Metabolic Pathway Analysis for Nutrient Removal of 3 the Consortium between C . vulgaris and P . 4 aeruginosa 5

Anthropogenic activities have increased the amount of urban wastewater discharged into 18 natural aquatic reservoirs confining in them a high amount of nutrients and organics contaminants. 19 Several studies have reported that an alternative to reduce those contaminants is using consortiums 20 of microalgae and endogenous bacteria. In this research, a genome-scale biochemical reaction 21 network is reconstructed for the co-culture between the microalga Chlorella vulgaris and the bacterium 22 Pesudomonas aeruginosa. Metabolic Pathway Analysis (MPA), is applied to understand the metabolic 23 capabilities of the co-culture and to elucidate the best conditions in removing nutrients such 24 as Phosphorus (inorganic phosphorous and phosphate) and Nitrogen (nitrates and ammonia). 25 Theoretical yields for Phosphorus removal under photoheterotrophic conditions are calculated, 26 determining their values as 0.042 mmol of PO4/ g DW of C. vulgaris, 19.53 mmol of inorganic 27 Phosphorus /g DW of C. vulgaris and 4.90 mmol of inorganic Phosphorus/ g DW of P. aeruginosa. 28 Similarly, according to the genome-scale biochemical reaction network the theoretical yields for 29 Nitrogen removal are 10.3 mmol of NH3/g DW of P. aeruginosa and 7.19 mmol of NO3 /g DW of 30 C. vulgaris. Thus, this research proves the metabolic capacity of these microorganisms in removing 31 nutrients and their theoretical yields are calculated. 32


Introduction
Diverse human activities have increased the amount of urban wastewater effluents discharged into natural aquatic reservoirs, confining in them a high amount of nutrients and organics contaminants such as NH +  4 , NO − 3 and PO 3− 4 .These compounds have been identified as the main cause leading to eutrophication in natural aquatic reservoirs.Therefore, finding new strategies for secondary wastewater treatments have received an important attention to decrease the amount of these compounds before being discharged into the water bodies [1].
Microalga offers a promising approach to remove and to re-use nutrients such as Nitrogen (N) and 0.5 to 1.3 % for P [2].The advantages of using microalgae for this purpose include the low cost for the growing process by using solar energy; the metabolic capability of microalgae which can use endogenous Carbon sources and, the possibility of recycling those assimilated nutrients as a fertilizer, avoiding a sludge handling problem [3].In addition to the wastewater effluent treatments, microalgae can be also used for biodiesel production [4] and even as a food source [5].Thus, making the secondary wastewater treatment more affordable and sustainable [6,7].
Nevertheless, a pure culture of microalgae is not always maintained.Microalgae always coexist with endogenous bacteria which are able to thriving in natural aquatic systems [8].Hence, it is natural that some simultaneous interactions must exist between these microorganisms; on one hand, bacteria are benefited from the exudates of microalgae, like oxygen and starch and, on the other hand, the growth of microalgae is promoted by bacterial products such as Carbon dioxide (CO 2 ), inorganic substances and some growth factors [9,10].Therefore, natural interactions between microalgae and bacteria are considered as an innovative technology to improve the wastewater nutrient removal.
There are some experimental studies which have been working with different species of microalgae and bacteria for urban and industrial wastewater treatments [3,11].The consortium of C. vulgaris and P. putida, has demonstrated a good simultaneous nutrients removal (ammonium and phosphate) and organic contaminants in synthetic municipal wastewater, compared with the axenic cultures [12,13].
Lananan, et al. 2014 [14], reported a removal up to 99.15 % of the total Phosphorus concentration in domestic wastewater treatment, using the co-culture of Chlorella with an effective microorganism (EM-1).Moreover, it also has been reported the co-culture of C. vulgaris with Azospirillum brasilense in cellular immobilization increases the ammonia and the Phosphorus removal [9].Again, the co-culture of Chlorella with other bacteria removed up to 80 % of total N presents in animal feed wastewater production.However, studies with pure cultures have not effect on Nitrogen or Phosphorus removal in industrial wastewater [15].The above studies prove that the consortium of microalgae-bacteria is a better biological system to remove nutrients than pure cultures of these microorganisms.Pseudomonas is a common bacteria present in wastewater and mentioned in many studies [16,17].However the metabolic activity and capability of these microorganisms can be altered by varying the culture conditions in the wastewater processes, including those associated with the microflora, particularly the α-Proteobacteria group, as Pseudomonas.To the knowledge of the authors, there is a lack of studies regarding the interaction between these microorganisms -[ C. Vulgaris -P.aeruginosa]-, their metabolisms, the upper and lower bounds for nutrients removal according to their biochemical network and the possible metabolic phenotypes.
Currently, most of the genomic information from one specific microorganism is available from biological databases which is collect from high-throughput technologies, describing the metabolisms and components such as genes, proteins and metabolites.From those databases, it is possible to reconstruct genome-scale biochemical reaction networks for microorganisms and then analyze them using metabolic engineering tools [18].Varma et al. (1993) [19], were the first authors in modeling a metabolic network from an entire organism (E.coli), obtaining the optimal carbon flux distribution using Flux Balance Analysis (FBA).Metabolic Pathway Analysis (MPA), is another technique used to analyze genome-scale metabolic networks, to find their phenotypic capabilities calculating a set of systematically independent and unique Extreme Pathways (ExPas), [20].Extreme pathways (ExPas) are mathematically derived vectors that can be used to characterize the phenotypic potential of a defined metabolic network [20,21].ExPas describe the conversion of substrates into products, while creating all byproducts needed to maintain the systemic elemental balance and the cofactor pools at steady state [18,20].By calculating the ExPas from a metabolic network, it is possible to explain the active metabolisms in a particular pathway and the theoretical yields of products with respect to the sources of carbon or nutrients.Thus, calculating and analyzing ExPas from the metabolic network of the consortium between C. vulgaris and P. aeruginosa, it is possible to estimate their phenotypic potential under different schemes.Our research group has been working in the evaluation of nutrients removal by using different microorganisms and the consortium between them [13].Consequently, this project provides a fundamental approach to enhance our understanding of biological system where microalgae and bacteria coexist as it occurs in most wastewater treatment.

