LITERATURE REVIEW
2.4 Biological Processes
2.4.2 Biological Denitrification
The microbial reduction of nitrate to gaseous nitrogen, a method used in the treatment of nitrate contaminated groundwater, is termed dissimilatory denitrification or nitrate respiration. Biological denitrification of drinking water is often favoured considerably due to the lower running costs on a large scale to other methods. In this process, nitrate is used instead of oxygen as a terminal electron acceptor in presence of an electron donor, for energy generation. Nitrate is finally converted to innocuous nitrogen gas through a series of four steps (NO3− —> NO2− —> NO —> N2O—> N2). Each step is catalyzed by different functional enzymes which includes nitrate reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide reductase (Nos) respectively (Rich & Myrold, 2004). Denitrifiers, a phylogenetically diverse group of facultative anaerobic bacteria are found widely in the environment and display a variety of different characteristics in terms of metabolism and activities (Mateju et al., 1992;
Soares, 2000).Nearly 130 species of bacteria including archaeabacteria can reduce NO3− to N2. The ability of denitrification is also reported in certain fungi (Zumft, 1997). Major genera of denitrifying bacteria includes Achromobacter, Alcaligenes, Azospirillum, Bacillus, Chrombacter, Corynebacterium, Desulfovibrio, Flavobacterium, Halobacterium, Methanomonas, Moraxella, Paracoccus, Propionibacterium, Pseudomonas, Spirillum, Thiobacillus, and Xanthomonas (Mateju et al., 1992; Myrold, 1998). The majority of denitrifiers are heterotrophic but several autotrophic denitrifying bacteria have been identified, using H2 (Karanasios et al., 2010) or iron (Jha & Bose, 2005) and various reduced-sulphur compounds (H2 S, S, S2 O3, S4O6, SO3) as electron donors (Zhang et al., 2012). Many organic or inorganic compounds including liquids, solids, and gaseous (CH4 and H2) can be used as electron donor/carbon source for denitrification (Mateju et al., 1992). Major factors affecting the choice of a carbon source are: cost, pretreatment, denitrification rates, kinetics, degree of utilization, sludge
production, handling and storage safety/stability (ÆsØy et al., 1998). Solid substrates include wheat straw, plant pruning, reed, birch wood, and biodegradable polymers etc.
Recently, water-insoluble biodegradable polymers such as Poly 3-hydroxybutyrate (PHB), polylactic acid (PLA), polycaprolactone (PCL) and polylactic acid/Poly (3- hydroxybutyrate-co-3-hydroxyvalerate) blend (PLA/PHBV) have been tested for denitrification in drinking water (Xu et al., 2011b). A wide spectrum of liquid organic carbon sources such as methanol, ethanol, glucose, glycerol, acetic acid, and lactic acid have been employed in water treatment (Akunna et al., 1993; Mohseni-Bandpi et al., 2013). The use of liquid or gaseous compounds are preferred and advantageous due to their easier uptake by bacterial cells and faster degradation, resulting in higher denitrification rates (Mohseni-Bandpi & Elliott, 1998). Methanol, ethanol and acetate are the most commonly used carbon sources for denitrification processes related to drinking water treatment (Calderer et al., 2010; Khardenavis et al., 2007). Methanol, being cheapest used in full-scale wastewater treatment plants but not used in drinking water treatment because of its toxicity in treated water (Jensen et al., 2012). However, ethanol is a better alternative to escape methanol toxicity since it is largely produced in some countries from sugarcane and costs less than other carbon sources, and there is no set permissible limit for ethanol in potable water. The acetic acid was found to be more effective in removal of nitrate as it reduces chlorine demand by preventing the growth of non-denitrifying biomass (mostly coliforms) which may cause clogging in reactors (Silverstein, 1997), more yield for denitrifiers, higher denitrification rate, high buffering capacity, low intermediate nitrite in the effluent, moreover, readily metabolised than methanol and glucose (Mohseni-Bandpi & Elliott, 1998). Therefore, use of acetic acid is more suitable over methanol and other fermentable substrates that may result in formation of alcohols and carboxylic acids. In a recent study, Yang et al. (2012) reported preferential use of acetate and citrate with minimal intermediate products accumulation and high nitrate reduction by Pseudomonasstutzeri D6. Heterotrophic denitrification is preferred over autotrophic as slower growth rate, complex process control and need of post treatment for degasification and biomass removal (Mohseni-Bandpi et al., 2013).
