3.1 Performances of the different carbon sources for denitrification and acclimation
In this study, sludge six carbon sources (methanol, ethanol, glycerol, glucose, sucrose, and starch) were used to denitrify and acclimate sludge. The carbon sources were observed to exhibit varying denitrification response. Ethanol- and glycerol-acclimated sludges exhibited denitrification effect within one week, and were acclimated within four weeks, whereas the glucose- and sucrose-acclimated sludges required eight weeks to complete the denitrification and acclimation processes. When methanol was used as the carbon source, the acclimation process was longer, as nine weeks was required to achieve denitrification. In addition, the starch-acclimated sludge did not exhibit notable denitrification effect until 12 weeks, which can be attributed to the difficulties in the hydrolysis of starch under anoxic conditions.
The concentrations of COD, NO3−-N, and NO2−-N in the sludges acclimated by the various carbon sources are shown in Fig. 1. Except the starch-acclimated sludge, the sludges acclimated by other carbon sources exhibited notable denitrification effect. The yield (YH) was calculated using the ASM1 model as follows:
$$\frac{Ss}{{S}_{NO}}=\frac{2.86}{1-{Y}_{H}}$$
The saccharides carbon sources exhibited the highest sludge yield, whereas methanol exhibited the lowest sludge yield (Table 1), which is consistent with the results reported in previous study.
Table 1
Yield of different carbon sources-acclimated sludge.
Carbon | methanol | ethanol | glycerol | glucose | sucrose |
YH | 0.11 | 0.40 | 0.46 | 0.58 | 0.60 |
3.2 Denitrification performances of alcohols- and saccharides-acclimated sludge under VFA carbon sources conditions
To evaluate the practicability of additional carbon sources, the influent carbon sources (VFAs) should also be considered. A significant decrease in the denitrification efficiency of influent carbon after the addition of external carbon sources indicates that the denitrification process will rely entirely on external carbon sources, which will increase the use of external carbon sources and result in the waste of the influent carbon sources. Consequently, this results in a “carbon dependence” problem in practical WWTPs. Therefore, it is essential to consider the VFAs carbon compatibility of external carbon.
The denitrification performances of alcohols (methanol, ethanol, glycerol)- and saccharides (glucose, sucrose, starch)-acclimated sludges under VFAs conditions are shown in Fig. 2. Compared to the seed sludge (Fig. 2g), the denitrification effect of VFA carbon sources was completely lost in the methanol-acclimated sludge (Fig. 2a), which may be attributed to the unique metabolic characteristics of methylotrophy bacteria. In contrast, the denitrification capacity of the VFA carbon sources was preserved in the ethanol-acclimated sludge, and the denitrification rate of propionate increased from 4.68 mg-N/gMLVSS*h to 8.46 mg-N/gMLVSS*h (Fig. 2b). In addition, the denitrification effect of the VFA carbon sources was improved in the glycerol–acclimated sludge, in which the denitrification rate of acetate, propionate, and butyrate increased to 13.16, 12.02, and 9.80 mg-N/gMLVSS*h, respectively (Fig. 2c).
The denitrification ability of the VFA carbon sources was preserved in the saccharides-acclimated sludge(Fig. 2d, e, f), which can be attributed to the conversion of carbohydrates to Co-A through glycolysis, and the subsequent entry into the tricarboxylic acid cycle (TCA), which is the same metabolic pathway as that of VFA carbon sources; however, the metabolic pathway of carbohydrates is more complex. The denitrification rate of VFAs in the saccharides-acclimated sludge was different (Fig. 1). The denitrification rate of acetate was significantly increased in the glucose-acclimated sludge to 14.13 mg-N/gMLVSS*h. In addition, the denitrification rate of propionate in the sucrose-acclimated sludge increased to 7.08 mg-N/gMLVSS*h, whereas those of acetate and butyrate decreased slightly to 7.02 and 5.52 mg-N/gMLVSS*h, respectively. The denitrification rate of acetate, propionate, and butyrate in the starch-acclimated sludge decreased to 3.83, 2.67, and 3.69 mg-N/gMLVSS*h, respectively. It should be noted that saccharides-acclimated sludge generally exhibits a high NO2-N accumulation when VFAs are used as the denitrifying carbon source (Fig.S1). The glucose-acclimated sludge exhibited a high NO2-N accumulation concentration for propionate, and denitrification was completed within 4 h. The sucrose-acclimated sludge exhibited a higher NO2-N accumulation concentration for VFAs, but denitrification was rapidly completed within 2 h. Starch-acclimated sludge exhibited a rapid NO2-N accumulation for acetate and butyrate, and denitrification was completed within 2 and 3 h, respectively.
