Design of a sustainable system for wastewater treatment and generation of biofuels based on the biomass of the aquatic plant Eichhornia Crassipes

Colombia’s continuous contamination of water resources and the low alternatives to produce biofuels have affected the fulfillment of the objectives of sustainable development, deteriorating the environment and affecting the economic productivity of this country. Due to this reality, projects on environmental and economic sustainability, phytoremediation, and the production of biofuels such as ethanol and hydrogen were combined. The objective of this article was to design and develop a sustainable system for wastewater treatment and the generation of biofuels based on the biomass of the aquatic plant Eichhornia crassipes. A system that simulates an artificial wetland with live E. crassipes plants was designed and developed, removing organic matter contaminants; subsequently, and continuing the sustainability project, bioreactors were designed, adapted, and started up to produce bioethanol and biohydrogen with the hydrolyzed biomass used in the phytoremediation process, generating around 12 g/L of bioethanol and around 81 ml H2/g. The proposed research strategy suggests combining two sustainable methods, bioremediation and biofuel production, to preserve the natural beauty of water systems and their surroundings.


The taxonomic level is (Eichhornia crassipes)
The collection point is the municipality of Mosquera, in the outskirts of Bogotá DC, located at the coordinates 4.682995, − 74.256673; this activity was carried out on June 15, 2022, by the researcher Uriel Fernando Carreño Sayago.The plant samples were also identified by the researcher at the Faculty of Engineering of Bogotá, D.C.The final biomass of the plants was used as input for composting processes at the Libertadores University, with the code LIB 021212.

Characterization of Eichhornia crassipes
The physicochemical characterization was carried out to identify the properties of the collected macrophytes, determining the structural carbohydrates and the lignin content.In addition, the quantification of the biomass matrix used was carried out considering the following parameters: (a) % hemicellulose, (b) % cellulose, (c) % lignin, and (d) ashes [25][26][27] .
An extract of benzene and ethanol with a 2:1 volume ratio was used to extract cellulose.Nitric acid and ethanol were used in a 1:4 ratio to extract hemicellulose.For the extraction of lignin, 12% hydrochloric acid was used, and for the determination of ashes, 72% hydrogen sulfide was used.

Phase 1. Assembly of the artificial wetland with the E. crassipes
The dimensions of the experimental model of phytoremediation are 100 cm long and 80 cm tall.This design is on a pilot scale and has 2.5 kg of E. crassipes (approximately 25 plants).Figure 1 shows the treatment system.The experiment was carried out in triplicate (showing the average in the results), evaluating DBO, total nitrogen, Kendal nitrogen and phosphorus for 10 days, taking samples before and after the treatment.About 150 L of domestic wastewater were treated.Then, a plastic mesh that floats was designed and built, as well as treatment compartments where the E. crassipes plants are located.
Once the composite sample of the original residual water was obtained, it was characterized using ex-situ physicochemical parameters such as biochemical oxygen demand (DBO5), phosphorus, ammoniacal nitrogen, and total nitrogen.The analysis was carried out in a credited laboratory in Bogotá, following the protocols presented in the standardized methods and established in the most recent editions of the standard methods for the analysis of water and wastewater of the American Public Health Association (APHA) and the American water services association (AWWA).The cost of this system is around two hundred dollars.

Phase 2. Production of ethanol and hydrogen
The design of the bioethanol and biohydrogen generation process consists of three bioreactors: a bioreactor to make the hydrolyzate, a bioreactor for fermentation generating bioethanol and dark fermentation.The following figure represents the joint process of hydrolysis and dark fermentation.
The design of the bioethanol and biohydrogen generation process has three bioreactors: a bioreactor to produce the hydrolyzate, a bioreactor for fermentation generating bioethanol, and the last bioreactor for dark fermentation.The following figure represents the joint process of hydrolysis and fermentations.

