Design, construction and optimization a flexible bench-scale rotating biological contactor (RBC) for enhanced production of bacterial cellulose by Acetobacter Xylinium.

In this research a bench scale rotating biological contactor (RBC) was designed and constructed to produce BC. The effects of variables including rotation speed of the disk, distance between disks, disk type and external aeration on BC productivity were investigated. Results showed that the highest weight of BC produced on the surface of integrated polyethylene discs which rotated at 13 rpm. It was also found that the highest amount of BC was obtained when the space between two adjacent discs was adjusted to 1 cm and the disk number was 16. An aquarium pump was used to investigate the impact of aeration on RBC made of 12 integrated polyethylene discs and operated at optimal rotation speed of 13 rpm. Disk spacing distance was adjusted to 1.5 cm to consider the possible increasing of the thickness of BC film by aeration. Wet weight and dry weight of BC resulted from aerated fermentation increased more than 64 and 47%, respectively as compared to non-aerated RBC. In comparison with static culture, wet weight and dry weight of BC produced in aerated RBC fermentation increased more than 90.7 and 71%, respectively. Nanoscale structure of produced bacterial cellulose was confirmed by SEM analysis.

Effect of Atomized Delivery of Nutrients on the Growth Characteristics and Microstructure Morphology of Bacterial Cellulose.

This work demonstrates a general strategy for introducing remarkable changes in matrix organization and, consequently, functional properties of bacterial cellulose (BC). BC-producing cells were induced, using a well-defined atomized droplet nutrient delivery (ADND) system, to form pellicles with a regular layered morphology that persists throughout the mat depth. In contrast, the morphology of mats formed by conventional static medium nutrient delivery (SMND) is irregular with no distinguishable pattern. ADND also resulted in larger meso-scale average pore sizes but did not alter the fibril diameter (∼70 nm) and crystallinity index (92-95%). The specific modulus and specific tensile strength of ADND mats are higher than those of SMND mats. This is due to the regularity of dense layers that are present in ADND mats that are able to sustain tensile loads, when applied parallel to these layers. The density of BC films prepared by ADND is 1.63-fold lower than that of the SMND BC film. Consequently, the water contents (g/g) of ADND- and SMND-prepared BC mats are 263 ± 8.85 and 99.6 ± 2.04, respectively. A model that rationalizes differences in mat morphology resulting from these nutrient delivery methods based on nutrient and oxygen concentration gradients is proposed. This work raises questions as to the extent that ADND can be used to fine-tune the matrix morphology and how the resulting lower density mats will alter the diffusion of actives from the films to wound sites and increase the ability of cells to infiltrate the matrix during tissue engineering.

Production and characterization of bacterial cellulose obtained by Gluconacetobacter xylinus utilizing the by-products from Baijiu production.

Bacterial cellulose (BC) has extensive application prospects in many fields in view of its unique characteristics. However, the large-scale applications of BC are severely limited because of relatively low BC productivity and high cost of culture medium. Herein, the distiller's grain enzymatic hydrolysate (DEH) and yellow water were successfully combined as an effective substitute (the best distiller's grains-yellow water medium, BDY medium) for traditional Hestrin-Schramm medium (HS medium) for BC production by Gluconacetobacter xylinus through the response surface methodology. The BC production in BDY medium was significantly enhanced to 7.42 g/l with BC conversion yield of 42.4% after 7 days static cultivation, which was 3.72-fold and 3.37-fold higher than that in HS medium, respectively. The structure and properties of BC membranes produced in HS and BDY medium were evaluated by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FE-SEM), thermogravimetric analysis (TGA) and hydrophilicity analysis. There was no significant difference between BC samples produced in the HS and BDY medium, indicating that BDY, as abundant and inexpensive substrates, can effectively replace HS medium to enhance BC production. The employment of distiller's grains and yellow water to BC production not only is conducive to achieve industrial production of BC, but also can effectively realize the recycling of waste from Baijiu distillery.

Grown Ultrathin Bacterial Cellulose Mats for Optical Applications.

