Insights into Agitated Bacterial Cellulose Production with Microbial Consortia and Agro-Industrial Wastes

Abstract

Bacterial cellulose (BC) is emerging as an attractive large-scale polymer due to its superior properties. The dominant static culture for BC fermentation by bacteria or microbial consortium results in low productivity. Agitated culture, as an industrially projected technique, has been widely investigated but exclusively for cellulose-producing bacterial strains. Addressing this concern and evaluating the potential of residues as feedstock, this study highlights the utilization of microbial consortium BA2 and seven agro-industrial wastes including cocoa husks, sugarcane bagasse and others. Remarkably, rice bran (RB) appears as a promising substrate, achieving 2.14 g/L (dry basis) and outperforming the traditional HS medium, evident from a 15-day fermentation. A complex interplay between oxygen availability, glucose consumption and BC yield was revealed; while orbital and magnetic stirring with forced air ventilation (AFV) showed low BC yields and early biomass saturation, 4.07 g/L (dry basis) was targeted by magnetic stirring at 100 rpm from the start using only headspace air. However, beyond controlled operating conditions, mechanical agitation and favorable cellulose adhesion to metal in the stirred tank bioreactor negatively affect BC yield. This pattern uncovers the need for a further approach to the design of bioreactors when the microbial consortium is considered.

1. Introduction

Bacterial cellulose (BC) is a multifunctional biomaterial with singular properties related to mechanical strength, high crystallinity and biodegradability [1]. Unlike plant cellulose, BC has a unique synthesis mechanism incorporating a refined self-assembly path [2]. The absence of hemicellulose, lignin and pectin allows relatively simple purification with NaOH solution and low energy consumption [1,2]. Conventionally, species of bacteria belonging to the genera Komagataeibacter, Acetobacter, Gluconacetobacter, Agrobacterium, Aerobacter, Achromobacter, Azotobacter, Sarcina, Salmonella and Rhizobium have demonstrated extracellular cellulose production [2,3]. A biotechnology approach widely applied to enhance BC yield is media formulation to replace the original Hestrin and Schramm (HS) broth. Several carbon sources have been utilized, from monosaccharides, oligosaccharides, alcohols and organics acids to sugared feedstock (industrial and agricultural wastes) [1,2]. BC has been produced through the fermentation of acidic, basic and enzymatic hydrolysates from different lignocellulosic materials with pure bacterial strains [4]. In recent years, a diverse range of hydrolysates have been investigated, including corncob and sugarcane bagasse with isolates of Komagataeibacter sp. [5], miscanthus, barley straw and pine tree with Gluconacetobacter xylinus ATCC 53524 [6], sweet sorghum with Acetobacter xylinum ATCC 23767 [7], and grass straw, grass husk, wheat husk and corncob with Bacillus cabrialesii [8]. However, cellulose film can be targeted as a byproduct of sugared tea infusion fermentation (commonly called Kombucha) due to the mediation of a symbiotic community of bacteria and yeast (SCOBY). The synergic microbial metabolism between acid acetic bacteria and yeast has demonstrated promising improvements in BC production [9]. Only a few attempts at accounting for microbial consortia and agro-industrial wastes, beyond different herbal infusions [10] and an additional nitrogen source [11], have been identified in the literature. In static fermentation, Medusomyces gisevii Sa-12 symbiotic culture produced BC from enzymatic hydrolysates of Miscanthus x giganteus [12].

Static culture is the dominant cultivation technique, whether bacteria or microbial consortia are assessed. This method results in a gelatinous BC pellicle forming on the surface, which promotes a high dependency on specific interfacial surface (SIS) and low productivity [1,2]. The influence of a surface-to-volume ratio suitable for scale-up process has been already investigated. According to Lončar [13] and Caicedo [14], biofilm production increases along with surface area, but a minimal medium height is also required since several cellulose layers will form and occupy part of the initial volume. Shavyrkina [15] produced the so-called world’s largest BCN sheets of 29 400 cm² using a Medusomyces gisevii Sa-12 symbiotic culture through a biofilm removal scheme to refresh the fermentation. Conversely, agitated culture has emerged as a suitable alternative for mass production as BC can be assembled like a fibrous suspension, irregular masses and pellets [1,3]. A high oxygen transfer rate is crucial to assure oxygen availability for bacteria metabolism [9,16]; thus, adequate agitation and aeration combination reduces the viscosity and increases the homogeneity of the broth [3]. Unlike agitated cell production by bacteria exclusively, a lack of potential agitation approaches to provide economic BC production by microbial consortium have been developed.

The spherelike BC particles produced by Acetobacter xylinum JCM 9730 have been characterized at different rotational speeds in shake flasks [17]. A spin filter attached to the agitator shaft was implemented in a 2 L stirred tank bioreactor to improve BC yield by Gluconacetobacter hansenii PJK [18]. High cellulose production by Acetobacter xylinum sp. sucrofermentans was reported through oxygen transfer rate upgrading in a 50 L internal loop airlift bioreactor [19]. A rectangular wire-mesh draft tube was developed for an airlift bioreactor to increase gas holdup and mixing capability during cellulose production by Acetobacter xylinum [20]. A rotatory disk bioreactor was designed to enhance BC production by Gluconacetoactor sp. RKY5 by means of eight rotatory disks at low rotational speeds [21].

This study aims to investigate strategies for enhancing BC yield in an agitated culture while overcoming oxygen adeptness between yeast and bacteria belonging to a microbial consortium. The primary challenge is to avoid yeast role suppression in metabolic symbiosis as an ethanol releaser through the fermentation pathway. To address this challenge, a systematic protocol has been developed encompassing various agitation techniques, including orbital shaking flasks, magnetic stirring flasks and a stirred tank bioreactor while agro-industrial wastes are incorporated.

