Biological Diversity in Aerated Facultative Lagoon Treating Kraft Cellulose Euent Through Bioaugmentation

Background: This study analyzes the microbiological diversity in an aerated facultative lagoon system with volumetric organic loading rates of 0.2 and 0.6 kgCODm -3 d -1 treating euent from the kraft pulp industry through bioaugmentation. The samples for the study of biological diversity were taken from a laboratory-scale lagoon at steady state treating kraft pulp euent and operated with 0.2 and 0.6 kgCODm -3 d -1 for 120 days. This analysis was performed by identifying the 16s DNA sequencing, through DNA extraction, polymerase chain reaction and agarose gel electrophoresis at 1%. Next, the autochthonous bacteria were named through statistical similarity obtained from the National Center for Biotechnology Information database. The lagoon performance was assessed based on the removal eciency of specics compounds. Results: In the biomass samples collected at steady state, 9 and 12 species of bacteria were identied and the species Bacillus cereus, Bacillus thuringiensis and Paenibacillus glucanolyticus found in this matrix presented signicant removal of the parameters in the kraft euent. In the treatment, it was possible to observe that the removal of organic matter above 50% for chemical demand and above 95% for biochemical demand. The specic compounds were not signicantly removed, but this is a characteristic of biological treatments. Conclusions: We found that the three referred species show great promise in the removal of specic parameters in a lagoon biological treatment system using bioaugmentation.


Background
Biological treatments comprise bioremediation processes, which are de ned by the American Society for Microbiology as the use of living organisms to reduce or eliminate environmental risks arising from the accumulation of toxic chemicals and other hazardous waste [1]. Within the bioremediation process, the following remediation classi cations can be found: natural attenuation, biostimulation and bioaugmentation.
Natural attenuation is a biological process that transforms pollutants into substances that are less harmful to the environment through biodegradation carried out by microorganisms [2].
Biostimulation is the process of improving the conditions of the environment for microorganisms through the addition of nutrients, oxygen, temperature adequacy, pH, aeration or reduction potential. Thefore, there is an improvement in the performance of microorganisms in the treatment process [2].
Bioaugmentation is related to the introduction of allochthonous bacteria, which are not native to the environment, or autochthonous bacteria, which are native to the environment, isolated or in consortium.
This process is based on the spontaneous and controlled action of microorganisms to increase their quantity, allowing them to degrade pollutants in the soil, water bodies, and in industrial and domestic e uents [1]. The microorganisms used can also act in synergy with the local native species, without interfering with natural biogeochemical processes [3].
According to [4], the application of biotechnological processes involving microorganisms in consortium or individually has been on the increase. In this sense, the great motivation of researchers involved in biodegradation studies is the continuous search for versatile microorganisms capable of e ciently degrading many pollutants at a low operating cost [5].
Among the biological systems used in the treatment of e uent from the pulp and paper industry, aerated facultative lagoons (AFL) stand out [6][7][8]. In Brazil, such systems are widely used due to climatic conditions, their simple maintenance, low cost and greater stability against shock loads than other systems, such as activated sludge [9,10].
Before 2007, the bioaugmentation technique was not used in Brazil since it depended on the agreement and authorization of government agencies and environmental inspection agencies, such as the Companhia de Tecnologia de Saneamento Ambiental [1] (CETESB  [11].
Thus, bioaugmentation started to be allowed in Brazil, more speci cally in the State of São Paulo, following the speci c guidelines presented in the referred document. Among the standards to be followed, CETESB's technical standard No. L1.022 stands out for referring to the use of biotechnological products, which consist of microorganisms intended for the treatment of liquid e uents, solid waste and soil and water remediation [11].
Bioaugmentation can also be used with genetically modi ed organisms to improve the treatment system.
However, since these are allochthonous microorganisms, there is a Brazilian legislation that regulates their use: Biosafety Law No. 11.105/05, in effect since March 24, 2005 [12].
The autochthonous bacterial species, commonly present in biological treatments of cellulose e uents, can live in extreme environments of temperature, pH, biochemical oxygen demand (BOD 5 ), chemical oxygen demand (COD) and low oxygen concentration [13]. Therefore, due to such characteristics, they are of signi cant importance to the treatment of kraft e uent [14].
The objective of this paper is to analyze the microbiological diversity in an AFL system treating e uent from the kraft pulp industry with volumetric organic loading rates (OLR) of 0.2 and 0.6 kgCODm -3 d -1 through bioaugmentation. [1] Environmental Sanitation Technology Company.

