL for three reactors 2a,

2b, and 2c .Maximum cell densities were 1.24x107 cells/

mL, 1.46x107 cells/mL and 1.72x107 cells/mL respec-

tively. To overcome some of the problems that appeared

from using pure NO2 gas in case 1, NO2 calibration

gases of 5000ppm and 10000 ppm NO2 were used to

supply NOx for case 2. This allowed for accurate NOx

loading concentrations. Actual NOx Loading rates for

case 2 were 27 ppm, 57 ppm and 126 ppm for reactors

2a, 2b, and 2c respectively.

The data in Table 4 presents the removal of NOx for

the reactors (case 2). Inlet and outlet NOx in Table 4 is

the summation of measured NO and NO2 concentrations

in the inlet and outlet streams. Reactor 2a got an aver-

age NOx removal of 59% reactor 2b got an average 96%,

and reactor 2c was able to remove an average of 95%

of inlet NOx. The lag phase was reduced to less than 24

hours due to delay in NOx loading, and log phase was

attained between 24 and 48 hours. NOx loading started

at 48 hours, and three reactors showed continued growth,

but with a signi cant decline in growth rate (Fig. 2).

Total suspended solids (TSS) for initial and  nal sam-

ples were taken to quantify algal growth and to esti-

mate the nitrogen content of the cells. The results were

summarized in the Table 5. Total suspended solid results

show that 37 fold average mass growth was accom-

plished over the 190-hour run. For nitrate and nitrite

concentrations, liquid samples from case 2 were ana-

lyzed, and the results were shown in Fig.3. Nitrate was

completely drained in all three reactors before NOx was

loaded into the system at 49 hours. Nitrogen source

available for algal growth only after that point was from

dissolved NO2. Initial and  nal total organic nitrogen

content of the algal cultures was used to estimate the

uptake of nitrogen by algae and to determine the nitro-

gen content of the cells. These analyses summarized in

the table: the cells in reactors 2a, 2b, and 2c were found

to contain 6.2%, 4.1%, and 7.9% nitrogen respectively.

Kethineni Chandrika et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS NOX REMOVAL USING DUNALIELLA SALINA ALGAE IN PHOTOBIOREACTORS 679

A mass balance of nitrogen was conducted to assess the

effect of NOx through the system. In reactor 2a, the mass

balance alleged for 88% of the observed NOx removal,

as slightly more nitrogen was found in the cells and

growth medium than entering the system.

For reactors 2b and 2c, only 45% and 36% respec-

tively of the nitrogen that entered the system was found

in the cells and growth medium. NOx feed stream: Load-

ing NOx at a particular concentration tested to be very

dif cult for the  rst case, as can see by more standard

deviations in the table 6:. In the  rst case, NOx was

given from a pure NO2 cylinder, and vapors from this

liquid were pumped using peristaltic pumps into the

reactors. To overcome large deviations in concentration,

case 2 was operated using calibration gases. Data analy-

sis considered only in case 2 because of the NOx feed

was not consistent during case1.Nitrogen mass balance

data summarized in Table 6. The only difference in the

three reactors in case 2 was the loading rates of NOx.

NOx removal: In case 2, NOx removal rates are 59%,

95%, 96% for reactors 2a, 2b, and 2c respectively. Only

59% NOx removal was achieved in reactor 2a, which

had the lowest NOx loading rate. NOx loading rates were

different in three reactors. Aqueous nitrate and nitrite

concentrations in the reactors are shown in Figure3.

Cell growth: The average speci c growth rate for the

 rst 2 days in reactors 2a, 2b, and 2c was 0.03, 0.06,

0.12 respectively, the average speci c growth rates for

the same period in reactors 1a, 1b were both negative,

the growth curves of case 2 presented in Fig. 3, after

loading NOx, growth rates started to decline in all three

reactors and never achieved the value as before NOx

loading, and this declining growth is proportional to the

NOx concentration entering the system. Approximately

48 hours of NOx loading, the inlet concentrations of

NOx do not affect algal growth. Algae took nearly 48

hours to adopt new nitrogen source.

Nitrate /Nitrite: The algae was able to take nitrate in

solution before the loading of into the reactors. After 48

hours Nitrogen source is available only from NOx. The

steady fall in NO3- concentrations and NO2- accumula-

tion in reactor 2c suggests that algae preferred NO3- as

its nitrogen source over NO2-.

CONCLUSION

The primary purpose of this study was to test the

hypothesis that Dunaliella can grow on nitrogen from

dissolved NO2 as its only nitrogen source for cell syn-

thesis. Dunaliella grew used only nitrate /nitrite gener-

ated by the NOx dissolution for cell synthesis, reaching

a maximum cell density of 1.75x107cell/ml. A reactor

with lower NO2 loading concentrations resulted in lower

NOx removal rates, for this reactor, nitrate was not accu-

mulated as ef ciently as a similar with grated NOx load-

ing. In case 2: cell growth of mass between 1850mg/L

to 198mg/L. Nitrogen was removed from gaseous NOx

at a rate of 0.06-0.45 mg N/mg cell growth. Assuming

a 700 MW natural gas  red power plant can produce up

to 1,70,000m3/h of  ue gas with approx. 50ppm NOx

concentrations, growth of a minimun110 kg algal cell/h

would be required to treat this stream.

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