2. Materials and Methods

2.1. Sugar Recovery from Agro-Industrial Wastes

2.1.1. Agro-Industrial Wastes

Seven complex lignocellulosic substrates were used: cocoa husks (CHs), sugarcane bagasse (SB), pineapple bagasse (PB), rice bran (RB), pineapple husks (PHs), mango husks (MHs) and corn stover (CS).

CHs and SB were obtained from a farm located in Puerto Quito, Pichincha province. PB, PHs and MHs were provided as waste from the production of fruit pulp by the company PROALVA, located in Quito. RB was donated by INTIAGRO CORPORACION ANDINA DE NEGOCIOS S.A.S., located in Guayaquil. The CS was collected on a farm in Valle de los Chillos, Quito.

CHs and RB were received as dry materials. SB, PB, PHs, MHs and CS were received wet and immediately dried in a forced air oven at 70 °C until excess water was removed. All substrates were stored in sealed bags at room temperature until pretreatment.

2.1.2. Chemical Characterization

A proximate analysis of agro-industrial wastes was conducted, focusing especially on carbohydrate determination, to optimize the culture medium for bacterial cellulose production. This targeted approach ensures the efficient utilization of carbohydrates, crucial for maximizing the yield of bacterial cellulose. Total carbohydrates were determined by difference with the other components (moisture, ash, fat and protein). Crude fiber was also determined. The experimental procedures were carried out by using standard AOAC methods: moisture (925.10), ash (923.03), fat (920.85), protein (2001.11) and crude fiber (ICC113). For cocoa husks, specific AOAC methods were used: moisture (931.04), ash (972.15), fat (970.22), protein (970.22) and crude fiber (930.20).

2.1.3. Agro-Industrial Waste Pretreatment

The pretreatment and the particle sizes of the materials were like those indicated by Sánchez [22], Pattra [23], Santoso [24], Najafpour [25], Siacor [26] and Chen [27]. All dry substrates, except RB, were ground in an ALPINE 160-UDL hammer mill. The materials were then sieved in a Bertel electromagnetic shaker, model B-AGIT, with different mesh sizes depending on the material. The particle sizes of the substrates for the subsequent tests were between 425 and 600 µm for SB and CS, less than 425 µm for CHs, between 300 and 425 µm for RB, and less than 250 µm for PHs, MHs and PB.

2.1.4. Hydrolysate Preparation

The hydrothermal treatment of complex materials in the presence of H₂SO₄ was conducted under previously reported conditions in the literature as indicated in

Table 1. Hydrothermal treatment conditions for sugar release from complex materials.

Parameters	Agro-Industrial Wastes
	CH	SB	PB	RB	MH	PH	CS
Phase	1	1	1	1	1	1	1
Solid/liquid ratio	1:05	1:15	1:10	1:10	1:10	1:10	1:10
Temperature (°C)	121	121	121	121	121	121	121
Sulphuric acid concentration (% v/v)	3	1	2.5	1	4.0	2.5	1.5
Incubation time (min)	15	60	60	30	60	60	60
Particle size (µm)	<425	425–600	<250	300–425	<250	<250	425–600
Reference	[22]	[23]	[24]	[25]	[26]	[24]	[27]

. A control without acid was included in each process. The entire hydrolysate stock was prepared in a single run and stored frozen at −20 °C until used.

The hydrolysates were recovered by vacuum filtration [26] in a Büchner funnel using a LABDIN model Rocker 140 vacuum pump. The pH of the filtrate was adjusted to 6.0 [28] with NaOH 10 M, the reducing sugar content was quantified, and the filtrate was frozen and stored at −14 °C until further use.

The yield ($\mathrm{Ysm}$) of the hydrothermal process with and without acid was determined by Equation (1) as the ratio between the reducing sugars released ($RS$) and the dry material employed for hydrolysis ($DM$).

$$\mathrm{Ysm}={\displaystyle \frac{RS}{DM}}$$

(1)

2.2. Fermentation

2.2.1. Microbial Consortium and Inoculum Production

The assays were carried out with the microbial consortium BA2, isolated previously by Dávalos and Cerda-Mejía [29]. BA2 was activated in 100 mL flasks with 50 mL of Hestrin–Schramm (HS) [30] medium under static conditions for 15 days at 25 °C in a HYSC model SI-64 incubator. The formed BC layer was transferred to 5 mL HS medium and vortexed for 1 min to release the attached microorganisms. The HS medium composition (w/v) was glucose 2%, peptone 0.5%, yeast extract 0.5%, disodium phosphate 0.27% and citric acid 0.115% [30]. The cell suspension was quantified by the McFarland scale and used as an inoculum for fermentation assays.

2.2.2. Culture Medium from Agro-Industrial Wastes and Fermentation Conditions

The culture medium was formulated using the hydrolysates of the complex substrates as suggested by Castro [31] with some modifications. The hydrolysates were diluted with distilled water to 20 g/L of reducing sugars, supplemented with the components of the HS medium except glucose, and adjusted to pH 6.0. Control fermentation was carried out in HS medium with the composition previously mentioned.

Fermentation was performed in 100 mL flasks with 50 mL of sterile culture medium (121 °C for 15 min) and an initial cell concentration of 1 × 10⁶ CFU/mL on the McFarland scale. The flasks were placed in a HYSC incubator, model SI-64, at 25 °C for 15 days without shaking. For each hydrolysate, 12 flasks were used, 6 with the culture medium and 6 with the HS control.

Fermentations with their corresponding controls were carried out in triplicates (3 independent runs).

2.3. FTIR Characterization

The Fourier Transform Infrared Spectroscopy technique was used to verify that the product obtained from the process was BC. The FTIR analysis was performed using JASCO FT/IR-6800 infrared spectroscopy (FTIR) equipment. A dry sample was placed into the compartment. The spectrum was recorded in the range of 4000 to 450 cm⁻¹ with a resolution of 2 cm⁻¹ and 25 scans.

