**3. Impact of salinity on specific denitrification rate**

Specific denitrification rates (SDNRs) are usually expressed in the mass of nitrate removed within a unit time in regard to one unit of reactor volume, biomass, biofilm surface, or fixed-film media bed. There had been conflicting reports about the effects of salinity on specific denitrification rates.

Osaka et al. [2] studied two suspended biomass systems fed with acetate acid and methanol, respectively, and found that acetate-fed process attained high nitrate removal at 0–10% NaCl, whereas methanol was shown effective for nitrate removal at 0–3% NaCl without sacrificing efficiencies. Nitrate removal efficiencies were close to 100% at a mass loading of 0.15 g NO3 -N/g MLSS/day or a volumetric loading of 0.75 kg NO3 -N/m3 /day. This study was carried out in a manner that allowed enough time (at least 20 days) for microbial communities to adapt to a higher salinity with a 1% incremental change.

Similar to the observation by Osaka et al. [2], the denitrification rate with methanol as a carbon source was unaffected by sodium chloride up to 2% in a fluidized bed biofilm reactor with media carriers encapsulated with mixed denitrification cultures [3].

SDNR was 0.06 g NO3 -N/g MLSS/day for a freshwater system without salt spiking; SDNR appeared not to be affected (similar to 0.06 g NO3 -N/g MLSS/day) for a system with 5 g/L salt spiking, and it only slightly decreased to 0.048 g NO3 -N/g MLSS/day for an acclimated system with 30 g/L salt addition [4]. In fully acclimated systems (two bench-scale sequencing batch reactors operated in parallel for 4 months), as complete denitrification occurred, the maximum specific nitrate reduction rate was 1.2 g NO3 ± N/g MLSS/day at a wastewater TDS concentration of 4.8% with acetate as a carbon source and the denitrification rate was decreased to 0.456 g NO3 ± N/g MLSS/day at 18% TDS [5]. These studies suggest that acclimated (to saline water) systems appeared less sensitive to salinity increase.

The maximum nitrification and denitrification rates were 0.05 and 0.036 g NO3 -N/g VSS/day, respectively, in a down-flow hanging sponge reactor treating phenol (electron donor for denitrification) and ammonia wastewater. The system had been acclimated for 1100 days with 10.9 g/L chloride before the study where a dominant species, *Azoarcus*-like species, was found [6]. The maximal denitrification rate achieved with ethanol mixture (industrial byproduct) (0.64 g N-NOx /g VSS/d) was much higher than the rate reached with methanol mixture (industrial byproduct) (0.11 g N-NOx /g VSS/d) at sulfate concentrations of 1.5–2% after 450 days of operation [7]. Pure culture *Pseudomonas stutzeri* in a packed bed bioreactor achieved high denitrification rate of 0.84 kg NO3 -N/(m3 /day) or 0.025–0.13 g NO3 -N/g biomass/day at 10 g/L salinity [8]. The strain PAD-2 (closely related to *M. alkaliphilus*) in genus *Marinobacter* of γ-*proteobacteria* exhibited higher denitrification rates at concentrations of 3–6% than at other salinities of 12–18% w/w [9].

depleted before the denitrification unit. Under these situations, external supplementation of organic substances (electron donors) is usually needed to generate dedicated microbial communities. Generally, an external carbon source, such as methanol, ethanol, acetic acid, glycerol, sugar, or molasses, is used as a supplement. Another commercial product worth mentioning is MicroCTM. It is manufactured by Environmental Operating Solutions Inc. (Bourne, MA) and is an environmentally benign, proprietary wastewater treatment chemical containing a mixture of organic compounds, mainly glycerol. It contains 670,000 mg/L chemi-

**Carbon source COD/N Yield (gVSS/gCOD)** *μ***max (d−1)** *kD* **(mgN/gVSS-h)**

2 (20°C)

4.8 (25°C)

3.5 (19°C)

32 (15°C) 91 (20°C)

46 (15°C) 139 (20°C)

13.6

Methanol 4.1–4.5 0.23–0.25 0.77 (15°C)

Ethanol 5.9 0.25–0.28 1.89 (15°C)

Acetate 5.7 0.35 1.2 (13°C)

**Table 1.** Kinetic information of selected carbon sources [1].

2 Nitrification and Denitrification

Glucose 8.9 0.38 3.8

The stoichiometric reaction C/N ratio, yield, specific growth rate, and Arrhenius temperature factor are different for different carbon sources, some of which are summarized in **Table 1**. It should be advised that these parameters were obtained with municipal wastewater under different acclimation and feeding conditions and microbial compositions, and the use of these