3.3 Denitrification performances of alcohols- and saccharides-acclimated sludge under alcohols and saccharides conditions
Commonly used external carbon sources for actual wastewater denitrification, such as alcohols (e.g., methanol and ethanol) and sugars (e.g., glucose and sucrose), can achieve stable denitrification effect after a period of adaptation. The addition of alcohols (methanol, ethanol, and glycerol) and saccharides (glucose, sucrose, and starch) to the seed sludge did not induce a notable change in the denitrification rate (Fig. 3g). Therefore, the denitrification rates of the acclimated sludge differed in the presence of different alcohols and saccharides, and the relationship between the metabolic characteristics of different carbon sources was explored.
The denitrification performances of alcohols (methanol, ethanol, glycerol)- and saccharides (glucose, sucrose, starch)-acclimated sludge are shown in Fig. 3. The single carbon acclimated sludge exhibited notable denitrification effect on multiple carbon sources. Both methanol and ethanol exhibited significant denitrification effect in the methanol-acclimated sludge (Fig. 3a), with denitrification rates of 14.42 mg-N/gMLVSS*h and 9.65 mg-N/gMLVSS*h, respectively. In addition, methanol and ethanol exhibited significant denitrification effect in the ethanol-acclimated sludge (Fig. 3b) with denitrification rates of 7.80 mg-N/gMLVSS*h and 22.23 mg-N/gMLVSS*h, respectively. Furthermore, as methanol and ethanol can be used for denitrification in the methanol- and ethanol-acclimated sludge, there is a potential correlation between the functions of methanol- and ethanol-acclimated sludges. The glycerol-acclimated sludge exhibited significant denitrification effect on methanol, ethanol, glycerol, and glucose (Fig. 3c) with denitrification rates of 11.99, 12.93, 15.42 and 4.73 mg-N/gMLVSS*h, respectively. This indicated the ability of glycerol to denitrify both carbohydrate and alcohol carbon sources after acclimation, and possible similarity in the metabolic pathway of glycerol and those of alcohols and saccharides.
The denitrification effects alcohols and saccharides carbon sources in saccharides-acclimated sludge were similar. Glycerol, glucose, and sucrose exhibited denitrification effects in the glucose- and sucrose-acclimated sludge (Fig. 3d, e). The denitrification rates of glycerin, glucose, and sucrose in the glucose-acclimated sludge were 8.73, 6.30, and 7.41 mg-N/gMLVSS*h, respectively. This indicated that the denitrification rates of glycerol and sucrose in the glucose-acclimated sludge were higher than that of glucose despite acclimation by glucose. The denitrification rates of glycerol, glucose, and sucrose in sucrose-acclimated sludge were 1.49, 4.14, and 4.03 mg-N/gMLVSS*h, respectively. Compared to that in the glucose-acclimated sludge, the denitrification effect of glycerol in the sucrose-acclimated sludge was not significant. Ethanol, glycerol, and glucose exhibited significant denitrification effect in the starch-acclimated sludge (Fig. 3f); however, owing to the difficulties in the short-term hydrolysis of starch, the starch did not exhibit a denitrifying effect in the sludge.
There was a high accumulation of NO2-N during the denitrification of the saccharides-acclimated sludge (Fig.S2), and the NO2-N accumulation of glucose was the most significant. The glucose-acclimated sludge exhibited high NO2-N accumulation when glucose, sucrose, and glycerol are used for denitrification. The concentration could be above 20 mg/L and lasted for 5. This indicates that the denitrification of glucose-acclimated sludge by alcohol and saccharide carbon sources was not sufficient. When glucose and sucrose are used for denitrification, the sucrose-acclimated sludge exhibited high NO2-N accumulation, and compared to the glucose-acclimated sludge, the denitrification could be completed in 4 h.