2.4. BC Production in a Stirred Tank Bioreactor (STB)

2.4.1. Operating Conditions Setup

Two sets of orbital stirring tests were carried out, one with standard HS medium [30] and the other with culture medium containing double the peptone concentration, called modified HS. Flasks of 250 mL volume were used with 100 mL of sterile culture medium (121 °C for 15 min) at pH 6.3 and an initial cell concentration of 1x10⁶ CFU/mL on the McFarland scale. Julabo SW22 and Selecta UNITRONIC thermostatic baths with 8 to 22 L capacity were used to maintain the temperature at 25 °C. Two runs at 100 rpm were performed for the standard and modified HS medium each.

To simulate the flow pattern in a STB, magnetic stirring tests at 100 rpm were conducted. Two agitation frequencies were studied, stirrer action from the beginning of fermentation (AM₀) and stirring from the fifth day (AM₅). Oxygen was supplied exclusively from the headspace air (AELC) available in 250 mL flasks with 100 mL of standard HS medium [30] at pH 6.3. Type-C PTFE magnetic stir bars (25 mm long) were employed in order to replicate the standard ratio between impeller and tank diameters (0.33) stated in STB.

The oxygenation role was assessed through forced aeration, where 500 mL kitasates coupled with a 0.2 µm Whatman Uniflo air filter were used with 250 mL of standard HS medium. Similarly, Type-C PTFE magnetic stir bars (40 mm long) were used. The flasks and kitasates along with plate magnetic stirrers were placed in a HYSC incubator, model SI-64, at 25 °C for 15 days. One replicate was carried out for all experiments.

2.4.2. Oxygen Uptake Rate (OUR)

The growth rate of microbial consortium BA2 in the broth was indirectly determined through oxygen uptake rate (OUR). The BC film was removed from the flasks and kitasates after 5, 10 and 15 days fermentation, and the dissolved oxygen (DO) concentration was measured with a polarographic DO sensor, Hamilton model VisiFerm. Each data set was registered every 20 sec over 10 min. OUR was calculated by Equation (2) from the variation of dissolved oxygen concentration ($\Delta C_L/\Delta t$); meanwhile, the oxygen transfer rate in the liquid–gas interface ($OTR$) was determined by Equation (3) accounting for oxygen solubility at 25 °C (${C_L}^{*}$).

$$OUR=OTR-{\displaystyle \frac{\Delta C_L}{\Delta t}}$$

(2)

$$OTR=k_{L}a\left({C_L}^{*}-C_L\right)$$

(3)

Mass transfer coefficient ($k_{L}a$), which denotes the oxygen transfer capability in the flask, was determined by Equation (4). This correlation was employed by Liu [32] for Phaffia rhodozyma shaked culture at 250 rpm and 20 °C, and contemplates the shaking speed ($N$), liquid culture volume ($V_L$) and the nominal flask size ($V_o$).

$$k_{L}a=0.141 N^{0.88}{\left({\displaystyle \frac{V_L}{V_o}}\right)}^{-0.8}$$

(4)

2.4.3. Fermentation in STB

BA2 was activated in five flasks of 250 mL with 100 mL of standard HS medium [30] under static conditions for 15 days at 25 °C in a HYSC model SI-64 incubator. The formed BC layer was transferred to 3 mL HS medium and vortexed for 3 min with 1 min rest to effectively release the entrapped microorganisms. The cell suspension was used as an inoculum with an initial optical density (OD) of at least 0.1.

A 1.5-L Infors HT bench-top Bioreactor Minifors 2 series was employed with one Rushton 6-blades impeller and a ring sparger at the bottom. Fermentations were performed at 25 °C, 100 rpm with 500 mL of medium and non-sparged air. Four culture media were tested for BC production: (1) standard HS [30], (2) modified HS doubled in peptone concentration, (3) complex HS with the hydrolysate with the highest BC yield in static conditions instead of glucose and (4) enriched HS which refers to the complex HS doubled in peptone concentration. A 100 mL flask for static culture control was incorporated in all fermentations.

At the end of each fermentation, medium density ($\rho$) was measured using Alcolyzer Beer ME equipment Anton Paar, model DMA 4500, with 5 mL culture medium previously centrifuged at 8000 rpm for 5 min. Concurrently, medium viscosity ($\mu$) was determined in a borosilicate glass Ostwald Viscometer College Pattern, model AC-464V. Agitation power ($P$) and Reynolds number ($N_{Re}$) were calculated by Equations (5) and (6), respectively [33], accounting for medium density and viscosity, impeller diameter ($D_a)$, agitation speed ($N$) and a power number ($Np$) equal to 4 for a Rushton 6-blades impeller at turbulent flow.

$$P=Np \rho N^3{D}_a^5$$

(5)

$$N_{Re}={\displaystyle \frac{N D_a^2 \rho }{\mu }}$$

(6)

2.5. Reducing Sugars Determination

Reducing sugars were measured in duplicates every 5 days for 15 days fermentation, and in STB’s case every 24 h. The 3,5-dinitrosalicylic acid (DNS) method [34] was used to quantify reducing sugars. A total of 250 μL of the DNS reagent was added to 500 μL sample and placed in a water bath at 95 °C for 15 min. The sample was then centrifuged at 8000 rpm for 5 min. The supernatant was diluted tenfold, and the absorbance was measured at 540 nm in a Biotek model Epoch 2 microplate spectrophotometer. Absorbance values were interpolated to a calibration curve to determine reducing sugar concentration.

The same method was employed when glucose was the only carbon source in the culture medium.

2.6. BC Recovery

The formed BC, when no layer was targeted, was recovered by vacuum filtration using a Büchner funnel and a LABDIN model Rocker 140 suction pump. The recovered BC was placed in a 0.1 M NaOH solution with a 1:20 solid–liquid ratio and incubated at 90 °C for 20 min to remove impurities and residual cells present in the polymer [35]. Then, several washes with distilled water were performed until a neutral pH was reached. The BC was dried at 50 °C in a forced air oven (Pol-eko 115ECO) for around 6 h or until a constant weight was reached.