Specific denitrification rates (SDNRs) are usually expressed in the mass of nitrate removed within a unit time in regard to one unit of reactor volume, biomass, biofilm surface, or fixed-film media bed. There had been conflicting reports about the effects of salinity on specific denitrification rates. Osaka et al. [2] studied two suspended biomass systems fed with acetate acid and methanol, respectively, and found that acetate-fed process attained high nitrate removal at 0–10% NaCl, whereas methanol was shown effective for nitrate removal at 0–3% NaCl without sacrificing efficiencies. Nitrate removal efficiencies were close to 100% at a mass loading of 0.15 g

out in a manner that allowed enough time (at least 20 days) for microbial communities to

Similar to the observation by Osaka et al. [2], the denitrification rate with methanol as a carbon source was unaffected by sodium chloride up to 2% in a fluidized bed biofilm reactor



/day. This study was carried


cal oxygen demand (COD) with a specific gravity of 1.22 g/mL at 25°C.

**3. Impact of salinity on specific denitrification rate**


with media carriers encapsulated with mixed denitrification cultures [3].

adapt to a higher salinity with a 1% incremental change.

appeared not to be affected (similar to 0.06 g NO3

parameters should be with care.

NO3

SDNR was 0.06 g NO3

On the contrary to the above studies, it was concluded that denitrification rates were severely affected with salt spiking. At 1.52% of salt spiking, a specific denitrification rate decreased by half from 0.7 to 0.35 kg NO3 -N/m3 /day [10]. In another study by Ucisik and Henze [11], it was found that a specific denitrification rate decreased with an increasing chloride concentration in a suspended growth system fed with acetate, and the maximal specific denitrification rate decreased from 1.2 kg NO3 -N/m3 /day at 0.48% chloride down to 0.04 kg NO3 -N/m3 /day at 9.67% chloride. However, this study may still have suffered from insufficient acclimation time, as at each chloride concentration level, the microorganisms were only allowed to acclimatize for 4–5 days. The spiking of salt sharply reduced the microbial activity in an activated sludge system seeded with municipal sludge. When salt concentrations were below 10 g/L NaCl, microorganisms were able to acclimatize in several weeks and achieve the same initial activity as in raw sludge samples; when the salt concentration was above 30 g/L NaCl, the acclimatization process was slow [12]. A mathematical model was developed to predict the SDNR at different salt spiking levels where a salt inhibition constant was identified to be 1.52% (SDNR was reduced by half) [10].

**Table 2** summarizes SDNR in high-salinity wastewater and SDNR varied from 0.75 to 4.8 kg NO3 -N/m3 /day or 0.025 to 1.2 g/g biomass/day, depending on the salinity levels, carbon sources, and temperature. It appeared that biofilm systems had relatively higher volumetric denitrification rates as compared to the suspended growth systems. A maximal denitrification rate of 4.8 kg NOx -N/m3 media bed/day(sintered fly ash) was achieved in a fluidized bed reactor; 2.5 kg NO3 -N/m3 /day was achieved with a reactor filled with sponge cubes for microbial attachment; and 0.84 kg NO3 -N/m3 /day was achieved in a packed bed reactor (with clinoptilolite). These observations of high rates were perhaps attributed to higher specific surface area of carrier media and higher biomass density. Furthermore, in a biofilm reactor filled with cellulose triacetate carriers encapsulated with mixed denitrification cultures, an exceptionally high denitrification rate of 11 kg/m3 media bed/day was achieved [3].


Introductory Chapter: Effects of Salinity on Biological Nitrate Removal from Industrial Wastewater http://dx.doi.org/10.5772/intechopen.69438 5


VSS, volatile suspended solids; MLSS, mixed liquor suspended solids; TDS, total dissolved solids; COD, chemical oxygen demand.

**Table 2.** Denitrification rates under different conditions.

In summary, it appeared that denitrification efficiency will drop upon an initial increase of salinity and can be sustained if biomass is properly acclimated and adapted to corresponding salinities, and rates were comparable to that at low-salinity concentrations. However, in fullscale installations, this effect may be pronounced during the initial period of commissioning, which usually required the designer to provide enough redundancy for the process, or seed the process with an acclimated culture obtained elsewhere to speed up the process.