Hydrolysis of Eichhornia Crassipes
The hydrolyzate bioreactor is made of glass, with a capacity of 6 L; it has a hose for the evolution of gases, pH, and temperature sampling, and it was placed in a heater with magnetic stirring at 120 rpm at a temperature of 30° C. In this bioreactor, 2.5 kg of dried and crushed Eichhornia crassipes was taken and mixed with distilled water 27,28 .The cost of this system is around one hundred dollars.

Hydrolysis alkali
The samples of the E. crassipes were set to react in 1% (w/v) caustic soda (NaOH) at a temperature of 30° C for 12 h; then, the samples were washed with tap water until reaching the pH value of the water 27,28  ) was added at a temperature of 60° C, for 12 h.The samples were washed with tap water until reaching the pH value of water.The content of reducing sugars was determined by the Dinitro Salicylic Acid (DNS) method, which indirectly quantifies the consumption of substrate.6 L of Eichhornia crassipes hydrolyzate solution were obtained for the continuation of biofuel production 27,28 .

Fermentation bioreactor
Glassware 2.5 L. The hydrolyzed plant material of E. crassipes (1 kg) was washed and taken to the alcoholic fermentation bioreactor, where 150 g of Saccharomyces cerevisiae was added; the pH should be around 6.0.The bioreactors were hermetically sealed with rubber septa and aluminum stoppers.During the hydrolyze fermentation, the tests of the ethanol percentages are carried out 28,29 .The experiment was carried out in duplicate (showing the average in the results).The cost of this system is around one hundred dollars.

Production of biohydrogen
The dark fermentation bioreactor is made of glass, with a capacity of 4 L. It has a lid for gas release, pH, and temperature sampling, and it was placed in a heater with magnetic stirring at 120 RPM at a temperature of 30° C. The bioreactor was hermetically sealed with rubber septa and aluminum stoppers.Bird manure was used as raw material to carry out the hydrogen production process.After that, they were put to a temperature of 100° C in an oven to deactivate microorganisms that do not benefit the production process of this biofuel.500 g of the hydrolyzate from the E. crassipes biomass were taken to the bioreactor, where it was mixed with distilled water, and 500 g of the inoculum (bird manure) was added; the initial pH was adjusted to 5.5.The bottle holes were purged with nitrogen for 5 min to ensure the anaerobic condition.At each time interval, the biogas volume was measured by the plunger displacement method.Hydrogen gas was determined by gas chromatography using a TCD detector on a GC-Agilent 7890 chromatograph.The optimum temperature for hydrogen production is 30° C. The experiment was carried out in duplicate (showing the average in the results).The cost of this system is around two hundred dollars.
The results of the different tests were determined with the Gompertz equation (Eq.( 1)) where, α latency time, R m Maximum rate of H 2 production, H max Maximum production potential. (1)

Result of characterizations chemistry
The E. crassipes collected in the wet bodies had a hemicellulose content of 33% and 30% cellulose; lignin was lower, with 9%, and ash content was high, with 23% due to the contamination inherent to the plant.In Table 1 is the composition of the biomass of E. crassipes.
6][27][28][29] who averaged 18% in hemicellulose and 25% in cellulose.The presence of these two polysaccharides favors biofuel production 30 .The presence of lignin also makes the biomass of this plant a process extra of hydrolysis before the bioethanol and biohydrogen production process 31 .

Analysis of phytoremediation
In the system of phytoremediation, the waste water had a very strong odor; however, through the phytoremediation process, there was a gradual reduction of it.Figure 2 shows the percentages of removals in the wetland with E. crassipes.