A facile and effective method is described for the biosynthesis of ultrathin bacterial cellulose (BC) mats, which are green, inexpensive, lightweight, and flexible. Physical properties studied include thickness, morphology, reflectance, transmittance, and crystallinity index. BC mat thickness was varied by controlling the depth of the culture broth so that films with predictable thickness, between 113 and 1114 nm, were produced. These BC films have similar fiber morphology to corresponding mm thick BC films prepared under static culture conditions. To increase BC film hydrophobicity, surface trihexylsilylated BC (THSBC) mats with DSavg 0.015 were prepared. Both native and THSBC mats were investigated as antireflection coatings for silicon substrates. The 328 ± 42 nm thick BC mat demonstrated broadband, interference type antireflection over a spectral range of 500-1800 nm. Different reflection properties obtained as a function of BC film orientation reveals that engineered density gradients can be used to manipulate BC optical properties. Thus, optical quality and environmental friendly ultrathin BC films are promising biomaterials for next-generation optoelectronic devices.

Comparison of tolerance of four bacterial nanocellulose-producing strains to lignocellulose-derived inhibitors.

BACKGROUND: Through pretreatment and enzymatic saccharification lignocellulosic biomass has great potential as a low-cost feedstock for production of bacterial nanocellulose (BNC), a high value-added microbial product, but inhibitors formed during pretreatment remain challenging. In this study, the tolerance to lignocellulose-derived inhibitors of three new BNC-producing strains were compared to that of Komagataeibacter xylinus ATCC 23770. Inhibitors studied included furan aldehydes (furfural and 5-hydroxymethylfurfural) and phenolic compounds (coniferyl aldehyde and vanillin). The performance of the four strains in the presence and absence of the inhibitors was assessed using static cultures, and their capability to convert inhibitors by oxidation and reduction was analyzed. RESULTS: Although two of the new strains were more sensitive than ATCC 23770 to furan aldehydes, one of the new strains showed superior resistance to both furan aldehydes and phenols, and also displayed high volumetric BNC yield (up to 14.78 ± 0.43 g/L) and high BNC yield on consumed sugar (0.59 ± 0.02 g/g). The inhibitors were oxidized and/or reduced by the strains to be less toxic. The four strains exhibited strong similarities with regard to predominant bioconversion products from the inhibitors, but displayed different capacity to convert the inhibitors, which may be related to the differences in inhibitor tolerance. CONCLUSIONS: This investigation provides information on different performance of four BNC-producing strains in the presence of lignocellulose-derived inhibitors. The results will be of benefit to the selection of more suitable strains for utilization of lignocellulosics in the process of BNC-production.

Preparation of an inoculum of Gluconacetobacter xylinus without mutants in shaken culture.

AIMS: A high-quality inoculum of Gluconacetobacter xylinus is important to produce bacterial cellulose (BC), a versatile biomaterial. This work aims to develop a method of preparing an inoculum of this bacterium with high cell density and without mutants. METHODS AND RESULTS: Inocula of G. xylinus ACCC 10220 without and with cellulase or carboxymethyl cellulose (CMC) were prepared in shaken culture. BC pellets and BC-negative mutants were present in the inoculum without additives but absent in the inoculum with additives. Based on BC weights statically produced in fresh BC-producing media initiated by different seed culture, the 24-h-shaken inoculum with 1·50% (w/v) CMC was the best because of high biomass and absence of mutants. The BC weights in fresh media inoculated by the 96-h-static inoculum and 24-h-shaken CMC inoculum at 7% (v/v) were 0·70 and 1·05 g l(-1) , respectively, implying significant difference (P < 0·01) in BC weights. However, structure properties of the two BC samples, including the crystallinity index, mass fraction of cellulose Iα , degree of polymerization (DP) and micromorphology were slightly different. CONCLUSIONS: The 24-h-shaken CMC inoculum was the most suitable for a starter culture of BC. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel method of preparing G. xylinus inoculum in shaken culture was developed, featuring high biomass, absence of mutants and no BC entanglements. Cellulase or CMC added into the medium completely suppressed mutation of G. xylinus, and CMC facilitated to form colloidal BC with the low DP in shaken culture, indicating less BC stress to cells. These findings suggested the mutation could be induced by BC stress, and not by shear stress commonly accepted.

Bacterial cellulose production by Gluconacetobacter xylinus by employing alternative culture media.

