Effects of CMC addition on bacterial cellulose production in a biofilm reactor and its paper sheets analysis

Bacterial cellulose (BC) can be grown into any desired shape such as pellicles, pellets, and spherelike balls, depending on the cultivation method, additives, and cell population. In this study, Acetobacter xylinum (ATCC 700178) was grown in the production medium with different concentrations of carboxylmethylcellulose (CMC) and were evaluated for BC production by using a PCS biofilm reactor. The results demonstrated that BC production was enhanced to its maximum (～13 g/L) when 1.5% of CMC was applied, which was 1.7-fold higher than the result obtained from control culture. The major type of the produced BC was also switched from BC pellicle to small pellets. The ratio of BC pellets in suspension increased from 0 to 93%. Fourier transform infrared (FTIR) spectroscopy demonstrated that CMC was incorporated into BC during fermentation and resulted in the decreased crystallinity and crystal size. The X-ray diffraction (XRD) patterns indicated that CMC-BC exhibited both lower crystallinity (80%) and crystal size (4.2 nm) when compared with control samples (86% and 5.3 nm). The harvested BC was subjected to paper formation and its mechanical strength was determined. Dynamic mechanical analysis (DMA) results demonstrated that BC paper sheets exhibited higher tensile strength and Young's modulus when compared with regular paper.     

Enhanced pullulan production in a biofilm reactor by using response surface methodology

Pullulan is a linear homopolysaccharide that is composed of glucose units and often described as α-1, 6-linked maltotriose. In this study, response surface methodology using Box–Behnken design was employed to study the effects of sucrose and nitrogen concentrations on pullulan production. A total of 15 experimental runs were carried out in a plastic composite support biofilm reactor. Three-dimensional response surface was generated to evaluate the effects of the factors and to obtain the optimum condition of each factor for maximum pullulan production. After 7-day fermentation with optimum condition, the pullulan production reached 60.7 g/l, which was 1.8 times higher than the result from initial medium, and was the highest yield reported to date. The quality analysis demonstrated that the purity of produced pullulan was 95.2%, and its viscosity was 2.5 centipoise (cP), which is higher than the commercial pullulan and related to its molecular weight. Fourier transform infrared spectroscopy (FTIR) also suggested that the produced exopolysaccharide was pullulan.     

不同培养方式对细菌纤维素产量和结构性质的影响

考察了自行筛选的Acetobacter xylinum NUST4.2在静置培养和发酵罐培养获得的细菌纤维素(BC)的产量、基本结构和性能的差异。结果表明:静置培养时产纤维素7.5g/L,产率为0.052g/L/h,在机械搅拌发酵罐中培养3d产量达3.13g/L,产率达0.043g/L/h;SEM分析显示静置培养和发酵罐培养得到的纤维素均具有网状结构,但静置获得的纤维素丝带相互缠绕且层状重叠,更加致密,丝带更细;FT-IR分析知搅拌不改变纤维素的化学结构,但能减弱分子间氢键,和XRD结合分析可知静置培养的纤维素具有更高结晶指数,更高Iα含量和更大晶粒尺寸,但不改变晶型,仍为纤维素I型,说明搅拌会干扰纤维素初始纤丝的结晶,有利于形成更小的晶粒和较Iα稳定的Iβ。与棉纤维素相比,静置培养获得的纤维素的热稳定性更好,而发酵罐培养获得的纤维素则阻燃性更好。     