Biochemical oxygen demand
The variation of the gross DBO of the influent and effluent samples of the systems can be observed in Fig. 2. The average removal efficiencies for DBO were 90%.The availability of oxygen in the wetland corresponds to the photosynthesis process carried out by the E. crassipes, being the amount of this plant essential to guarantee aerobiosis 22 .Aerobic conditions are necessary to reduce contamination by organic matter.The availability of oxygen is a design criterion for wetlands with E. crassipes, being essential for the biochemical removal of organic matter.34 In a water treatment system from a tannery, were obtained removal efficiencies of 88% with E. crassipes plants in DBO 33,34 ; they achieved a higher DBO removal efficiency (92.3%) in wetlands planted with Phragmites australis and Canna indica.Although the presence of chromium was minimal, it can still affect the efficiency www.nature.com/scientificreports/ of nutrient elimination.Heavy metals like chromium can inhibit root oxygenation and seriously impact the elimination of nitrogen and phosphorus [35][36][37] .To ensure effective treatment, it is important to separate domestic wastewater from industrial wastewater 38,39 .

Nitrogen
The elimination of 50% of the nitrogen obeys to the fact that the plant incorporates nitrogen for its growth and subsequent reproduction 40 .The nitrogen present in domestic wastewater is organic and is transformed by hydrolysis into ammonia, followed by a chemical oxidation of the ammonia to nitrite and nitrate, the form in which it is assimilated by the plant 41 .The activity of certain anaerobic bacteria present in the roots of the plant leads to denitrification, which consists in the reduction of the nitrate ion to gaseous nitrogen, which is released to the atmosphere.The efficiency of Kjeldahl nitrogen is close to 55% in this type of wetland with E. crassipes because the plant adsorbs this nutrient mainly as ammonium and nitrate.A mixture of both forms is usually beneficial.These two forms of nitrogen differ in the way they are converted to amino acids in their metabolism in the plant.
The ammonium is metabolized in the roots and requires more oxygen, while the nitrate metabolism takes place in the leaves due to the oxygenation of the water, and there is a mutual benefit between the treatment and the plant.Also, ammonium and nitrate uptake affect the root environment differently from another nutrient uptake 42,43 .

Phosphorus
The efficiencies report 60% because the removal process of this nutrient depends to a great extent on the bacteria present in the plant.Dissolved organic phosphorus, particulate organic phosphorus, and insoluble phosphorus are not available to plants unless they are transformed into soluble inorganic phosphorus 41,44 .In the E. crassipes wetland, these transformations can occur through the intervention of bacteria associated with the roots and in biofilms in the sediments.Once solubilized by these microorganisms, it can be assimilated by the plant for its growth and reproduction; being this process the treatment that the wetland has to reduce the phosphorus in the water 45,46 .The biomass used in the previous phytoremediation process was used in this biofuel production process (see Fig. 3).

Hydrolysis results
There was a continuous production of sugars through acid hydrolyzation, alkaline hydrolyzation, and a combination between both processes.The best sugar production yield was the alkaline hydrolyzation, since it obtained a production of 140 g/L, and the acid hydrolyzation obtained a low production of around 60 g/L.The mixture between the two processes gave a result of 220 g/L.In the yield of sugar production, the alkaline hydrolyzation is the one that has the best performance with the E. crassipes plant 47,48 .

Production of bioethanol
Figure 4 shows a higher ethanol production for the E. crassipes sample.When carrying out the mass balance, it was established that the production of ethanol from hydrolyzed biomass of E. crassipes is profitable, with an amount of 12,200 (mg/l) in 48 h, with a conversion of 90% of the sugars into ethanol, conversion results similar to Bioethanol production in Ref. 49 .
In the Table 2, show the resume of yield the biomass, where different investigations were taken in order to compare the results obtained.
The biomass of the cellulose is a promising source of biofuel production; processes must be optimized to make better use of these resources.For example, Ref. 50 used alkaline pretreated sugarcane bagasse using Zymomonas mobilis and Pichia stipitis in the fermentation, achieving a yield and ethanol productivity of 36,000 mg/l, (it/ they).Also 51 used lignocellulose biomass to generate bioethanol, with a yield of 20,000 mg/l using genetically modified yeasts.The cassava pulp yield was 15,000 mg/l of bioethanol 52 , peel has a yield of 12,000 mg/l 53 , and 22,000 mg/l were obtained from potato peel wastes 54 .But all these biomasses are not by-products, let alone having been used in other sustainable processes.The production of bioethanol with the E. crassipes plant after a www.nature.com/scientificreports/phytoremediation process makes this process viable.The projects [55][56][57][58][59][60][61][62][63] have interesting results in the productions of this biofuel, but with a more specialized process and increased cost, which makes production on a larger scale unfeasible.Although the production of bioethanol is not as high as in other investigations, the E. crassipes plant is a waste product and is also a biomass that was previously subjected to a phytoremediation process, which may have depleted the cellulose content of the plant.