Bacterial cellulose (BC) is used in different fields as a biological material due to its unique properties. Despite there being many BC applications, there still remain many problems associated with bioprocess technology, such as increasing productivity and decreasing production cost. New technologies that use waste from the food industry as raw materials for culture media promote economic advantages because they reduce environmental pollution and stimulate new research for science sustainability. For this reason, BC production requires optimized conditions to increase its application. The main objective of this study was to evaluate BC production by Gluconacetobacter xylinus using industry waste, namely, rotten fruits and milk whey, as culture media. Furthermore, the structure of BC produced at different conditions was also determined. The culture media employed in this study were composed of rotten fruit collected from the disposal of free markets, milk whey from a local industrial disposal, and their combination, and Hestrin and Schramm media was used as standard culture media. Although all culture media studied produced BC, the highest BC yield-60 mg/mL-was achieved with the rotten fruit culture. Thus, the results showed that rotten fruit can be used for BC production. This culture media can be considered as a profitable alternative to generate high-value products. In addition, it combines environmental concern with sustainable processes that can promote also the reduction of production cost.

Effects of aromatic compounds on the production of bacterial nanocellulose by Gluconacetobacter xylinus.

BACKGROUND: Bacterial cellulose (BC) is a polymeric nanostructured fibrillar network produced by certain microorganisms, principally Gluconacetobacter xylinus. BC has a great potential of application in many fields. Lignocellulosic biomass has been investigated as a cost-effective feedstock for BC production through pretreatment and hydrolysis. It is well known that detoxification of lignocellulosic hydrolysates may be required to achieve efficient production of BC. Recent results suggest that phenolic compounds contribute to the inhibition of G. xylinus. However, very little is known about the effect on G. xylinus of specific lignocellulose-derived inhibitors. In this study, the inhibitory effects of four phenolic model compounds (coniferyl aldehyde, ferulic acid, vanillin and 4-hydroxybenzoic acid) on the growth of G. xylinus, the pH of the culture medium, and the production of BC were investigated in detail. The stability of the phenolics in the bacterial cultures was investigated and the main bioconversion products were identified and quantified. RESULTS: Coniferyl aldehyde was the most potent inhibitor, followed by vanillin, ferulic acid, and 4-hydroxybenzoic acid. There was no BC produced even with coniferyl aldehyde concentrations as low as 2 mM. Vanillin displayed a negative effect on the bacteria and when the vanillin concentration was raised to 2.5 mM the volumetric yield of BC decreased to ~40% of that obtained in control medium without inhibitors. The phenolic acids, ferulic acid and 4-hydroxybenzoic acid, showed almost no toxic effects when less than 2.5 mM. The bacterial cultures oxidized coniferyl aldehyde to ferulic acid with a yield of up to 81%. Vanillin was reduced to vanillyl alcohol with a yield of up to 80%. CONCLUSIONS: This is the first investigation of the effect of specific phenolics on the production of BC by G. xylinus, and is also the first demonstration of the ability of G. xylinus to convert phenolic compounds. This study gives a better understanding of how phenolic compounds and G. xylinus cultures are affected by each other. Investigations in this area are useful for elucidating the mechanism behind inhibition of G. xylinus in lignocellulosic hydrolysates and for understanding how production of BC using lignocellulosic feedstocks can be performed in an efficient way.

[Preparation for and study on the property of medical bacterial cellulose].

Bacterial cellulose (BC) was prepared by Acetobacter xylinum in static culture. After purified by chemical treatment, the microstructure, chemical structure, crystal structure and mechanical property of BC were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffractometry (XRD) and tensile strength measurement respectively, and compared with those of the imported bacterial cellulose wound dressing served as control sample (XBC). The results indicated that the diameter of the BC was (22 +/- 9) nm, and the crystallinity index was 89.71%. The tensile strength and the Young's mouduls of BC were significant higher than XBC both in wet and dry states. The biocompatibility of BC and XBC were evaluated by cytotoxicity test, delayed contact sensitization study in the Guinea Pig and skin irritation test. The results showed that BC had reliable biocompatibility as well as XBC. With the unique nanostructure, high crystallinity, high mechanical strength, and reliable biocompatibility, BC produced in our country as well as XBC can be used as a safe biomaterial for the medical applications.