3.4 Insights into the metabolic similarity of denitrification carbon sources
3.4.1 Analysis of the microbial community structure in different carbon-acclimated sludge
The denitrification rate of VFAs, alcohols, and saccharides in different carbon acclimated sludge is shown in Fig. 4, and the relationship between the denitrification rates of different carbon sources can be observed in this image. Microbial community is the functional entity driving the degradation and transformation of pollutants. Therefore, the community structure of activated sludge can reflect the functional characteristics of the system, and similar microbial compositions will exhibit more similar functions. The relative abundance of Patescibacteria phylum in the glucose-acclimated sludge and glycerol-acclimated sludge was 30.69 and 22.01%, respectively, which was significantly higher than those in other groups (Fig. 5a). Studies have the ability of the predominant Patescibacteria to improve the nitrogen- and phosphorus-removal function of sludge, and it often co-exists with denitrification bacteria in groundwater, which plays an important role in the nitrogen removal process (Herrmann et al., 2019; Hosokawa et al., 2021). Compared to the seed sludge, the alpha diversity of the acclimated sludge was significantly decreased, which can be attributed to the elimination of aerobic bacteria during anoxic acclimation (Fig. 5c). The methanol methanol-acclimated sludge exhibited the highest chao1 index, but the lowest shannon index, indicating the methanol significant enrichment of specific bacterial communities in the sludge, as well as the presence of several rare species, resulting in a high species richness but low diversity. Bray Curtis distance revealed that the ethanol-acclimated sludge, glucose-acclimated sludge, and glycerol-acclimated sludge exhibited similar microbial community structure (Fig. 5d) and a high denitrification rate to many carbon sources.
3.4.2 Metabolic pathway analysis of the methanol-acclimated sludge
As one of the one carbon (C1) compounds, methanol exhibits special characteristics in the metabolism of microorganisms (Anthony, 2011; Baytshtok et al., 2009; Mokhayeri et al., 2006), and methylotrophic bacteria are a diverse group of microorganisms that possess a significant number of specialized enzymes (Schrader et al., 2009). There are two types pathways for methanol metabolism in microorganisms: assimilation pathway and dissimilation pathway (Yurimoto et al., 2005). The assimilation pathways include the serine and ribulose monophosphate (RuMP) pathways, and the dissimilation pathway generally refers to the oxidation pathway of methanol, including glutathione-dependent and folate-dependent pathways. All these pathways require the oxidation of methanol to formaldehyde by methanol dehydrogenase (mdh1) before the next reaction.
The methane metabolism pathway consists of serine and RuMP pathways. Figure 6a indicates that the enzymes related to the RuMP pathway and the serine pathway are highly expressed in the methanol-acclimatized sludge. In the RuMP pathway, formaldehyde was condensed with Ribulose 5-P (Ru5P) to produce Hexulose 6-P (Hu6P), which was catalyzed by 3-hexulose-6-phosphate synthase (hxlA). Thereafter, Hu6P was converted to fructose 6-phosphate (Fu6P) under the action of 6-phospho-3-hexuloisomerase (hxlB). Subsequently, Fu6P was cracked to form Fructose 1,6-bisphosphate (F1,6P), which was catalyzed by fructose-bisphosphate aldolase (FBA) to form Glyceraldehyde 3-phosphate (GAP). The GAP entered the EMP pathway to form pyruvate, and was completely metabolized in the glyoxylate cycle. In the serine pathway, after methanol was oxidized to formaldehyde, 5,10-methylene-THMPT was generated and catalyzed by 5,6,7,8-tetrahydromethanopterin hydrolyase (fae), after which 5,10-methylene-THMPT was converted to Formyl-MFR under the influence of methylenetetrahydromethylpterin dehydrogenase (mtd), methylenetetrahydromethylpterin cyclization hydrolase (mch), and other enzymes. Formyl-MFR was catalyzed by Formyl methoxyfuran dehydrogenase (fwdA) to produce formate, which was synthesized to Methylene H4F through a series of reaction. Thereafter, Methylene H4F and glycine were combined to synthesize serine under the influence of glycine hydroxymethyltransferase (glyA). Serine was converted to hydroxypyruvate under the action of serine-pyruvate transaminase (AGXT) in the serine metabolism pathway, after which it entered the glyoxylate cycle for complete metabolism via the hydroxypyruvate → glycerol → 2-phosphate → glycerate → phosphoenolpyruvate → oxaloacetate metabolism.