Method
Collection, biological treatment and performance analysis of aerated facultative lagoon Aerated facultative lagoon treatment system The industrial e uent used for continuous treatment in the AFL system was kindly provided by an unbleached kraft pulp mill based in the metropolitan region of Curitiba, in the state of Paraná, Brazil. The aforementioned mill treats its e uents through biological systems with aerated facultative lagoons and tertiary treatment to reach discharge criteria.
The continuous biological treatment used in this research was carried out in a bench-scale AFL reactor, as shown in the scheme in Figure 1, in transparent acrylic material with 1 L of useful volume and a sedimentation zone, to which the OLRs of 0.2 and 0.6 kgCODm −3 d −1 were applied for a period of 120 days and separated into the two lagoons. Aeration was promoted by air pumps with a owrate of 35 L/h and the dissolved oxygen was measured in parallel with the temperature inside the reactor, in the aeration zone and in the sedimentation zone [15].
For the composition of the AFL system biomass, we used sludge from the bottom of the aerated lagoon of the mill that provided the e uent for the study. The biomass was inoculated to a nal concentration of 70 mgVSSL −1 , an intermediate value among those used in biological systems [16].
The e ciency of the treatment system was assessed as a function of the reduction in the following parameters: BOD 5 , COD, total organic carbon (TOC), lignin-derived compounds, total phenolic compounds (TPC), aromatic compounds, color and turbidity [17][18][19], whose analyses were performed at the Multiuser Laboratory of Chemical Analysis of the Federal Technological University of Paraná (LAMAQ-UTFPR).

Microbiological analysis a) Isolation, cultivation and gram staining
The microbiological analyses to identify the groups of bacteria that were present were carried out at the Microbiology Laboratory of the Federal University of Paraná (UFPR). The isolated and analyzed samples came from the nal stabilization period of each OLR in the lagoons.
Thus, to separate the cultivated bacteria, we used the technique of isolation of colonies grown in culture media. The method is based on the seeding of microorganisms on the surface of solid culture media in Petri dishes [20]. The culture media used were nutrient agar and Luria-Bertani medium. After the seeding, the dishes were incubated in an oven at 37°C, where they remained for 24 hours in the absence of light and CO 2 .
The isolated bacteria were morphologically characterized through the characteristics of the bacterial colonies, classifying them as Gram-negative and Gram-positive [21]. Gram stain slides were analyzed under an optical microscope at 400x magni cation to classify bacteria into Gram-negative and Grampositive.

b) DNA extraction
In order to analyze the microbiological diversity, DNA extraction from the bacteria was initially performed by adapting the method employed by [22], which consists of three phases: membrane lysis, cleaning of contaminants (proteins and other macromolecules) and DNA precipitation.

c) Polymerase chain reaction (PCR)
After extracting DNA from the bacteria, we performed a polymerase chain reaction, known as PCR.
Initially, the reaction was prepared and later, the ampli cation of the DNA strands was carried out in a thermocycler, following the technique adapted from [22].

d) Agarose gel electrophoresis at 1%
After PCR ampli cation, the products were analyzed through the technique of agarose gel electrophoresis at 1%, which had been previously prepared, and the samples were placed inside the electrophoresis tank at 108 V for approximately 1 hour. The bands generated were evaluated through the PhotoDoc-It™ Imaging System. e) Genetic sequencing Finally, the DNA fragments corresponding to the 16s DNA strand were puri ed and sequenced in the Laboratory of Biochemistry of the Department of Biological Sciences -UFPR. Such a sequence was analyzed with the help of the database of the National Center for Biotechnology Information (NCBI) to obtain statistical similarity with NCBI-BLAST. In this respect, the statistical similarity result with a value above 97% reveals a speci c species of bacteria [23].

f) Phylogenetic tree
The alignments of the nitrogenous bases, which were previously obtained in the genetic sequencing step, were done through the bootstrap method using the MEGA software in order to provide greater reliability to the result according to the genetic evolution of the species, which were later displayed in a phylogenetic tree. When the number of replicas is 100, the species whose replica is closest to 100 is the one identi ed in the sample, originating from a common ancestor [24].