Dry BC weight was measured in an analytical balance Radwag AS 310.X2 with a readability of 0.1 mg. BC to glucose yield (Yps) was calculated by Equation (7) as the ratio between BC production and the reducing sugars consumed at the end of the fermentation. $M{c}_{DB}$ is the BC dry weight, $V_m$ is the culture medium volume, and $R{S}_0$ and $R{S}_f$ are the initial and final reducing sugars concentrations, respectively.

$$\mathrm{Yps}={\displaystyle \frac{M{c}_{DB}/V_m}{R{S}_0-R{S}_f}}$$

(7)

3. Results and Discussion

3.1. BC Production from Agro-Industrial Wastes

3.1.1. Characterization and Hydrolysis of Agro-Industrial Wastes

Table S1 (supplementary material) shows the results of total carbohydrates and crude fiber on a dry basis for each material and the crude fiber to total carbohydrates ratio (CF/TC). Total carbohydrates are the major component in all materials, which range around data reported in the literature. Sugarcane bagasse registered 98% [36], mango peels and pineapple peels from different varieties contained approximately 90% [37,38], cocoa bean shell reached 63% [39] and rice bran from different crops registered 60 to 70% [40].

In plants, total carbohydrates comprise fiber and nonfiber carbohydrates. Nonfiber carbohydrates include cell wall components such as pectin substances and mixed linkage β-glucans and cell-built-in components like starch, fructans and other sugars [41], all of them sources of simple sugars. In contrast, for lignocellulosic materials, CF/TC depends on several factors. For instance, in mango peels from different varieties this parameter ranges from 23 to 48% [42], peels from fully ripe fruits registered 19.5% [43], pineapple peels for different crops contain from 8 to 22% [38], and rice brans resulting from grains with different grinding stages registered 25 to 31% [44]. In this sense, for the agro-industrial wastes CH, RB, PHs, PB, MHs, SB and CS, the parameter CF/TC ranges from 19 to 50%; in particular, that for MHs, PHs and RB reach 19.5%, 20.4% and 19.4%, respectively. Consequently, the remaining components of total carbohydrates without crude fiber represent 50 to 81%, and likely become a source of simple sugars. Thus, its extraction may require less intense treatments.

On the other hand, crude fiber in lignocellulosic materials is composed primarily of cellulose, hemicellulose and lignin [45]. Cellulose and hemicellulose are sources of simple sugars, and under severe hydrolysis conditions, the sugars released along with lignin are also sources of microbial inhibitors such as furan derivatives—furfural and hydroxymethylfurfural (HMF)—certain weak acids and phenolic compounds [46]. In this context, higher fiber content may result in hydrolysates with higher levels of simple sugars and microbial inhibitors, depending on hydrolysis conditions.

Overall, treatments with higher acid concentrations lead to hydrolysates with higher sugar and inhibitor content. Lenihan [47] reported an acceleration in the hydrolysis of potato peels when the concentration of phosphoric acid was increased from 2.5 to 10% (w/w). This resulted in a concomitant rise in the concentration of sugars from 8 to 55% (w/w) (T = 135 °C) and the concentration of inhibitors (HMF, furfural and acetic acid) from 4.5 to 7% (w/w) (T = 200 °C). Concentrated acid hydrolysis, involving concentrations greater than 25% w/w and temperatures below 100 °C [48], can result in sugar concentrations up to 80 g/L (80 °C, 80% w/w H₂SO₄) [49]. However, the elevated inhibitor content of these hydrolysates (up to 6 g/L HMF) requires the implementation of detoxification processes to reduce their concentration [50]. Conversely, when the hydrolysis conditions are less intense (comparable to those employed herein) with dilute acid (up to 4% w/w) at temperatures below 200 °C, the resulting hydrolysates display reduced levels of sugars, while also exhibiting diminished concentrations of inhibitors [51]. For instance, sugarcane bagasse hydrolyzed with 1.2% (w/w) H₂SO₄ in a 1:10 solid–liquid ratio for 2 h at 121 °C yields 7.2 g/L of glucose, 17.1 g/L of xylose, 2 g/L of arabinose and 1.4 g/L of furfural [52]. Defatted rice bran treated with 2% (v/v) H₂SO₄ in a 1:8 solid–liquid ratio for 2 h at 90 °C exhibits 13.36 g/L glucose, 1.88 g/L xylose, 1.13 g/L arabinose and 0.004 g/L furfural [53].

Of the seven processed materials, the presence of acid in hydrothermal treatment had a positive effect on the release of reducing sugars; the Ysm with acid is higher in all except MHs compared to the control without acid (

Table 2. Reducing sugars (g/L) released by the hydrothermal treatment of different agro-industrial wastes.

Materials	With Acid	Without Acid	Acid Contribution	With Acid Ysm (g/g)	Without Acid Ysm (g/g)
CH	28.57	6.19	22.38	0.14	0.03
SB	23.20	6.30	16.90	0.35	0.09
PB	54.74	51.46	3.28	0.55	0.51
RB	27.01	1.71	25.30	0.27	0.02
MH	41.54	51.96	NA	0.42	0.52
PH	45.75	44.86	0.89	0.46	0.45
CS	41.94	18.74	23.20	0.42	0.19

). However, the contribution is significant (more than 50%) only in CHs, SB, RB and CS. The hydrothermal treatment without acid released reducing sugars from all materials, particularly PB, MHs and PHs. In PB and PHs, the sugars released by the acid effect were only 3.28 g/L and 0.89 g/L, respectively. In MHs, acid treatment resulted even in a lower concentration of reducing sugars than the control without acid. This indicates that the acid effect in PB, MHs and PHs, under tested hydrolysis conditions, is not relevant to reducing sugars release and its use can be avoided. In PB, MHs and PHs, most of the sugars released may come from carbohydrates other than those contained in crude fiber, which comprise about 80% of total carbohydrates.