Stoichiometric matrix S
The  The Figure 1 represents a novel way to describe graphically the obtained matrix S from the reconstructed metabolic model at genomic scale of this particular consortium of microalgae-bacteria.
The abscissas axis represents the internal and exchange fluxes and the ordinates axis denotes the metabolites in order of appearance in the stoichiometric model.It is also represented in Figure 1 the different metabolism for each microorganisms.Hence, in Figure 1 can be noticed in which biochemical fluxes, the external nutrients are incorporated and, also it is possible to relate those fluxes with a metabolism belonging to a particular microorganism.Second, in the metabolism of P. aeruginosa (from reaction 148 to 273), the Piext ( ) enter as part of the oxidative phosphorylation for ATP synthesis and after it is incorporated in twenty-eight biochemical reactions belonging to the metabolisms of glycolysis, Krebs cycle and synthesis of acetic acid (Figure 1).In 33 % of the total calculated ExPas, it was found the maximum theoretical inorganic phosphorous removal by the bacteria which was 4.90 mmol Pi − Pa/ g DW of P. aeruginosa.Even so, this maximum yield by the bacteria can occur either in a photoautotrophic or photoheterotrophic scheme of the consortium, its value is less than the one obtained by the microalgae.
The phosphate specie (PO4ext) which is in green circle in Figure 1 ( ), is assimilated by C. vulgaris (PO4ext→PO4 − Cv) as a part of the substrate for biomass synthesis (flux number 147), meaning a removal of this nutrient in 90.43% of the ExPas, when there is microalgae biomass production 59 PO4ext m mol / g DW of C. vulgaris (Figure 2).

Extreme pathways analysis for Nitrogen species removal
Nitrogen nutrient is represented in two nitrogenous species in the metabolic network, as nitrate