Results And Discussion
Performance of the treatment system Figure 2 presents the organic matter removal data in terms of BOD 5 and COD in the two lagoons.
In Figure 2 (a) and (b), it can be observed that the average BOD 5 removal values were greater than 90%, with a maximum removal of 94% in the two systems, which is in line with the literature that suggests that aerated lagoon systems can vary between 50-95% in the removal of BOD 5 in e uent from the pulp and paper industry [25,26].
In relation to COD, it can be observed that at both loading rates, there was removal varying between 40-60% during the 120 days of operation. In general, the removal level was similar to that obtained by [25] using the same volumetric organic loading rate employed in this research for an aerated facultative lagoon.
The TOC removal analysis had an average of 49% for the AFL with an OLR of 0.2 kgCODm −3 d −1 and 41% removal for the one with an OLR of 0.6 kgCODm −3 d −1 . The results obtained at these loading rates were similar to the result obtained by [8] in an aerated facultative lagoon. Figure 3 shows the data on the removal of speci c compounds, namely: total phenolic compounds, lignin compounds, aromatic compounds, lignosulphonic compounds, in addition to the parameters of color and turbidity.
It is possible to observe in Figure 3 (a) that the TPC had an increase during the AFL treatment with an average of 26%, and in Figure 3 (b) there was a TPC removal of approximately 11%. Some studies with kraft e uent have shown an increase in total phenolic compounds in aerated biological systems [19,25,[27][28][29].
In relation to the other speci c compounds of the kraft pulp e uent, it was observed that the removal of lignin compounds was approximately 13% and 27% at the OLRs of 0.2 and 0.6 kgCODm −3 d −1 , respectively. For the aromatic compounds, the removal average was 16% and 18% at the OLRs of 0.2 and 0.6 kgCODm −3 d −1 , respectively. The lignosulphonic compounds had an average removal of 8% at both OLRs.
Possible increments of speci c compounds derived from lignin in aerated lagoons were also observed by [25,28], and were related to biotransformation processes of high-molecular-weight molecules during biological treatment in these systems.
As shown in Figure 3 (a) and (b), it is possible to verify that there was no expressive color removal, reaching 4% and 10% at the OLRs of 0.2 and 0.6 kgCODm −3 d −1 , respectively. According to [8,29], the increase in color may be related to the process of biotransformation of chromophore units and the condensation of color-forming compounds without mineralization of the e uent. In other studies, low color removal was also veri ed during treatment through aerated lagoons [7,29].
In relation to turbidity removal, the system showed an average removal of 94% and 87% at the OLRs of 0.2 and 0.6 kgCODm −3 d −1 . In general, the AFL system, in both phases, showed signi cant removal in this parameter, indicating potential for clari cation of the e uent in the AFL sedimentation zone. Table 1 shows the bacteria identi ed at the steady state of the two applied OLRs.
The genetic sequencing of the bacteria was performed through a comparison of information from the NCBI database and statistical similarity analysis, naming the bacteria in the sample by their high similarity with the microorganisms in the database. The total number of microorganisms identi ed was 9 species of bacteria at the OLR of 0.2 kgCODm −3 d −1 and 12 species at the OLR of 0.6 kgCODm −3 d −1 .
In the literature, two aspects were observed regarding the study of biological diversity. The rst for those observed in biological treatment systems which are related to reactor performance, especially regarding speci c parameters such as phenolic compounds and color; and the second those from studies in which there was isolation of bacteria with subsequent bioaugmentation treatment utilizing these selected groups to improve the removal of speci c parameters from cellulose e uents.
[33] used Bacillus thuringiensis and veri ed a removal in terms of COD and TPC of 61% and 64%, respectively.
The three identi ed species (Bacillus cereus, Bacillus thuringiensis and Paenibacillus glucanolyticus) show great promise in removing speci c parameters in an AFL biological treatment system using bioaugmentation.
Phylogenetic tree Figures 4,5 and 6 represent the phylogenetic tree of the species Bacillus cereus, Bacillus thuringiensis and Paenibacillus glucanolyticus, identi ed at the two OLRs employed in the AFL, whose data were obtained using the MEGA software and the results from the application of the Bootstrap method closest to 100 indicates the species of bacteria present in the sample.
The microorganisms identi ed in the kraft e uent were also found in other studies with bacteria, such as the ones by [6, [30][31][32]34]. The species with the greatest potential for removing the speci c parameters of pulp and paper e uents is Bacillus cereus, especially regarding color removal in cellulose e uent treatment, as analyzed by [30,36].

Conclusions
In this study, we analyzed the microbiological diversity in an aerated facultative lagoon system treating kraft pulp industry e uent at OLRs of 0.2 and 0.6 kgCODm −3 d −1 utilizing bioaugmentation treatment.
We found that the identi ed species, namely Bacillus cereus, Bacillus thuringiensis and Paenibacillus glucanolyticus, show great promise in the removal of speci c parameters in an AFL biological treatment system using bioaugmentation.   Phylogenetic tree of the species Bacillus cereus Note: The phylogenetic tree was obtained using the union of neighbors with Bacillus megaterium as an outer group, obtaining a replica of 97 for the Bacillus cereus.

Figure 5
Phylogenetic tree of the species Bacillus thuringiensis Note: The phylogenetic tree was obtained using the union of neighbors with Bacillus megaterium as an outer group, obtaining a replica of 93 for the Bacillus thuringiensis.

Figure 6
Phylogenetic tree of the species Paenibacillus glucanolyticus Note: The phylogenetic tree was obtained using the union of neighbors with Bacillus subtilis as an outer group, obtaining a replica of 100 for the Paenibacillus glucanolyticus.