Response surface methodology-based improvement of the yield and differentiation of properties of bacterial cellulose by metabolic enhancers.

This study aims to examine the effect of ethanol and lactic acid on the production of bacterial cellulose, and determine the optimal composition of a co-supplemented culture using response surface methodology. Both ethanol and lactic acid, when added separately or jointly, affected the yield and properties of the biomaterial. Optimization resulted in an increase of 470% in the yield, compared to the Schramm-Hestrin medium. Culture growth profiles, substrate consumption and by-products generation, were examined. The growth rate was increased for cultures supplemented with lactic acid and both lactic acid and ethanol, while the production of gluconic acid was diminished for all modified cultures. The properties of BNC, such as the structure, crystallinity, water holding capacity and tensile strength, were also determined. BNC produced in optimal conditions is more porous and characterized by wider fibers. Despite a decrease in crystallinity, by the addition of ethanol, lactic acid and both additives, the ratio of cellulose Iα was almost unchanged. The stress, strain, young modulus and toughness were improved 2.8-4.2 times, 1-1.9 times, 2.4-3.5 times and 2.5-6.8 times, respectively. The new approach to improving BNC yields and properties presented here could contribute to more economical production and wider application of this biopolymer.

Influence of 1-methylcyclopropene (1-MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture.

AIMS: Bacterial cellulose is an extracellular polysaccharide secreted by Acetobacter xylinum, which has become a novel material increasingly used in food and medical industries. However, its broad application is limited by its low yield and high cost. 1-Methylcyclopropene (1-MCP) is a potent inhibitor to either exogenous or endogenous ethylene during the biological senescence of plants, which has been broadly applied in commercial preservation of fruits and vegetables. The purpose of this study was to investigate the effects of 1-MCP on both the growth of Acet. xylinum and its cellulose production to demonstrate the potential enhancement of bacterial cellulose yield. METHODS AND RESULTS: Three groups of samples were fermented under agitated culture with 125 rev min(-1) rotational speed. To the culture media, 0.14 mg of 1-MCP contained in 100 mg dextrose powder was added on assigned days or on the first culture day only. Results from the measurement of bacterial cell concentration and bacterial cellulose yield at the end of a 12-day culture demonstrated that cultures excluding 1-MCP displayed a higher cell concentration and a lower cellulose production, while cultures containing 1-MCP produced 15.6% more cellulose (1-MCP added on day 1) and 25.4% (1-MCP added on each assigned day) with less biomass. CONCLUSIONS: 1-MCP was able to affect the growth of Acet. xylinum cells and resulted in increasing bacterial cellulose yield up to 25.4% over controls, which did not contain 1-MCP. SIGNIFICANCE AND IMPACT OF THE STUDY: This was the first study to use the growth inhibitor of plants to investigate its effects on bacterial growth and production. It also demonstrated a significant enhancement of bacterial cellulose yield by the addition of 1-MCP during the common agitated culture of Acet. xylinum.

Production and characterization of Gluconacetobacter xylinus bacterial cellulose using cashew apple juice and soybean molasses.

Bacterial cellulose (BC) has been largely used in biomedical and technological fields. The use of agro-industrial byproducts as alternative source of carbon and nitrogen in culture media reduces the BC cost production, adds value to the byproducts and minimizes the environmental impact. In this study, the use of cashew apple juice and soybean molasses were evaluated to produce BC by Gluconacetobacter xylinus in comparison to the usual Hestrin and Schramm medium (HS). BC produced in static cultivation was characterized by X-ray diffraction, Fourier transform infrared spectroscopy and thermogravimetric analysis. The BC production (4.50 g L-1) obtained from the medium using cashew apple juice as carbon source (20 g L-1) with soybean molasses as nitrogen source (10 g L-1) was superior than HS medium (4.03 g L-1). Morphological analysis showed that bacterial celluloses produced with agro-industrial byproducts combined were similar to those found for the pellicle obtained from HS medium.

Robust All-Cellulose Nanofiber Composite from Stack-Up Bacterial Cellulose Hydrogels via Self-Aggregation Forces.