On the other hand, PB, MH and PH hydrolysates achieved without acid could have low microbial inhibitors, mainly of aromatic nature, generated during lignin degradation in the presence of acid [54]. Rogalisnki [55] reported similar yields of glucose and xylose during the hydrolysis of ray straw in water at 190 °C for 2 h with and without H₂SO₄ (0.07% w/w). In the same work, the yield of furan derivatives was higher in the presence of acid.

In this study, the purpose of hydrothermal treatment, under specific conditions for each material, is to allow a reducing sugars concentration higher than 20 g/L, the minimum value required to initiate the fermentation process. Thus, hydrolysates of CHs, SB, RB and CS with acid pretreatment, and hydrolysates of PB, MHs and PHs without acid pretreatment were used.

3.1.2. Fermentation

The fermentation process started with 20 g/L of reducing sugars in all culture media: CH, SB, PB, RB, MHs, PHs, CS and HS control. In all cultures, sugar consumption was gradual and did not exceed 50% for most materials after 15 days. For the HSs (control), sugar consumption was only 30% (Figure 1).

Under static conditions a 100% sugar consumption is rarely reported. Komagataeibacter xylinus, a typical cellulose-producing bacterial strain, consumed 67% of the glucose and only 33% of the fructose when fermenting cocoa mucilage exudates for 15 days [56]. On less complex media, K. xylinus consumed less than 50% of glucose or glycerol after 7 days of fermentation [57] and about 45% of xylose after 14 days of growth [58].

In terms of BC production (Figure 2, Table S2), the culture media from all agro-industrial wastes (except SB) generated a significantly higher amount of cellulose than the HS control medium after 10 and 15 days of fermentation, despite the possible presence of growth inhibitors in the hydrolysates. The inhibitory effect of furan derivatives, certain organic acids and phenolic compounds on Bacillus subtilis depends on their concentration in the culture medium. Furfural, 5-HMF, acetic acid, benzoic acid, ferulic acid, vanillic acid, vanillin, p-coumaric acid, 4-hydroxybenzoic acid and syringaldehyde have a marked inhibitory effect at concentrations greater than or equal to 0.2 g/L, 0.1 g/L, 0.75 g/L, 0.5 g/L, 0.5 g/L, 2 g/L, 0.3 g/L, 0.5 g/L, 2 g/L and 0.075 g/L, respectively [59].

For lignocellulosic residual hydrolysates obtained under analogous conditions to those used in this study, furan derivative values of 0.1 g/L furfural and 0.84 g/L HMF were reported for corn stover [52]; 0.12 g/L furfural for sugarcane bagasse [23]; 0.052 g/L furfural and 0.009 g/L HMF for mango seed husk [26]; 0.02 g/L furfural for rice hulls [25]; less than 0.001 g/L furfural and 0.03 g/L HMF for cocoa bean hulls [60]; and 0.18 g/L furfural and 0.26 g/L HMF for pineapple peels [24].

Moreover, as all agro-industrial waste hydrolysates were diluted 1.1 to 2.5-fold to normalize the reducing sugar content to 20 g/L, lower concentrations of furan derivatives inhibitors in the culture media should be expected compared to those indicated above. Particularly, for PB, MH and PH hydrolysates, even lower furan derivates could be detected since non-acid pretreatment was conducted. In this sense, the likely presence of inhibitory compounds, such as furan derivatives, at concentrations below inhibitory levels would explain the sugar consumption and BC production in all media by BA2.

Higher sugar consumption and BC production compared to the HS control (which only had glucose as a carbon source) as evidenced in the culture media from agro-industrial wastes (except SB) may be due to the presence of compounds beyond sugars that promote the microbial growth and formation of BC by the BA2 consortium. Nitrogen sources as well as its concentration affect BC production in different ways [28]. The higher concentration of free amino nitrogen, the lower the BC production [61]. An increase in yeast extract from 0.2% to 0.6% (w/v) [62], in peptone from 1% to 2% (w/v) [63] or supplying a more complete amino acid profile improves biopolymer production [64]. Therefore, it is possible that during the hydrolysis process, assimilable nitrogen has been released to the hydrolysates, in addition to the nitrogen provided by the components of the culture medium. For rice bran, its use as an alternative nitrogen source in HS medium to produce BC by Acetobacter xylinum has been demonstrated [65].

However, despite the possible presence of growth factors favoring the BA2 consortium, sugar consumption did not exceed 50% for most substrates, and BC production stabilized at day 10 (

Table 3. BC yield (dry basis) by BA2 from reducing sugars obtained using agro-industrial wastes.

Materials	Day 10			Day 15
	Reducing Sugars Consumed (g/L)	BC (g/L)	Yps (g/g)	Reducing Sugars Consumed (g/L)	BC (g/L)	Yps (g/g)
HS	5.83 ± 1.54	0.32 ± 0.11	0.061 ± 0.031 a	6.41 ± 1.20	0.30 ± 0.13	0.049 ± 0.022 a,b
CH	6.01 ± 0.44	1.50 ± 0.42	0.251 ± 0.069 c,d	8.08 ± 0.94	1.30 ± 0.62	0.164 ± 0.079 c,d
PB	6.90 ± 2.39	1.37 ± 0.15	0.213 ± 0.063 b,c	8.89 ± 1.71	1.29 ± 0.33	0.143 ± 0.015 c,d
MH	6.69 ± 0.49	1.02 ± 0.30	0.151 ± 0.036 a,b,c	8.79 ± 1.14	1.03 ± 0.57	0.113 ± 0.056 b,c,d
SB	6.67 ± 0.77	0.45 ± 0.16	0.067 ± 0.020 a	10.29 ± 0.59	0.31 ± 0.02	0.031 ± 0.004 a
RB	6.19 ± 1.30	2.13 ± 0.42	0.362 ± 0.139 d	8.18 ± 0.88	2.14 ± 0.37	0.261 ± 0.029 e
PH	7.95 ± 0.97	2.23 ± 0.93	0.277 ± 0.097 c,d	11.27 ± 1.54	2.04 ± 1.00	0.175 ± 0.067 d
CS	9.27 ± 0.21	0.84 ± 0.55	0.090 ± 0.059 a,b	11.81 ± 0.40	1.02 ± 0.58	0.087 ± 0.053 a,b,c

Each value is expressed as mean ± SD (n = 3). Different letters as superscript in a column indicate a significant difference (p-value < 0.05).