Enhanced human lysozyme production in biofilm reactor by Kluyveromyces lactis K7

Lysozyme is an antimicrobial compound, which has been used in pharmaceutical and food industries. Chicken egg is the commercial source of lysozyme. However, human lysozyme is more effective and safer than egg-white lysozyme. Human milk is an important source for human lysozyme, but it is not feasible to provide the needed lysozyme commercially. Biofilm reactors provide passive immobilization of cells onto the solid support, which may lead to higher productivity. The aim was to evaluate the fermentation medium composition for enhanced human lysozyme production by Kluyveromyces lactis K7 in biofilm reactor with plastic composite supports. Yeast nitrogen base was selected as the best nitrogen source when compared to the yeast extract and corn steep liquor. Moreover, inhibition effect of NaCl and NH₄Cl at the concentrations of 25 and 50 mM was observed. Three factors Box–Behnken response surface design was conducted and the results suggested 16.3% lactose, 1.2% casamino acid, 0.8% yeast nitrogen base as optimum medium composition for maximum human lysozyme production. Overall, the human lysozyme production by K. lactis K7 was increased to 173 U/ml, which is about 23% improvement in biofilm reactor and 57% improvement compared to the suspended-cell fermentation.     

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

Optimization of the production of bacterial cellulose using multivariable linear regression analysis

The biosynthesis of bacterial cellulose by Acetobacter xylinum was optimized by numerically finding the maximum of an arbitrarily chosen second order polynomial model function of several variables (describing the dependence of the cellulose production on the concentrations of the medium components), using multivariable linear regression analysis. The chosen function appeared to describe the analyzed correlation sufficiently well. Consequently, three to six stages of optimization made the determination of the optimum medium compositions possible for 16 days of fermentation at 30°C in a medium based on fructose (wt%: fructose, 3.68; yeast extract, 5.02; (NH₄)₂NO₃, 0.001; KH₂PO₄, 0.3; MgSO₄ × 7 H₂O, 0.05; resulting in a cellulose production equal to 0.505 wt.% – namely 5.6 times higher than before the optimization) and for 7 days fermentations at 30°C in a medium based on sucrose and ethanol (wt.%: sucrose, 5.0; ethanol, 1.36; yeast extract, 1.27; (NH₄)₂SO₄, 0.5; KH₂PO₄, 0.3; MgSO₄ × 7 H₂O, 0.05; resulting in a cellulose production equal to 0.251 wt.% – namely 1.5 times higher than before the optimization).     

Establishing a laboratory model of dental unit waterlines bacterial biofilms using a CDC biofilm reactor

In this study, a laboratory model to reproduce dental unit waterline (DUWL) biofilms was developed using a CDC biofilm reactor (CBR). Bacteria obtained from DUWLs were filtered and cultured in Reasoner’s 2A (R2A) for 10 days, and were subsequently stored at ?70°C. This stock was cultivated on R2A in batch mode. After culturing for five days, the bacteria were inoculated into the CBR. Biofilms were grown on polyurethane tubing for four days. Biofilm accumulation and thickness was 1.3 × 10⁵ CFU cm^?2 and 10–14 μm respectively, after four days. Bacteria in the biofilms included cocci and rods of short and medium lengths. In addition, 38 bacterial genera were detected in biofilms. In this study, the suitability and reproducibility of the CBR model for DUWL biofilm formation were demonstrated. The model provides a foundation for the development of bacterial control methods for DUWLs.     

Statistical optimization of bacterial cellulose production by Leifsonia soli and its physico-chemical characterization

Bacterial cellulose has multiple applications in various industries such as food, biomedical, textile due to its uniqueness of being a better bio-compatible coating agent, binding material, etc. In this study, optimization of the culture medium for producing BC from Leifsonia soli was carried out by selecting different parameters. Five significant factors such as maltose, pH, incubation days, soy whey and calcium chloride were estimated through ANOVA based response surface methodology. Maximum cellulose production (5.97 g/L) was obtained where maltose 1 % (w/v) supplemented with 0.8 % (v/v) soy whey and calcium chloride 0.8 % (w/v) at pH 6.5 for 7 days of incubation. In addition, assurance of cellulose production from bacteria was done by using High-performance liquid chromatography analysis. Further, the structure and purity of obtained cellulose were examined by SEM and elemental analysis where it was observed that the sample holds the value of carbon 44.1 ± 0.20 % and hydrogen 6.2 ± 0.3 %, respectively. This study concludes that the addition of maltose and soy whey could be used as carbon, nitrogen sources and calcium chloride was used as an additive for the bacterial cellulose production compared to the Hestrin Schramm medium. In addition, the calculated water holding capacity of the sample was found to be 73 %.     