The main metabolic pathways of methanol involved seven important metabolic pathways: methane metabolic pathway, serine metabolic pathway, cyanamino acid metabolic pathway, pentose phosphate pathway, glycolysis, nitrogen metabolism, and glyoxylate cycle. Both the RuMP pathway and the serine pathway of methanol eventually enters the metabolic pathway of glyoxylate cycle, whereas the tricarboxylic acid cycle metabolism was not significant in the metabolism of methanol, indicating that the glyoxylate cycle was more important in the metabolism of methanol. Ethanol can be used for denitrification in the methanol-acclimated sludge owing to the entry of ethanol into the glyoxylate cycle metabolism.
The difficulties in the use of other types of carbon sources for denitrification in methanol-acclimated sludge can be attributed to the unique metabolic mode of methanol. Moreover, owing to the lack of enzymes related to fatty acid metabolism pathway, VFAs, which do not exhibit notable denitrification effect, cannot be easily metabolized by the methanol-acclimated sludge (Fig. 6d).
3.4.3 Metabolic pathway analysis of ethanol-acclimatized sludge
Ethanol-acclimated sludge exhibits various metabolic pathways (Fig. 6a,d), including glycolysis pathway, TCA cycle, pyruvate metabolism, glyoxylate cycle, propionate metabolism, and fatty acid metabolism. The main metabolic pathway of ethanol is the TCA cycle. First, ethanol is converted to acetaldehyde by alcohol dehydrogenase (exaA), after which it is converted to acetate under the influence of aldehyde dehydrogenase (ALDH). Acetate generated acetyl-P catalyzed by acetic acid kinase (ackA), after which it is converted to Acetyl-CoA through phosphate acetyltransferase (E2.3.1.8) and entered TCA cycle. In addition, acetyl-CoA can enter the glyoxylate cycle. Acetyl-CoA and glyoxylate synthesized malate catalyzed by malate synthase (aceB), and then complete the glyoxylate cycle according to the metabolic pathway of: malate → oxaloacetate → citric → isocitrate → glyoxylate.
In addition to TCA cycle and glyoxylate cycle, the ethanol-acclimated sludge also exhibited an active propionate metabolic pathway, which confirmed the significant improvement in the denitrification rate of propionate in the ethanol-acclimatized sludge. After the entry of propionate into the metabolic pathway, propionyl-CoA was generated via catalysis by propionate CoA transferase (pct), which could be converted into succinyl-CoA, after which it enters the TCA cycle. In addition, glutathione metabolism and folate-carbon pool were detected in the ethanol-acclimated sludge, which can be attributed to the dissimilation metabolic pathway of methanol. This suggested that ethanol-acclimated sludge can effectively use methanol for denitrification. After the introduction of methanol into the ethanol-acclimated sludge, first, it was oxidized to formaldehyde by alcohol dehydrogenase. Thereafter, formaldehyde was oxidized to formate with the participation of glutathione or tetrahydrofolate. Formate enters into the serine cycle and glyoxylate cycle, and could also be directly oxidized and decomposed into CO2.
3.4.4 Metabolic pathway analysis of glycerol-acclimated sludge
Glycerol is mainly involved in the glycolysis pathway and TCA cycle of microorganisms. Glycerol is converted to α-glycerol phosphate by glycerol kinase (glpK), and then catalyzed to glycerone-P by glycerol-3-P dehydrogenase (glpA). The glycerone-P was converted into pyruvate during glycolysis, after which it enters the TCA cycle to complete decomposition. In addition to the oxidation pathway, glycerol-acclimated sludge also exhibited significant active fatty acid biosynthesis and degradation metabolism. The acetyl-CoA produced in TCA cycle could participate in fatty acid biosynthesis metabolism through fabB enzyme, after which it is completely degraded during the fatty acid degradation metabolism. fabB enzyme (3-oxyacyl - [acyl carrier protein] synthase I) is a type of fatty acid synthetase, which can synthesize acetyl-CoA into long-chain fatty acids. ACADM (acyl-CoA dehydrogenase), fadN (3-hydroxyacyl-CoA dehydrogenase) and fadA (acetyl coenzyme A acyltransferase) participated in the degradation and metabolism of fatty acids, which were also highly expressed in the glycerol-acclimated sludge. Therefore, the glycerol-acclimated sludge exhibited strong fatty acid metabolism ability, which improved the denitrification rate when VFAs were used as carbon source.