). There may have been other limiting factors, such as surface tension, which prevents the diffusion of O₂ and nutrients through the BC layer [66], or the lack of enhancers such as acetic acid [36], ethanol or lactic acid [67]. Despite the fact that cellulose-producing microbial consortia are characterized by the symbiotic activity of yeasts, which convert sugars to ethanol, and acetic acid bacteria, which use it to produce acetic acid [9], the concentration of these enhancers may have been lower than necessary to regulate the metabolism of cellulose-producing microorganisms in BA2 [67], which would explain the lower consumption of reducing sugars.

Among all the materials, RB is distinguished by its increased BC production (2.14 g/L) and significantly higher yield (0.261 g/g) after 15 days of fermentation (Table 3). This performance is related to the potential presence of sucrose in the RB hydrolysate [68] (a fermentable sugar useful for BC production) and the potential additional nitrogen input [65], making this material a good candidate for BC production.

Conversely, under conditions like those tested in this work, BC production by BA2 on certain substrates is similar and even exceeds that reported in the literature. After a 10-day fermentation of enzymatic hydrolysates of corncob (pretreated with liquid hot water) supplemented with yeast extract (5 g/L), peptone (5 g/L), citric acid (1. 1 g/L) and Na₂HPO₄ (2.7 g/L) adjusted to pH 6.0, with Komagateibacter sp. strains CCUG73629 and CCUG73630 yielded approximately 1 g/L and 0.4 g/L of BC, respectively [5]. Rice bran extract enriched with peptone (3 g/L) and D-mannitol (25 g/L) fermented with Acetobacter xylinum for 7 days produced about 0.25 g/L [65]. Pineapple peel juice fermented for 13 days with Gluconacetobacter swingsii sp. yielded 2.8 g/L BC [31]. These values, in comparison to 0.84g/L, 2.13g/L and 2.23g/L BC obtained after 10 days of fermentation using CS, RB and PHs, respectively, are indicative of the important role of the strain in BC production. The potential synergy of the BA2 consortium could be an advantage over the use of bacteria alone, which in many cases require the addition of BC enhancers in the culture medium, such as ethanol or lactic acid [69].

The strategies to produce BC from wastes of different industries aim to improve yields and BC properties by testing new materials or finding new ways to produce BC from already known materials [70]. Previous reports on BC production from complex substrates demonstrate similar or even higher yields compared to the classical HS medium.

Voon [71] showed equal BC production in enzymatic sago byproduct hydrolysates and HS medium with two bacterial strains, with Beijerinkia fluminensis and Gluconacetobacter xylinus reaching 0.5 g/L and 1.5 g/L BC, respectively. Pacheco [72] revealed similar BC production (6 g/L) in cashew exudates and HS medium. Besides, higher BC production compared to standard HS medium has been reported from date syrup (5.8 g/L) [73], enzymatic hydrolysates of corn cob (1 g/L), sugarcane bagasse (0.7 g/L) [5], root (2.25 g/L), stalk (1.75 g/L), and leaf sweet sorghum (2.5 g/L) [7].

3.1.3. FTIR Characterization

The FTIR spectra of the BCs obtained from the seven complex lignocellulosic substrates and micronized BC from HS medium control are presented in Figure S1 (supplementary material). All spectra show two main domains: one at high wavenumbers (between 3500 cm⁻¹ and 2800 cm⁻¹) and the other at low wavenumbers (between 1700 cm⁻¹ and 650 cm⁻¹) [74]. The peaks observed at high wavenumbers are characteristic of the stretching vibration of the -OH and -CH bonds in polysaccharides. The broad peak of 3334 cm⁻¹ is characteristic of the stretching vibration of the hydroxyl group in polysaccharides [75]. This peak also includes intermolecular and intramolecular hydrogen bond vibrations in cellulose [76]. The peak at 2896 cm⁻¹ is attributed to the vibration due to the stretching of the -CH group of the polysaccharides. Typical peaks assigned to cellulose are observed in the region between 1650 cm⁻¹ and 900 cm⁻¹. The peak located at 1643 cm⁻¹ corresponds to the vibration of water molecules absorbed in cellulose [75]. The absorption bands at 1428, 1366, 1315, 1031 and 898 cm⁻¹ belong to the stretching and bending vibrations of -CH₂ and -CH, -OH and -CO in cellulose [77]. The peak around 1420–1430 cm⁻¹ is associated with the crystalline structure of cellulose, while, at 898 cm⁻¹, the peak is assigned to the amorphous region in cellulose [75]. The absence of peaks between 1600 cm⁻¹ and 1500 cm⁻¹ and between 1740 cm⁻¹ to 1700 cm⁻¹ indicates the absence of lignin. These peaks correspond to the C=C aromatic skeletal vibration and to the acetyl or uronic ester groups of hemicelluloses, respectively [74]. This assessment revealed that all the materials obtained were cellulose.

3.2. BC Production in STB

3.2.1. Effect of Orbital Stirring and Increase of the Nitrogen Source

In shaken flasks oxygen availability is particularly high since the gas–liquid transfer area comprises the surface exposed to the surrounding air and the film on the flask wall [78]. This tendency promotes biomass generation along with high glucose consumption (>90%) and the consequent low product yield (6.53 mg/g) (

Table 4. Submerged fermentation outcomes with orbital stirring for BC production at 100 rpm and 25 °C with the BA2 consortium.

Parameter	Standard HS	Modified HS
Glucose consumption (%)	96.45 ± 0.57	94.90 ± 1.32
BC concentration (g/L)	0.126 ± 0.015	0.306 ± 0.071
Yps (mg/g)	6.53 ± 0.91	16.12 ± 0.22

). The eminent turbidity of the broth and disaggregated cellulose at the bottom of the flask (Figure 3a) reveal the predominance of BA2 consortium growth over BC production with a low glucose concentration after 15 days of fermentation.