Enhanced bacterial cellulose production by Gluconacetobacter xylinus via expression of Vitreoscilla hemoglobin and oxygen tension regulation

Applied Microbiology and Biotechnology - Oxygen plays a key role during bacterial cellulose (BC) biosynthesis by Gluconacetobacter xylinus. In this study, the Vitreoscilla hemoglobin (VHb)-encoding...     

Production of bacterial cellulose by Acetobacter xylinumwith an air-lift reactor

Bacterial cellulose was produced by Acetobacter xylinum subsp. surcrofermentans BPR2001 in a 50 liter air-lift reactor using fructose as the main carbon source. When air was supplied, the production of the cellulose was only 2.3 g/l in 80 h but when O -fortified air was supplied, the cellulose concentration increased to 5.63 g/l in 28 h and the productivity of the cellulose in an air-lift reactor with O -fortified air supply was comparable to that in a mechanically agitated jar fermenter.     

Effect of addition of water-soluble polysaccharides on bacterial cellulose production in a 50-L airlift reactor

Bacterial cellulose (BC) production was carried out in a batch cultivation of Acetobacter xylinum in a 50-L internal loop airlift reactor by addition of water-soluble polysaccharides into the medium. When 0.1% (w/w) agar was added, BC production reached 8.7 g/L compared with 6.3 g/L in the control, and duration of the cultivation period to reach the maximum concentration of BC was almost half of that without addition of polysaccharides. During cultivation, BC was formed into pellets whose size was smaller when the productivity of BC was higher, indicating that increase in the relative viscosity by addition of polysaccharides hindered formation of large clumps of BC and increase in the volumetric oxygen transfer coefficient at high flow rate led to increase in BC productivity.     

Bifurcational and dynamical analysis of a continuous biofilm reactor

A dynamical model of a continuous biofilm reactor is presented. The reactor consists of a three-phase internal loop airlift operated continuously with respect to the liquid and gaseous phases, and batchwise with respect to the immobilized cells. The model has been applied to the conversion of phenol by means of immobilized cells of Pseudomonas sp. OX1 whose metabolic activity was previously characterized (Viggiani, A., Olivieri, G., Siani, L., Di Donato, A., Marzocchella, A., Salatino, P., Barbieri, P., Galli, E., 2006. An airlift biofilm reactor for the biodegradation of phenol by Pseudomonas stutzeri OX1. Journal of Biotechnology 123, 464-477). The model embodies the key processes relevant to the reactor performance, with a particular emphasis on the role of biofilm detachment promoted by the fluidized state. Results indicate that a finite loading of free cells establishes even under operating conditions that would promote wash out of the suspended biophase. The co-operative/competitive effects of free cells and immobilized biofilm result in rich bifurcational patterns of the steady state solutions of the governing equations, which have been investigated in the phase plane of the process parameters. Direct simulation under selected operating conditions confirms the importance of the dynamical equilibrium establishing between the immobilized and the suspended biophase and highlights the effect of the initial value of the biofilm loading on the dynamical pattern.     

Phenol and sulfide oxidation in a denitrifying biofilm reactor and its microbial community analysis

A microbial consortium attached onto a polyethylene support was used to evaluate the simultaneous oxidation of sulfide and phenol by denitrification. The phenol, sulfide and nitrate loading rates applied to an inverse fluidized bed reactor were up to 168 mg phenol–C/(l d), 37 mg S^2?/(l d) and 168 mg NO₃^?–N/(l d), respectively. Under steady state operation the consumption efficiencies of phenol, sulfide and nitrate were 100%. The N₂ yield (g N₂/g NO₃^?–N) was 0.89. The phenol was mineralized resulting in a yield of 0.82 g bicarbonate–C/g phenol–C and sulfide was completely oxidized to sulfate with a yield of 0.99 g SO₄^2?–S/g S^2?. 16S rRNA gene-based microbial community analysis of the denitrifying biofilm showed the presence of Thauera aromatica, Thiobacillus denitrificans, Thiobacillus sajanensis and Thiobacillus sp. This is the first work reporting the simultaneous oxidation of sulfide and phenol in a denitrifying biofilm reactor.     