Glutathione metabolism pathway, nicotinate, and nicotinamide metabolism and ABC transport vehicle were also highly expressed in the glycerol-acclimated sludge. The glutathione generated in the glutathione metabolism pathway could participate in the oxidation pathway of methanol. Therefore glycerol-acclimated sludge exhibited denitrification capacity for methanol. In the metabolism of nicotinate and nicotinamide metabolism, L-Aspartate could synthesize NAD+ through L-aspartate oxidase (nadb), nicotinate-nucleotide pyrophosphorylase (nadC), and NAD+ synthetase (E6.3.5.1), which can enhance the efficiency of electron transport, thus improving the denitrification process. Glycerol transport system permease protein (glpP) in the metabolism of ABC transport vehicle was the key protein responsible for the absorption of extracellular glycerol, which was significantly enhanced in the glycerol-acclimated sludge, by microorganisms,. Therefore, other carbon sources-acclimated sludge cannot use glycerol for denitrification in the absence of glpP.
Glycerol and ethanol also participated in metabolism. Ethanol could generate acetate after dehydrogenation, after which it enters into the metabolism of TCA cycle. The related enzymes were contained in glycerol-acclimated sludge, which can effectively use ethanol for denitrification, whereas the ethanol-treated sludge cannot use glycerol for denitrification, which may be attributed to the lack of glycerol kinase and glpP.
3.4.5 Metabolic pathway analysis of saccharides-acclimated sludge
Saccharides carbon sources are mainly involved in glycolysis metabolism and TCA metabolism. Starch is a polymer polysaccharide made from glucose, and sucrose is a disaccharide composed of one molecule of glucose and one molecule of fructose. The metabolism of starch, sucrose, and glucose are similar owing to their similar structural composition. Starch is hydrolyzed to glucose by α-amylase (AMY) and glucan 1,4-α-glucosidase (susB). Starch metabolic pathway was characteristically expressed in the seed sludge, but not in the starch acclimated sludge, indicating the difficulties in realizing the hydrolysis of polysaccharides in anoxic acclimated environment. Sucrose was metabolized by the hydrolysis of β- fructofuranosidase (INV) and α-glucosidase to produce one molecule of glucose and one molecule of fructose. Thereafter, glucose was catalyzed by glucokinase (glk) to produce D-6-phosphoglucose, whereas fructose was catalyzed by glucokinase (E2.7.1.4) to produce D-6-fructose phosphate, which was catalyzed by glucose-6-phosphate isomerase (GPI) to produce D-6-phosphoglucose. Lastly, D-6-phosphoglucose enters the glycolysis pathway and TCA cycle for complete metabolism.
Generally, glucose is formed during the glycolysis of saccharides, but the formation of glucose differs. Therefore, there is a certain similarity in the denitrification of saccharides carbon sources. Both the glucose- and sucrose-acclimated sludges can utilize both sucrose and glucose for denitrification. The difference is that glucose- acclimated sludge exhibits a higher denitrification rate. This may be attributed to the shorter metabolic pathway and faster electron generation rate of glucose. In addition, the glucose-acclimated sludge can effectively use glycerol for denitrification. This may also be because most of the metabolic pathways of glycerol are concentrated in the glycolysis process, and the common metabolic pathways enables the utilization of the two carbon sources by the respective acclimated sludge. In addition, the saccharides-acclimated sludge retained the denitrification capacity of VFAs carbon sources. The glucose-acclimated sludge significantly improved the denitrifying metabolic capacity of sodium acetate, whereas the sucrose-acclimated sludge improved the denitrifying metabolic capacity of sodium propionate. This is because saccharides and VFA carbon source have a common TCA cycle, and saccharides-acclimated sludge also retained the hydrolysis of VFA.
The saccharides-acclimated sludge exhibited a high NO2-N accumulation when saccharides are used for denitrification. When glucose, sucrose, and glycerol were used for denitrification, the glucose-acclimated sludge could not reduce NO2-N in 5 h. Several metabolic pathways related to biosynthesis were highly expressed in the glucose-acclimated sludge, such as ribosome and gene replication. During the denitrification process, the reduction of NO3-N to NO2-N was faster, and the reduction of NO2-N becomes a rate-limiting reaction. Owing to the electronic competition of biosynthesis, the reduction of NO2-N during denitrification process is blocked, resulting in a large amount of NO2-N accumulation.