All-cellulose composites are usually prepared by removing impurities and using a surface-selective dissolution approach, which detract significantly from their environment-friendly properties. In this paper, we report an environment-friendly approach to fabricate all-cellulose nanofiber composites from stack-up bacterial cellulose (BC) hydrogels via self-aggregation forces of the hydrogen bond by water-based processing. Structural and mechanical properties of BC-laminated composites have been investigated. The results indicated that BC composites possess the structure of all nanofibers, a tensile strength of 116 MPa, and a storage modulus of 25 GPa. Additionally, the interfacial shear strength and tensile strength of piece-hot-press BC demonstrate the strong self-aggregation forces of BC nanofibers. Thus, BC-laminated composites will be attractive in structural material.

Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524.

AIMS: To determine the effect of carbon sources on cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, and to characterize the purity and structural features of the cellulose produced. METHODS AND RESULTS: Modified Hestrin Schramm medium containing the carbon sources mannitol, glucose, glycerol, fructose, sucrose or galactose were inoculated with Ga. xylinus strain ATCC 53524. Plate counts indicated that all carbon sources supported growth of the strain. Sucrose and glycerol gave the highest cellulose yields of 3.83 and 3.75 g l(-1) respectively after 96 h fermentation, primarily due to a surge in cellulose production in the last 12 h. Mannitol, fructose or glucose resulted in consistent rates of cellulose production and yields of >2.5 g l(-1). Solid state (13)C CP/MAS NMR revealed that irrespective of the carbon source, the cellulose produced by ATCC 53524 was pure and highly crystalline. Scanning electron micrographs illustrated the densely packed network of cellulose fibres within the pellicles and that the different carbon sources did not markedly alter the micro-architecture of the resulting cellulose pellicles. CONCLUSIONS: The production rate of bacterial cellulose by Ga. xylinus (ATCC 53524) was influenced by different carbon sources, but the product formed was indistinguishable in molecular and microscopic features. SIGNIFICANCE AND IMPACT OF THE STUDY: Our studies for the first time examined the influence of different carbon sources on the rate of cellulose production by Ga. xylinus ATCC 53524, and the molecular and microscopic features of the cellulose produced.

Solid matrix-assisted printing for three-dimensional structuring of a viscoelastic medium surface.

Gluconacetobacter xylinus (G. xylinus) metabolism is activated by oxygen, which makes the formation of an air-medium interface critical. Here we report solid matrix-assisted 3D printing (SMAP) of an incubation medium surface and the 3D fabrication of bacterial cellulose (BC) hydrogels by in situ biosynthesis of G. xylinus. A printing matrix of polytetrafluoroethylene (PTFE) microparticles and a hydrogel ink containing an incubation medium, bacteria, and cellulose nanofibers (CNFs) are used in the SMAP process. The hydrogel ink can be printed in the solid matrix with control over the topology and dimensional stability. Furthermore, bioactive bacteria produce BC hydrogels at the surface of the medium due to the permeability of oxygen through the PTFE microparticle layer. The flexibility of the design is verified by fabricating complex 3D structures that were not reported previously. The resulting tubular BC structures suggest future biomedical applications, such as artificial blood vessels and engineered vascular tissue scaffolding. The fabrication of a versatile free-form structure of BC has been challenged due to restricted oxygen supplies at the medium and the dimensional instability of hydrogel printing. SMAP is a solution to the problem of fabricating free-form biopolymer structures, providing both printability and design diversity.

Improved bacterial nanocellulose production from glucose without the loss of quality by evaluating thirteen agitator configurations at low speed.

