Kinetic parameters of Lactobacillus acidophilus growth in the lactic fermentation of non-centrifugal cane sugar agroindustry wastes

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Introduction
In the agroindustrial sector, a wide variety of wastes are generated from agricultural, livestock, forestry, and related processes; most of these wastes are not treated, and are not used, being incinerated or improperly disposed of, generating environmental pollution problems (Azelee et al., 2020).In Mexico, the state of Veracruz is the largest supplier of agroindustrial products, which during processing generate 7 million tons of waste per year, with sugarcane cultivation generating the largest amount with 5.3 million tons per year (Nava-Pacheco et al., 2019).
Within the sugar agroindustry, non-centrifuged sugar, or non-centrifugal cane sugar, is a sweetener obtained from sugarcane juice, and manufactured in small agroindustries in developing countries (Velásquez et al., 2019).Noncentrifugal cane sugar production participates with 2.3% of the national production of sweeteners from sugarcane, with an average of 115 thousand tons per year (Juárez et al., 2018).
The typical non-centrifugal cane sugar production process includes a juice clarification stage that generates a black colloidal liquid residue, called cachaza, which traditionally has poor or no management practices, generating bad odors, and ecological risk (Mendieta et al., 2020a).In addition, approximately 500 L of wastewater are generated per ton of non-centrifugal cane sugar during the washing of molds, tables, and floors of the beating, and molding room; this water contains about 0.5% dissolved solids, mostly sugars that favor the growth of microorganisms that cause the eutrophication of bodies of water (García et al., 2007).
Anaerobic digestion and co-digestion have the disadvantage of requiring long times for the adaptation of microorganisms to the substrate, the application of treatment to the by-products generated in the process, and the generation of greenhouse gases (Náthia-Neves et al., 2018).While for the production of ethanol, hydrogen, and cellulose it is necessary to apply a pretreatment to the residue, to have greater control over the operating conditions of the process, and the use of chemicals at different stages of the process (Carrillo-Nieves et al., 2019;Zhang et al., 2021).
In order to generate value-added products from organic wastes, the use of emerging technologies involving the application of anaerobic microorganisms to carry out microbial fermentation using the carbohydrates present as a carbon source to produce lactic acid, ethanol, volatile fatty acids, and hydrogen has increased (De Groof et al., 2021).Microbial fermentation is a process in which the strain, culture medium, reactor, and operating conditions combine, and interact (Romero-Mota et al., 2023).
Among the anaerobic processes is lactic fermentation, which increases the nutritional properties, and functional value of substrates, increases their shelf life, and microbiological safety, and improves sensory characteristics, while decreasing the concentration of non-nutritional compounds, and sugar content (Jamnik et al., 2022).Lactic fermentation is carried out by a group of microorganisms called lactic acid bacteria (LAB), which grow in a pH range of 3.5 -10, and a temperature of 5 -45 °C, and are known for their ability to ferment carbohydrates (hexoses and pentoses) to lactic acid (Raj et al., 2022).One of the LAB species is the Lactobacillus acidophilus strain, which is one of the most recognized species for its commercialization in different foods, and supplements containing probiotics (Ozogul et al., 2022).This species ferments carbon sources such as glucose, lactose, maltose, and sucrose, making it have an affinity for substrates rich in these components (Estrada-García et al., 2023).
A very important aspect in the study of fermentation processes is the application of kinetic models, since they are important because they allow the implementation of strategies for process design, cost reduction, and optimization of operating conditions to obtain a desired product (Ajala et al., 2021).Thus, this work presents the study of the lactic fermentation process of SCS and MWW, residues generated during the production of non-centrifugal cane sugar, by using of L. acidophilus and the obtaining of the kinetic parameters of growth of this strain.

Collection of the residues
The SCS and MWW were collected from a mill located in the town of Zentla (19°05'24.9"N 96°48'04.3"W), municipality of Zentla, Veracruz.Subsequently, a solution of 150 g SCS/L was prepared using MWW as solvent.This concentration was established on the basis of reports by Gonzalez-Del Rosario et al. (2023), they found no differences in LA production during fermentation of 50, 100 and 150 g SCS/L solutions.This solution was then kept refrigerated at 4°C until use.

Physicochemical characterization of the residues
For SCS, MWW, and SCS solution, pH was determined using a Science Med model SM-3BW potentiometer, Total Solids (TS), and Total Volatile Solids (TVS) according to Standards Methods (APHA, WPCF, AWWA, 2017), carbohydrates by the anthrone-sulfuric colorimetric method with a Thermo Scientific spectrophotometer model Genesys 20 at a wavelength of 620 nm, total nitrogen and proteins by the micro Kjeldahl method using a conversion factor of 6. 25.All determinations were performed according to standard procedures.