Metabolic flux analysis of Gluconacetobacter xylinus for bacterial cellulose production

Metabolic flux analysis was used to reveal the metabolic distributions in Gluconacetobacter xylinus (CGMCC no. 2955) cultured on different carbon sources. Compared with other sources, glucose, fructose, and glycerol could achieve much higher bacterial cellulose (BC) yields from G. xylinus (CGMCC no. 2955). The glycerol led to the highest BC production with a metabolic yield of 14.7 g/mol C, which was approximately 1.69-fold and 2.38-fold greater than that produced using fructose and glucose medium, respectively. The highest BC productivity from G. xylinus CGMCC 2955 was 5.97 g BC/L (dry weight) when using glycerol as the sole carbon source. Metabolic flux analysis for the central carbon metabolism revealed that about 47.96 % of glycerol was transformed into BC, while only 19.05 % of glucose and 24.78 % of fructose were transformed into BC. Instead, when glucose was used as the sole carbon source, 40.03 % of glucose was turned into the by-product gluconic acid. Compared with BC from glucose and fructose, BC from the glycerol medium showed the highest tensile strength at 83.5 MPa, with thinner fibers and lower porosity. As a main byproduct of biodiesel production, glycerol holds great potential to produce BC with superior mechanical and microstructural characteristics.     

4-Chlorophenol degradation by a bacterial consortium: development of a granular activated carbon biofilm reactor

A bacterial consortium that can degrade chloro- and nitrophenols has been isolated from the rhizosphere of Phragmitis communis. Degradation of 4-chlorophenol (4-CP) by a consortium attached to granular activated carbon (GAC) in a biofilm reactor was evaluated during both open and closed modes of operation. During the operation of the biofilm reactor, 4-CP was not detected in the column effluent, being either adsorbed to the GAC or biodegraded by the consortium. When 4-CP at 100 mg l⁻¹ was fed to the column in open mode operation (20 mg g⁻¹ GAC total supply), up to 27% was immediately available for biodegradation, the rest being adsorbed to the GAC. Biodegradation continued after the system was returned to closed mode operation, indicating that GAC bound 4-CP became available to the consortium. Biofilm batch cultures supplied with 10–216 mg 4-CP g⁻¹ GAC suggested that a residual fraction of GAC-bound 4-CP was biologically unavailable. The consortium was able to metabolise 4-CP after perturbations by the addition of chromium (Cr VI) at 1–5 mg l⁻¹ and nitrate at concentrations up to 400 mg l⁻¹. The development of the biofilm structure was analysed by scanning electron microscopy and confocal laser scanning microscopy (CLSM) techniques. CLSM revealed a heterogeneous structure with a network of channels throughout the biofilm, partially occupied by microbial exopolymer structures. Received: 17 March 1999 / Received revision: 27 May 1999 / Accepted: 28 May 1999     

Continuous biocatalytic synthesis of epoxypropane using a biofilm reactor

Mixed culture methanotrophic attached biofilms immobilized on diatomite particles in a three-phase fluidized bed reaction system were developed. Methane monooxygenase (MMO) activity on diatomite particles increased as soon as the lag phase ended. More than 90% of the MMO activity in the fluidized bed was attached. A biofilm concentration of 3.3c3.7mg dry weight cell (dwc) per g dry solid (DS) was observed. Batch experiments were performed to explore the possibility of producing epoxypropane by a propene–methane co-oxidation process. The effect of methane on the epoxidation of propene and the effect of propene on the growth of methanotroph was also studied. In continuous experiments, optimum mixed gas containing 35 methane, 20 propene and 45% oxygen were continuously circulated through the fluidized bed reactor to deliver substrates and extract product. Initial epoxypropane productivity was 110–150 μmol/day. The bioreactor operated continuously for 53 days without obvious loss of epoxypropane productivity.     