Like electron donors, denitrifiers can also use wide array of electron acceptors DO, nitrate, sulphate , selanate, chromate, chlorate, perchlorate, arsenate (Chung et al., 2007; Nerenberg & Rittmann, 2002), bromate (Hijnen et al., 1999), and iron (III).
Similarly a diverse group of bacteria including SRBs, DAsRBs, DIRBs and PCRBs which includes Desulfovibrio desulfuricans (Dalsgaard & Bak, 1994) Desulfovibrio desulfuricans strain 27774 (Marietou et al., 2009), Chrysiogenes arsenatis gen. nov., sp.
nov.(Macy et al., 1996), JMM-4 (Santini et al., 2002), MIT-13, SES-3 strains (Newman et al., 1997), E1HT, MLS10T, BAL-1T, Desulfitobacterium hafniense DCB-2T and Desulfitobacterium frappieri PCP-1T (Niggemyer et al., 2001), arsenic reducers and Geobacter metallireducens is strictly anaerobic, dissimilatory iron-reducer (Murillo et al., 1999), Dechloromonas spp. Perchlorate reducer, can use nitrate as an electron acceptor.
Now days the N2O emissions from wastewater treatment plants are of great concern among world urban water authorities. NO and N2O can cause depletion of the ozone layer as its greenhouse effect is 300 times more potent than CO2 (Ravishankara et al., 2009). N2O is produced as an intermediate in the heterotrophic as well as in autotrophic nitrifying process, mainly ammonia-oxidizing bacteria (AOB) (Kampschreur et al., 2007). In contrast to AOB, the N2O produced by heterotrophic denitrifiers emits less N2O as it remains dissolved in absence of stripping and get time to reduced in to N2(Law et al., 2012). Other factors that promotes N2O emission are low nitrate levels, active stripping, aerated zones, DO (Ahn et al., 2010), change in operational conditions such as pH, electron donor limitation, increased nitrite concentrations, and DO (Law et al., 2012). Generally, higher nitrate concentrations lead to the complete extent of denitrification, with nitrogen gas as more desirable final product however, relative contributions could be dependent on process conditions.
Environmental Factors Affecting Denitrification
DO is having inhibitory effect on denitrification, as later is thermodynamically less favourable biochemical reaction. In fact, oxygen presence in water inhibits nitrous oxide reductase, thus limits nitrous oxide conversion to nitrogen gas (Baumann et al., 1996). Recently, Hocaoglu et al. (2011) observed complete nitrate removal within the DO range of 0.15-0.35 mg/L in an MBR, while it was inhibited at 0.5 mg/L DO.
The pH range preferred by heterotrophic denitrifiers for complete reduction of nitrate to nitrogen gas is considered to be between 6 and 8 (Rust et al., 2000). At low pH (pH < 5) inhibition is due to the accumulation of nitrite or N2O in solution (Glass &
Silverstein, 1998). High pH (pH 8.3) may also arrests the denitrification (Rust et al.,
2000), the optimal pH is site-specific because of the effects of acclimation and adaptation on the microbial biomass in the system.
Denitrifiers can be found in diverse environments from arctic regions to hot springs. In most scientific studies, temperature impact was more significant on denitrification. Denitrification rate gradually increases within the optimal temperature range from 25 to 40°C. However, denitrification has been observed in the range of 2–
50°C and beyond, microbial populations evolves to cope with specific environmental conditions on prolonged incubation such as high temperatures (Braker et al., 2010;
Sprent, 1987). However, the effect of temperature on denitrifying microbial communities is limited to a few studies. Hollocher and Kristjánsson (1992) found two denitrifiers Pseudomonas aeruginosa (mesophilic) and Bacillus (thermophilic) that grow at 70°C in water samples, able to reduce nitrate to N2.