Propagation of the Lactobacillus acidophilus strain in SCS solution.
The Lactobacillus acidophilus strain was obtained from the commercial product called Lyofast LA 3 (SACCO System), which is in lyophilized form.This strain is considered a probiotic and has been used in other fermentation processes.Luciano et al. (2018) studied the effect of the addition of L. acidophilus LA 3 on physicochemical parameters, sugar and organic acid content, phenolic profile, and sensory characteristics of mango and acai smoothies.In addition, it has the advantage of being easy to acquire and less expensive than strains from microbial collections. 1 g of the product was weighed and added to a flask with 100 mL of MRS broth (Man, Rogosa, and Sharpe) previously sterilized at 121 °C for 15 min and incubated at 37 ± 0.5 °C for 24 h.This flask was referred to as the seed flask.Then, in order to acclimate the strain to the substrate, 4 mL were taken from the seed flask and added to 36 mL of SCS solution, leaving it to incubate at the same temperature and time as the seed flask.This volume was later used to perform the lactic fermentation kinetics.

Lactic fermentation kinetics
A 0.5 L reactor with heating jacket was set up with a useful volume of 0.4 L. It was operated at a temperature of 37 ± 0.5 °C, continuous stirring at 120 rpm by using of a magnetic stirrer, the inoculum was added in a 1/10 ratio, and the fermentation time was 72 h (Gonzalez-Del Rosario et al., 2023;Romero-Mota et al., 2023).Samples were taken every 2 h during the first hours of fermentation and every 4 h during the rest of the process.For each sample, pH, carbohydrates (according to the described techniques for waste characterization), and cell density was determined using the pour plate technique on MRS agar (Peng et al., 2021), and titratable acidity according to the methodology described in the Mexican standard (COFOCALEC, 2014).

Obtaining the kinetic parameters
Cell growth kinetic parameters were determined with the modified Gompertz model described in Equation 1 and the Logistic model described in Equation 2 (Sharma et al., 2014): where  is ,   is the maximum specific growth rate of the microorganisms, A is the logarithmic increase of the population and  is the lag phase.The fit of the model with the experimental data was evaluated from the correlation coefficient 9  ; and the least squares error (MSE) percentage.

Physicochemical characterization of the residues
The composition of the residues generated during the production of non-centrifugal cane sugar is affected by different factors.In the case of the SCS, its composition is affected by the skill and experience of the personnel in charge of performing the clarification activity.While the MWW is affected by the type of washing process (spray or immersion), making the composition of the residues different from one mill to another (Gonzalez-Del Rosario et al., 2023).Table 1 shows the results obtained from the physicochemical characterization of the residues used in this study.The pH is a key factor that influences the fermentation process and its value depends on the type of microorganism used; in the case of the Lactobacillus genus, they develop at pH values in the range of 5 to 7 (Da Silva et al., 2021).It is observed that for the SCS, MWW and SCS solutions, the pH shows an acid trend, with values of 6.80, 5.60 and 6.70, respectively, being in the range for the development of the microorganism L. acidophilus.With respect to the TS content, when high values (19-25%) are present, mass transfer is affected by the low water content, causing an accumulation of by-products that inhibit the fermentation process (Ghimire et al., 2017).In the waste under study, SCS has a high content of TS (29.81%), so it is not convenient to ferment the waste without an adjustment in this parameter, thus, in the solution of 150 g SCS/L the content decreased to 5.12% due to the dilution process.As for the volatile solids content (TVS), which is indicative of the amount of organic matter in the waste, values above 75% were observed, mainly due to the origin of the waste, with 90.85% for SCS, 74.60% for MWW, and 89.61% for the 150 g SCS/L solution.
The SCS and MWW, being residues obtained from sugar cane processing, are rich in carbohydrates; thus, for SCS the content was 172.42 g/L and for MWW 12.48 g/L.In the case of the SCS solution, the carbohydrate content decreased due to the dilution carried out, having a value of 48.31 g/L.These values show that the residues are viable to be used as substrate to carry out lactic fermentation, since environments rich in carbohydrates are ideal for the development of LAB (Vinicius De Melo Pereira et al., 2020).Regarding the nitrogen and protein content, for SCS it is 1.33% and 8.35%, respectively while in MWW it is null, which may be because the facility from which the residue was obtained uses potable water for the mold washing operation and nitrogen, in the form of nitrites and nitrates, should be low.The presence of nitrogen in the substrate to be fermented is important because in the metabolism of microorganisms it acts as the main component of catabolic and anabolic processes (Rawoof et al., 2021).