The orbital mixing mechanism obviates the microorganism overlapping, breaks the boundary layers and promotes nutrient transfer [78], but poor oxygen transfer rate is frequently evidenced in shaken flasks with cotton plugs as closures, where $k_{L}a$ is estimated in 8.3 h⁻¹ for 250 mL (nominal volume) and 100 mL (filling volume) at 100 rpm [79]. Therefore, the required micro aerobic conditions in broth could be achieved after biomass production enhancement and consortium symbiosis for BC formation has been significantly affected.

Despite the unfavorable oxygen interplay, a 2.4-fold increase in BC production was accomplished as a double peptone in HS medium was adopted (Table 4). Hedge [80] found that peptone and yeast extract, as part of HS medium, have significant impacts on BC production. Thus, the optimal peptone concentration was estimated as 1.1704% (w/v) with a corresponding BC concentration of 16.943 g/L under static conditions while the other HS medium components remain steady. According to Zhang [63], the ratio of carbon to nitrogen leads to changes in the metabolic pathways, as glucose cannot be used exclusively to produce BC. Specifically, an excess of gluconic acid, a byproduct of metabolism, results in a substantial decrease in BC yield.

Apart from BC production improvement, peptone supplementation enhances polymer strength parameters as can be elucidated in Figure 3b, where disaggregated but more structured cellulose is shown. According to Betlej [11], an increase of 0.25% in peptone concentration results in an increase of tensile strength (948%), thickness (799%) and elongation (100%). The latter is correlated with the decrease in the degree of polymerization, which reflects that less molecules are building the polymer.

In this context, additional peptone in culture medium becomes a promising strategy in STB not only for BC yield but also for the upgrading of cellulose mechanical properties under higher shear forces than orbital shaking.

3.2.2. Effect of Magnetic Stirring and Different Oxygen Supply

As different agitation frequencies and types of aeration supply are conducted during submerged fermentation, a comparative approach of glucose consumption (Figure 4) and BC production (Figure 5) is presented.

In general, magnetic stirring promotes biofilm formation similar to static culture because the shape and size of the stirrer create a radial flow pattern with low shear forces. When headspace air (AELC) is used as the oxygen source, the glucose consumption rate decreases compared to orbital shaking. After 5 days of fermentation, 69% of the glucose has been used in contrast to the 93% registered in orbital fermentations. This trend indicates an enhancement in glucose assimilation for BC production (Figure 5). As biofilm reaches 1 mm thickness (around day 5 in a static culture), oxygen diffusion into the liquid medium is enabled. Thereafter, the resistance to oxygen transfer increases and most of the bacteria immobilized in the film assimilate it [60]. On the other hand, accelerated glucose consumption (75% after 10 days) without significant cellulose production (0.30 g/L) is demonstrated under static conditions up to 5 days of fermentation. Apparently, stirring after biofilm formation improves the rate of oxygen transfer into the broth and disrupts the oxidative metabolism of bacteria trapped in the cellulose layer.

Aeration onto the medium surface rather than deep into the medium has demonstrated a 25-fold rise in BC yield with symbiotic Medusomyces gisevii [16]. However, this aeration mechanism (kitasato), along with agitation, appears to be effective when no agitation is used until the fifth day of fermentation. Progressive glucose consumption is evident (Figure 4), with a direct effect on BC production (2.69 g/L). After 15 days of fermentation, 72.1% of the glucose is consumed, not much different from the 52.2% recorded on day 5. Forced air with ventilation (AFV) clearly compensates and even surpasses the increase in oxygen transfer rate derived from magnetic stirring on day 5 resulting in enhanced BC production. In contrast, a fully agitated culture with forced air causes a rapid glucose depletion (93%) that is not reflected in BC production (scarce concentration of 0.044 g/L). In this case, excessive oxygen transfer was promoted, leading to maximum biomass generation and an early steady-state fermentation, where low cellulose formation should be expected.

The oxygen role during fermentation in the scenarios with the highest BC yield (AM0-AELC and AM5-AFV) is shown in

Table 5. Oxygen uptake rate by consortium BA2 (mg/L h) and oxygen concentration in the liquid medium (mg/L) during submerged fermentation.

Fermentation Days	Magnetic Stirring at Beginning AELC		Magnetic Stirring at 5th Day AFV
	CL0	OUR	CL0	OUR
5	7.23 ± 0.37	0.028 ± 0.003	8.26 ± 0.30	0.027 ± 0.002
10	6.69 ± 0.19	0.033 ± 0.001	7.25 ± 0.46	0.029 ± 0.001
15	6.04 ± 0.54	0.038 ± 0.003	7.10 ± 0.34	0.030 ± 0.001

. Oxygen concentration in the liquid HS medium experiment shows a slight decrease throughout 15 days of fermentation (16% AELC and 14% AFV). It contrasts with the previous anaerobiosis detected during Kombucha production after 24 h, where tea compounds alone induced soluble oxygen consumption [5]. This reduction is not ascribable to microbial mortality since the BC layer allows oxygen access for bacteria to remain on the surface and perform oxidative metabolism. According to Tran [81], when microbial consortium is accounted for in BC production, bacteria dominate yeasts in biofilms from day 3 and reach a maximum population on day 6. In this sense, OUR is categorically related to yeasts composing the consortium BA2, which dominated bacteria in the liquid medium. Thus, the intensification of OUR (Table 5) is a consequence of microbial growth since the vast majority of currently known yeast species could grow under oxygen limited conditions while partially fermenting glucose to ethanol and carbon dioxide [82].