Analysis of the best Extreme pathways analysis for nutrients removal
Considering only the ExPas that showed removal for the four nutrients species, there were obtained 864 feasible ExPa, 96 were for a photoautotrophic scheme and 768 for photoheterotrophic scheme.Nevertheless, with the announced ExPas it was necessary to reduce them even more.
Therefore, another parameter to consider in reducing the amount of ExPas, was the degradation of organic carbon presented in wastewater, in this case was represented as an external glucose (GLUext) in the model.This last criterion reduced to 336 feasible extremes pathways, considered as the best ExPas for nutrient removal.Thus, one of the best ExPa for nutrient removal by the co-culture is schematized in Figure 3; and it accounts for 246 biochemical flux reactions, 84 % of the total metabolic network.The above means that for the nutrient removal purpose, is not needed the entire metabolism machinery of this co-culture.The mentioned ExPa corresponds to a photoheterotrophic scheme, where there is organic carbon source as glucose (GLUext), and the inorganic carbon source is the endogenous product of bacterial respiration (CO2 − P); this behavior could represent a decrease in operation costs due to aeration or external Carbon dioxide supply in common wastewater treatment.
The glucose entry goes for both microorganisms, having a maximum removal of 0.93 mmol of glucose/ g DW of C. vulgaris and 13.85 mmol of glucose/ g DW of P. aeruginosa.Even so, most of the organic source is directed towards the bacteria at the glycolysis metabolism.This agree with experimental reports where the growth of a bacterium is related with glucose uptake at the expense of microalgae development [22].While the inorganic Carbon source comes from the internal respiration of bacteria, the total yield 34.07 mmol of CO 2 goes to the Calvin cycle in the microalga metabolism to be fixed into the triose glyceraldehyde 3 phosphate (G3P − c).This last metabolite goes for chlorophyll synthesis and it is also incorporated into the five step of glycolysis.In this particular ExPa, G3P − c is not needed as substrate to produce starch or maltose.The mentioned metabolites are only used as an important energy compound when there is no carbon dioxide or nutrients.The last idea can be reinforced because there is simultaneous C. vulgaris and P. aeruginosa biomass synthesis, so either microorganism is not in competence with each other.Therefore, the best obtained yields for phosphorous and Nitrogen removals with microalgae were due to its requirement during photosynthesys metabolism according to Figure 1, in fact it is important to mention that all the ExPas (2844) no matter the scheme, they presented inorganic phosphorous removal.On the other hand, bacteria only exhibited a phosphorous removal in 33.3 % of the total ExPas.

Reconstruction of a genome-scale biochemical reaction networks
The first step to rebuild a genome-scale metabolic network for the consortium of C. vulgaris and P. aeruginosa was to assemble the stoichiometric reactions base on their genome annotation.
Different metabolic databases exist to match the biochemical reactions with the specific genes for each microorganism.In this research, the reconstruction of the metabolic networks was made manually using the databases of BRENDA (BRaunschweig ENzyme DAtabase), NCBI (National Center of Biotechnology Information), MetaCyc, KEGG (Kyoto Encyclopedia of referenced literature as [23,24] and [5].These references contain genomic, genetic, enzymatic, taxonomic and biochemical information, available for a large number of microorganisms including Chlorella and Pseudomonas genres. The considered metabolisms for microalgae were those related with the autotrophy and photoheterotrophy schemes such as photosynthesis, chlorophyll synthesis ( Chla and Chlb), Calvin-Benson cycle, starch metabolism, glycolysis/gluconeogenesis and finally, the basic metabolism for biomass formation such as TCA cycle, fatty acids synthesis, triglycerides synthesis, oxidative phosphorylation, pentose phosphate pathway, protein synthesis (18 amino acids), nucleic acids synthesis, carbohydrate synthesis, glycerophospholipids and maintenance.These metabolisms were represented in the first 147 biochemicals reactions (Appendix A).
Otherwise, biochemicals reactions from 148 to 273 denoted the metabolisms for P. aeruginosa.This bacterium has a huge metabolic capacity, therefore for the purpose of this project, it was considered the metabolisms related with central metabolism in a prokaryotic cell such as: starch metabolism, glycolysis, TCA cycle, glyoxylate cycle, pentose phosphate pathway, oxidative phosphorylation, amino acid synthesis, nucleic acid synthesis, peptidoglycan synthesis, synthesis of fatty acids and biomass formation.In the same way metabolisms related with synthesis of acetic acid and polyhydroxyalkanoates.Moreover, it was included the transport and exchange fluxes at the end of these metabolic networks.
Additionally, to the information from the databases, our research group obtained experimentally the elementary composition of P. aeruginosa using an elemental analyzer (Fisons model 1108) [25].The results were employed to establish the biochemical reaction for the production of biomass (biochemical reaction 273, Appendix A).Metabolites such as CoA, NAD, NADP, FAD, ADP, and H 2 O, were omitted because of they are present in the same concentration as their analogous pairs such as AcCoA, NADH, NADPH, FADH, and ATP [26].All the stoichiometric coefficients have the units of mmol unless they were specified as grams.The nomenclature of compounds are given in Appendix B.