Lactic fermentation kinetics
Figure 1 shows the data of substrate consumption, LA production and cell density obtained during the lactic fermentation of the 150 g SCS/L solution.It can be observed that the highest consumption occurred during the first 24 h of the process.This represents a consumption of about 37% of the initial carbohydrates showing that the microorganism adapted to the SCS and MWW to perform the anaerobic bioconversion.For the lactic acid production parameter, it is observed that it begins to increase around 10 h, reaching its maximum value of 7.56 g LA/L.This value is similar to that reported by Guo et al. (2019), where they obtained a maximum production of 10.6 g LA/L during the study of glucose fermentation by applying nanobubble water.While Macedo et al. (2020) reported a production of 66.90 g LA/L during the fermentation of previously hydrolyzed cassava bagasse.The above values when compared with those obtained by other strains of the Lactobacillus genus are lower (Abedi et al., 2020).A possible explanation may be to the low nitrogen content present in the substrates, since L. acidophilus bacteria require significant amounts of amino acids since they do not follow the metabolic pathway via pentose-phosphate in a fully functional way, obtaining other by-products (Solval et al., 2019).
Regarding cell density, it was observed that the microorganism showed affinity for the substrate, mainly due to the high carbohydrate content.Microbial growth occurred around 8 h of the process, increasing from 6.96 to 7.47 log CFU/mL, then during the rest of the fermentation it remained stable, with an average value of 7.56 log CFU/mL.Romero-Mota et al. (2023) report values of 7.80 and 8.20 log CFU/mL, in the first 10 h during fermentation of sugarcane residues at 100 and 150 rpm, concluding that microbial growth and process yield is affected by agitation speed.As it is possible to observe, the cell density values are similar, since in the present study the agitation speed used was 120 rpm.
In relation to the kinetic parameters, both models presented a good fit to the experimental data, being better in the Logistic model with a value of   = . (Table 2), likewise, a value of   = .  / was reached with the Gompertz model, while with the Logistic model a value of   = .  / was obtained.These values are higher than those reported by Estrada- García et al (2023), who reported specific growth rates between . − .  / during the bioconversion of swine waste with L. acidophilus at different agitation speeds.On the other hand, Romero -Mota et al (2023) reported values between . − .  / in the anaerobic fermentation of agricultural waste using the same microorganism.This is due to the fact that SCS and MWW have a high carbohydrate content, and are solubilized, resulting in increased growth of L. acidophilus.Despite the above, it can be observed that the lag phase is high, 20.46 h for Gompertz and 25.39 h for Logistic, which indicates a long period of adaptation of the microorganism to the substrate.It is possible that this behavior is due to the low content of other nutrients that are essential for the development of the microorganism.Similar results were obtained by Akdemir et al. (2021) during the study of the survival of L. acidophilus in three different media, where the strain presented a higher specific growth rate (0.58 h -1 ) in white rice milk, but a lower lag phase (10.94 h) due to the low nutrient content of the medium.

Conclusions
The physicochemical characterization of SCS and MWW shows that these wastes possess carbohydrates and nitrogen, vital nutrients for the development of LAB.Since SCS has a high content of TS, it is necessary to carry out a dilution before the fermentation process, so the use of MWW as a diluent is useful for reducing the environmental impact of this waste.In the monitoring of the process, it was observed that both wastes promote the development of the microorganism L. acidophilus, having a maximum growth of 7.63 log CFU/mL, a removal of 50.32% of carbohydrates and a maximum production of 7.56 g LA/L.However, the kinetic parameters of cell growth were higher than those reported by other studies, indicating that the microorganism L. acidophilus needs more time to adapt to the residues generated in the production of non-centrifugal cane sugar, so it is necessary to explore strategies to reduce the value of these parameters in order to optimize the fermentation process.With this, lactic fermentation emerges as a viable option to be implemented as a new strategy for the utilization of the residues generated in the production of noncentrifugal cane sugar, allowing to valorize them and reduce their impact on the environment.

Figure 1 .
Figure 1.Substrate consumption, lactic acid production and cell growth during lactic fermentation of 150 g SCS/L solution.

Table 1 .
Physicochemical characterization of SCS, MWW and 150 g SCS/L solution.

Table 2 .
Kinetic parameters of L. acidophilus growth during lactic fermentation of 150 g SCS/L solution.