Magnetic stirring undoubtedly facilitates oxygen diffusion in the liquid medium ($k_{L}a$ = 16.89 h⁻¹) as well as forced air by the promotion of oxygen availability ($k_{L}a$ = 18.37 h⁻¹). BC film is definitely not the driver of anaerobiosis in the liquid. Therefore, magnetic stirring at 100 rpm from the beginning of the fermentation using only headspace air is the most appropriate alternative, leading to a BC concentration of 4.07 g/L (73% higher than static control).

3.2.3. Fermentation Performance in STB

The singularly high glucose consumption in standard and modified HS medium becomes representative when mechanical agitation is adopted, although lower BC production is targeted. Glucose assimilation is similar to magnetic stirrer tests up to 5 to 6 days of fermentation where about 55% was consumed (Figure 6). Thereafter, there is a drastic decrease with glucose almost depleted in 8 days. Despite the increase of the nitrogen source, a yield of 17.58 mg/g is obtained (

Table 6. Submerged fermentation outcomes for BC production in STB at 100 rpm and non-sparged air.

Parameter	Standard HS	Modified HS A	Complex HS B	Enriched HS C
BC concentration (g/L)	0.14	0.38	0.16	0.28
Yps (mg/g)	6.91	17.58	7.97	13.86
Reducing sugars consumption (%)	94.00	95.73	48.47	46.09

^A standard HS medium doubled in peptone concentration; ^B standard HS medium with the RB hydrolysate instead of glucose; ^C complex HS medium doubled in peptone concentration.

), which is 61 times less than the static control. The primary cause of this behavior may be the way cellulose is assembled in the bioreactor. In standard and modified HS medium, a tightly packed BC layer was formed due to the intrusion of a stirrer shaft, baffles and dissolved oxygen sensor into the interfacial surface. Most BC production is evidenced as pieces attached to metal accessories, including the Rushton impeller (Figure 7a). Therefore, the resistance to oxygen transfer to the broth decreases and biomass generation is promoted along with glucose consumption.

The bioreactor build-up material is a key feature that affects BC production. The stainless steel (AISI 316L) parts related to baffles, impellers and sensors within the borosilicate glass vessel restrain the biofilm formation. Skiba [83] reported a maximum layer thickness of 2 mm in an enameled iron vessel and 1.3 mm in glass, a maximum elastic moduli of 7300 MPa after 6 days in enameled iron and 16 days in glass closely linked to similar degrees of polymerization. These properties reflect an improved cellulose adherence to metal rather than glass.

The initial behavior of the RB hydrolysate replacing glucose under static conditions was not replicated when mechanical agitation was applied during fermentation. A first stage is distinguished up to 3 days with about 45% of reducing sugars consumed, then a second stage of a steady state is envisaged (Figure 6). After 8 days of cultivation with the complex HS medium, only 0.16 g/L BC was obtained (Table 6), 13-fold lower than the BC production after 10 days of static fermentation, although the reducing sugar consumption was similar. The likely additional nitrogen input related to the RB hydrolysate and the increase of the nitrogen source in the enriched HS medium certainly improve the BC yield up to 74%, but it is still 25-fold inferior compared to static fermentation.

In complex and enriched HS medium, no biofilm layer is observed, and cellulose is produced by adhesion exclusively to bioreactor walls and a little to metal accessories (Figure 7b). In this case, the specific interfacial surface (SIS) may be another factor influencing the interaction between oxygen transfer, reducing sugar consumption and cellulose formation. The bioreactor SIS (0.7854 m) is 35-fold and 65-fold larger than for 250 mL flasks and 500 mL kitasates, respectively. Tran [81] distinguished three regions in biofilm, a thick top layer containing viable and few non-viable bacteria, a middle layer composed of viable bacteria and yeast, and a thin bottom layer composed of numerous viable bacteria. Bacteria inhabiting the biofilm access reducing sugars primarily through the liquid/biofilm interface. Thus, a larger SIS inhibits reducing sugar assimilation by cellulose producing bacteria, although the biochemical dynamics are different for bacterial and SCOBY cultures [83].

BC production in bioreactors has been widely investigated for pure bacterial strains, where high BC yield is achieved under high shear forces [3]. This strategy is not feasible for consortium BA2 because high oxygen transfer rate promotes yeast formation. The rheological properties (viscosity and density) are quite similar to pure water, which involves low stirring power and Reynolds number (

Table 7. Submerged fermentation outcomes for BC production in STB at 100 rpm and non-sparged air.

Parameter	Standard HS	Modified HS A	Complex HS B	Enriched HS C
Viscosity at 20 °C ×${10}^{-3}$ (Pa·s)	1.087	1.213	0.939	1.002
Density (kg/m3)	1006.45	1011.02	1001.80	1007.06
Agitation power × ${10}^{-3}$(W)	1.908	1.917	1.899	1.909
Reynolds number	2469.14	2221.23	2864.11	2671.47

^A standard HS medium doubled in peptone concentration; ^B standard HS medium with the RB hydrolysate instead of glucose; ^C complex HS medium doubled in peptone concentration.

). In particular, since RB hydrolysate is a medium component, suspended solids can be detected and the Rushton impeller is unable to mix the culture broth, nor reduce sugar availability. Therefore, in addition to evaluation of operating conditions and medium formulation, analysis of bioreactor design is required to improve the synthesis of BC by microbial consortium.

4. Conclusions

The innovative use of seven agro-industrial wastes as nutrient sources, with outstanding rice bran (RB) BC production (2.14 g/L dry basis), marks a significant advancement over traditional HS medium matching cellulose structural integrity verified by FTIR. This research delineates the critical influence of fermentation conditions on BC yield, with a notable distinction between stirring methods and their impact on oxygen availability and glucose consumption during symbiotic metabolism of microbial consortium BA2. The strategy involving magnetic stirring at 100 rpm using only headspace air improves BC production by 73% compared to static culture. This study envisages the requirement of distinct bioreactor design since mechanical agitation and cellulose adherence to metal in stirred tank bioreactors negatively affect BC yield (67% lower than static control) despite the assurance of low shear forces and controlled oxygen transfer rate to liquid.