Thirteen agitator configurations were investigated at low speed in stirred-tank reactors (STRs) to determine if improved crude bacterial nanocellulose (BNC) productivity can be achieved from glucose-based media while maintaining high BNC quality using Komagataeibacter xylinus ATCC 23770 as a model organism. A comparison of five single impellers showed the pitched blade (large) was the optimal impeller at 300 rpm. The BNC production was further increased by maintaining the pH at 5.0. Among the single helical ribbon and frame impellers and the combined impellers, the twin pitched blade provided the best results. The combined impellers at 150 rpm performed better than the single impellers, and after optimizing the agitation conditions, the twin pitched blade (large) and helical ribbon impellers performed the best at 100 rpm. The performances of different agitators at low speed during BNC production were related to how efficiently the agitators improved the oxygen mass transfer coefficient. The twin pitched blade (large) was verified as providing the optimum performance by an observed crude BNC production of 1.97 g (L×d)-1 and a BNC crude yield of consumed glucose of 0.41 g g-1 , which were 2.25 and 2.37 times higher than the initial values observed using the single impeller respectively. Further characterization indicated that the BNC obtained at 100 rpm from the STR equipped with the optimal agitator maintained high degree of polymerization and crystallinity.

Performance of nanocellulose-producing bacterial strains in static and agitated cultures with different starting pH.

The impact of strain selection and culture conditions on bacterial nanocellulose (BNC) productivity and quality was investigated by using four strains, static and agitated cultures, and an initial pH in the range 4-6. With agitation, strain DHU-ATCC-1 displayed highest productivity [1.14 g/(L × d)]. In static cultures, DHU-ZGD-1186 exhibited superior BNC yield on consumed glucose (0.79 g/g), and lowest by-product formation with respect to gluconic acids [≤0.07 g/(L × d)]. By-product formation typically decreased in the order gluconic acid >2-keto-gluconic acid >5-keto-gluconic acid, and was lowest in cultures with high initial pH. The BNC from DHU-ZGD-1186 exhibited higher average viscometric degree of polymerization (DPv), higher crystallinity index, and higher tear index. In conclusion, both strain selection and cultivation conditions had an impact on BNC productivity and properties. Productivity, DPv, crystallinity, and mechanical strength of BNC from agitated cultures could be similar to or even higher than the corresponding values for static cultures.

Completely amorphous cellulose biosynthesized in agitated culture at low temperature.

The morphology and structure of the biosynthesized cellulose are related to the culture methods and conditions. In order to investigate the detail culture conditions, the Gluconacetbacter xylinum 1.1812 (ATCC 23767) strains were cultivated in static culture at 12 and 30 °C, and agitated culture at 12 °C. The cellulose samples were analyzed by FESEM, FTIR, CP/MAS 13C NMR, WAXRD and TGA. The cellulose membrane produced in static medium at 30 °C is made up of the microfibrils with a width of 60-90 nm, which has highest crystallinity index and the most content of cellulose Iα. The cellulose membrane produced in static medium at 12 °C is accumulated by the pellicles with a thickness of ~10 nm and a width of 700-3000 nm, which is cellulose I crystalline structure. The macroscopic sphere-like cellulose produced in agitated culture at 12 °C is composed of flat, strongly twisted cellulose bands with a width of 700-1200 nm, and reveals completely amorphous structure which exhibits only the diffuse X-ray diffraction pattern, lack of characteristic crystalline peaks. This work provides a new method to prepare amorphous cellulose.

Effect of in situ modification of bacterial cellulose with carboxymethylcellulose on its nano/microstructure and methotrexate release properties.

Bacterial cellulose/carboxymethylcelullose (BC/CMC) biocomposites with different DS-CMC (DS from 0.7 to 1.2) were developed in order to evaluate their impact as a drug delivery system. Biocomposites were loaded with methotrexate (MTX) as an alternative for the topical treatment of psoriasis. Scanning electron microscopy and atomic force microscopy showed that the CMC coated the cellulose nanofibers, leading to the decrease of the elastic modulus as the DS of CMC increased. BC/CMC0.9 exhibited the lower liquid uptake (up to 11 times lower), suggesting that the more linear structure of the intermediate substitute CMC grade (0.9) was able to interact more strongly with BC, resulting in a denser structure. All samples showed a typical burst release effect in the first 15min of test, however the BC/CMC0.9 biocomposite promoted a slight lowering of MTX release rates, suggesting that the DS of CMC can be considered the key factor to modulate the BC properties.

Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy.

A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to determine the composition and concentration in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards. This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation. Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass production on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 x 10(-3) [C-mol/C-mol substrate/h] in the batch process to 2.9 x 10(-3) [C-mol/C-mol substrate/h] in the fed-batch process described in this study.