Extreme Pathway Analysis (ExPas)
After rebuilding the genome-scale metabolic network for the consortium, the biochemical reactions were ordered in a matrix S with dimension m × n, whose rows (m) represent the mass balance for each metabolite (X), and n is the number of internal and exchange fluxes (ν) participating in each mass balance [21,27,28] stoichiometric matrix S has a dimension of 286 × 293, representing the metabolites and the set of internal fluxes and exchange fluxes, such as photons (Pho), external glucose (GLUext), sulfate (SO4ext), Magnesium (Mg), Potassium (K), iron (Fe), Calcium (Ca), Zinc (Zn), Copper (Cu), Manganese (Mn) and more important the nutrients which are studied in this article (PO4ext, Piext NO3ext and NH3ext).The outputs fluxes were biomass from each microorganism, polyhydroxyalkanoates, maltose, Carbon dioxide (COext) and oxygen production (O2ext).

Figure 1 .
Figure 1.Estequiometric matrix S, of co-culture of C. vulgaris and P. aeruginosa.Main metabolisms of microorganisms are show at the top of the figure; *Fi represents the exchange fluxes considered in the metabolic network -biochemical reactions from 273-293, see Appendix A-Piext is the inorganic phosphorous ( ), phosphate, PO4ext ( ); nitrate, NO3ext ( ) and ammonia, NH3ext ( ).

PreprintsFigure 3 .
Figure 3. Schematic representation of the best ExPa for nutrients removal under a photoheterotrophic condition for the consortium of C. vulgaris and P. aeruginosa.The numbers represent the yields for consuming or producing the indicated compounds with units of mmol of nutrient / g DW of microorganism, respectively.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 March 2019 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 March 2019 doi:10.20944/preprints201903.0002.v1
Peer-reviewed version available at Int.J.Mol.Sci.2019, 20, 1978; doi:10.3390/ijms200819782.2.Extreme pathways analysis for phosphorous species removalPhosphorous nutrient is presented in two forms; inorganic phosphorous (Piext) and phosphate (PO4ext).The inorganic phosphorous (red circle in Figure1,( )) is related with both microorganisms.First, it could enter as an external flux into endogenous inorganic Phosphorus (Pi − Cv) as part of the requirements for photosynthesis metabolism in the microalgae; subsequently, Pi − Cv takes part of other fifty-two biochemical reactions which are mainly related with glycolysis, Krebs cycle and oxidative phosphorylation.Therefore, Pi − Cv is one of the metabolites that shows a greater connectivity between the fluxes in the matrix, because it is mostly required by microalga as part of its anabolism as it can observed in the Figure2.For instance, the flux number 2, in red circle (Piext→Pi − Cv), is activated in 2844 (100 %) of the ExPas obtained.However, only 2572 ExPas correspond to an assimilation by microalga towards biomass generation.The rest, 273 ExPas are related with maltose production which is an endogenous organic compound destined for the growth of the P. aeruginosa.These last ExPas could suggest a commensalism interaction where microalgae metabolic machinery, works for bacteria supply Carbon in a photoautotrophic scheme.Until now, the active fluxes and their belonged metabolisms are elucidated.However, it is also possible to find theoretical yields and having a quantitative result like inorganic phosphorous removal with respect to biomass of each microorganism.For instance, the highest yield was 180.23 mmol Pi − Cv/ g DW of C. vulgaris, and it was discerned from 2572 calculated yields.The major ExPa removal corresponds with a pure culture of C. vulgaris in a photoheterotrophic scheme and it needs a requirement of 50.79 mmol of glucose/ g DW of C. vulgaris and 1252.06 mmol of photons/ g DW of C. vulgaris to be held.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 March 2019 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 March 2019 doi:10.20944/preprints201903.0002.v1
19.43 mmol of inorganic phosphorous/ g DW of C. vulgaris, 0.04 mmol of phosphate/ g DW of C. vulgaris and 7.19 mmol of nitrate/ g of DW of C. vulgaris.For bacteria nutrient removals, yields of 4.90 mmol of inorganic phosphorous/ g DW of P. aeruginosa and 10.30 mmol of ammonia / g DW of P. aeruginosa were obtained.The last results indicated a more efficient removal of inorganic phosphorous from microalgae than bacteria, Peer-reviewed version available at Int.J. Mol.Sci.2019, 20, 1978; doi:10.3390/ijms20081978