### **4. Halophilic cultures**

**Denitrification rate Acclimation and** 




4 Nitrification and Denitrification



> -N/m3 /day




day) at 10 g/L salinity; 0.025–0.13 g NO3

0.84 kg NO3

biomass.day)

day or 0.15 g NO3

N/g MLSS/day (10% salinity with acetic

0.75 kg NO3

1.2 kg NO3

0.7 kg NO3

2.5 kg NO3

0.8 kg NO3

0.64 g N-NOx

methanol

1.2 g NO3

4.8 kg NOx

11 kg NOx

kg NOx

media bed/day

g NO3

with ethanol; 0.11 g N-NOx

/g VSS/d with industrial waste

 ± N/g MLSS/ day at TDS 4.8%; 0.456

 ± N/g MLSS/ day at 18% TDS



> -N/m3 /day

at 10% salinity

at 4.8 g/L chloride; 0.04 kg NO3

at 96.7 mg/L chloride

for 0% NaCl and 0.35 for 1.52% NaCl

A slight drop in nitrogen removal, NR, and DNR was observed, when the salinity was increased from 4.2 to 9.8 g NaCl/L

acid)

**culture**

The saline concentration was steadily increased by 1% salinity with NaCl from 0%; at each salinity level, at least 20 days were maintained

At each chloride level, 4–5 days were allowed for acclimation

*Halomonas* sp. and *Marinobacter* sp.; seed sludge was acclimated for 3

acclimated for 1100 days prior to the

Reactors operated in parallel for 4 months

Media carriers encapsulated with mixed culture

The two-sludge plant was operated continuously for 450 days, using real, highstrength industrial wastewater

years

study

0.036 g/g-VSS/day *Azoarcus*-like species;

/g VSS/d

/day *P. pantotrophus and P. fluorescens*

**Carbon source Salinity System Reference**

0–100 g/L Suspended

Acetate 4.8–96.7 g/L Suspended

NaCl/L

Acetate 2 and 10% Sponge cubic

Up to 35 g/L Cl− and 17 g/L SO4 2−

1.5–2.0% SO4 2−

bed system (clinoptilolite)

growth system

growth system

system (1 cm plastic tubes)

media

Bacteria encapsulated in porous polyvinyl alcohol lenses

/L Down-flow

hanging sponge reactor

Suspended growth system

Suspended growth system

system

Fluidized bed system

Sequential batch biofilm reactor

[8]

[2]

[11]

[10]

[17]

[14]

[15]

[6]

[7]

[5]

[18]

[3]

*Pseudomonas stutzeri* Ethanol 10–40 g/L Packaged

Spiking Sugar 0–6% Packaged bed

Intrinsic COD 4.2–9.8 g

Biodegradable hydrocarbons Brenntaplus VP1

Industrial ethanol mix; industrial methanol mix

Phenol 10.9 g Cl−

Acetate 4.8, 16, and

Acclimated Acetic acid 45 g/L Cl− Fluidized bed

Methanol 0–30 g/L

18%

NaCl

Acetate and methanol

> Halophilic bacteria are microorganisms that do not need sodium chloride to grow but can grow in high-salinity environments. Halophilic bacteria are classified into three groups according to their response to sodium chloride concentrations: (i) the slight halophiles (most rapid growth at 2–5% NaCl), (ii) the moderate halophiles (most rapid growth at 5–20% NaCl), and (iii) the extreme halophiles (most rapid growth at 20–30% NaCl) [13].

> The phylogenetic analysis showed that the strains isolated from acclimated sludge (to saline water) had a high similarity to the genus *Alcaligenes* in β-*proteobacteria* and the genera *Vibrio, Pseudomonas, and Halomonas* in γ-*proteobacteria*. Genera *Halomonas and Marinobacter* in γ-*proteobacteria* were isolated [14]. α-*Proteobacteria* were also found [6]. *Azoarcus*-like species in β-*proteobacteria* was identified to conduct denitrification using phenol [6]. It was found that the dominant species shifted when salinity varied [14].

> Researchers used microorganisms *P. fluorescens* and *P. pantotrophus* for denitrification in saline water [15]. *P. stutzeri* in the packed bed bioreactor achieved a high denitrification rate at 10 g/L salinity [8], and the strain PAD-2 (closely related to *M. alkaliphilus*) in genus *Marinobacter* of γ-*proteobacteria* also exhibited high denitrification rates at concentrations of 3–6% [9]. The species

*M. aquaeolei* sp. *nov*. was found to grow under anoxic conditions in the presence of nitrate on succinate, citrate, or acetate, but not on glucose. It was also interesting that *H. campisalis* sp. *nov*. grew on acetate, lactate, glycerol, and ethanol but not on methanol [16].

**Table 3** summarizes the species capable of denitrifying under saline conditions. Most of the species were in the class of *γ-proteobacteria*. Species were found to even survive in a wide range of salinity as high as 23.4%.