Hydrogen productions
The hydrogen gas yield production remained almost constant for 12 days, after which it decreased to half its value when the initial content of the inoculum was consumed, (from 80 to 40 ml H 2 /g glucose).Figure 5 shows the results of biohydrogen production for 10 continuous days of productivity.
The specific hydrogen production rate reached its maximum value (81.3 ml H 2 /g) on day 4.The production should be maintained consistently over the next few days, with a target of around 80.0 ml H 2 /g until day 10.From day 12 onwards, the production should be reduced to reach a balance of 40 ml H 2 /g.In the trials to produce biohydrogen from E. crassipes, around 73 ml H 2 /g was also produced 64 .
Biohydrogen production has been carried out using various substrates.For instance, in a study by 65 , biohydrogen was produced from the saccharification of alfalfa, resulting in a yield of 55 ml H 2 /g.Table 3 provides a summary of the biohydrogen production process.
In more specialized process, as in the case of Ref. [67][68][69] , the celluloses were modified genetically and hydrogen production was better, achieving biohydrogen production yields above 100 ml H 2 /g.It  www.nature.com/scientificreports/biomass of E. crassipes and other lignocellulolytic modified materials or with other components can also increase the production of biohydrogen 16,[70][71][72][73] .A residue remains in this process, which is a mixture of the E. crassipes plant and poultry manure.This material is sanitized and has potential as a biofertilizer due to its physicochemical characteristics [74][75][76] .

Life cycle analysis (LCA)
The biomass generation of the E. crassipes plant is considerable, with an estimated yield of approximately 30 tons per year in the city of Bogotá 32,38,80 .This makes it an ideal raw material for the development of phytoremediation and bioenergy generation systems, including bioethanol and biohydrogen.Figure 6 presents a summary of the Life Cycle Analysis (LCA) of the biomass of E. crassipes.
The generated product could be used as a potential fertilizer, thus furthering this research and generating technical and economic feasibility.It is imperative to implement this project on sustainable farms in our country, especially where the aquatic plants come from, La Palma Cundinamarca, Colombia.

Conclusions
The research combined projects on environmental sustainability, phytoremediation, and biofuel generation.The E. crassipes plant material was used to treat domestic wastewater, which was then utilized as a source of ethanol and green hydrogen production in fermentation bioreactors.
The effectiveness of domestic wastewater treatment in removing organic matter represented by BOD was 90%.Total nitrogen removal was 50%; N t nitrogen removal was 40%, and phosphorus removal was 60%.These results suggest that these nutrients were assimilated as a food source by E. crassipes plants.The constructed wetland is designed to meet treatment needs, and it can be used in various environments, such as farms, homes, and apartments, as an environmentally sustainable solution due to its effectiveness, ease of installation, and low cost.ProduƟons of biohidrogen (mlH2/g)

Figure 3 .
Figure 3. Biomass used in phytoremediation and subsequent biofuel processing.

Figure 5 .
Figure 5. Production of biohydrogen with the biomass of E. crassipes.

Table 1 .
Composition of the biomass of E. crassipes.

Table 2 .
has been proven that the Production of bioethanol with the biomass of E. crassipes.Process of productions bioetanol.

Table 3 .
Process of productions of biohydrogen.