Influence of biomass production and detachment forces on biofilm structures in a biofilm airlift suspension reactor

The influence of process conditions (substrate loading rate and detachment force) on the structure of biofilms grown on basalt particles in a Biofilm Airlift Suspension (BAS) reactor was studied. The structure of the biofilms (density, surface shape, and thickness) and microbial characteristics (biomass yield) were investigated at substrate loading rates of 5, 10, 15, and 20 kg COD/m3. day with basalt concentrations of 60 g/L, 150 g/L, and 250 g/L. The basalt concentration determines the number of biofilm particles in steady state, which is the main determining factor for the biofilm detachment in these systems. In total, 12 experimental runs were performed. A high biofilm density (up to 67 g/L) and a high biomass concentration was observed at high detachment forces. The higher biomass content is associated with a lower biomass substrate loading rate and therefore with a lower biomass yield (from 0.4 down to 0.12 gbiomass/gacetate). Contrary to general beliefs, the observed biomass detachment decreased with increasing detachment force. In addition, smoother (fewer protuberances), denser and thinner compact biofilms were obtained when the biomass surface production rate decreased and/or the detachment force increased. These observations confirmed a hypothesis, postulated earlier by Van Loosdrecht et al. (1995b), that the balance between biofilm substrate surface loading (proportional to biomass surface production rate, when biomass yield is constant) and detachment force determines the biofilm structure. When detachment forces are relatively high only a patchy biofilm will develop, whereas at low detachment forces, the biofilm becomes highly heterogeneous with many pores and protuberances. With the right balance, smooth, dense and stable biofilms can be obtained. Copyright 1998 John Wiley & Sons, Inc.     

Biodegradation of acetonitrile by adapted biofilm in a membrane-aerated biofilm reactor

A membrane-aerated biofilm reactor (MABR) was developed to degrade acetonitrile (ACN) in aqueous solutions. The reactor was seeded with an adapted activated sludge consortium as the inoculum and operated under step increases in ACN loading rate through increasing ACN concentrations in the influent. Initially, the MABR started at a moderate selection pressure, with a hydraulic retention time of 16 h, a recirculation rate of 8 cm/s and a starting ACN concentration of 250 mg/l to boost the growth of the biofilm mass on the membrane and to avoid its loss by hydraulic washout. The step increase in the influent ACN concentration was implemented once ACN concentration in the effluent showed almost complete removal in each stage. The specific ACN degradation rate achieved the highest at the loading rate of 101.1 mg ACN/g-VSS h (VSS, volatile suspended solids) and then declined with the further increases in the influent ACN concentration, attributed to the substrate inhibition effect. The adapted membrane-aerated biofilm was capable of completely removing ACN at the removal capacity of up to 21.1 g ACN/m² day, and generated negligible amount of suspended sludge in the effluent. Batch incubation experiments also demonstrated that the ACN-degrading biofilm can degrade other organonitriles, such as acrylonitrile and benzonitrile as well. Denaturing gradient gel electrophoresis studies showed that the ACN-degrading biofilms contained a stable microbial population with a low diversity of sequence of community 16S rRNA gene fragments. Specific oxygen utilization rates were found to increase with the increases in the biofilm thickness, suggesting that the biofilm formation process can enhance the metabolic degradation efficiency towards ACN in the MABR. The study contributes to a better understanding in microbial adaptation in a MABR for biodegradation of ACN. It also highlights the potential benefits in using MABRs for biodegradation of organonitrile contaminants in industrial wastewater.     

Viscoelastic fluid description of bacterial biofilm material properties

A mathematical model describing the constitutive properties of biofilms is required for predicting biofilm deformation, failure, and detachment in response to mechanical forces. Laboratory observations indicate that biofilms are viscoelastic materials. Likewise, current knowledge of biofilm internal structure suggests modeling biofilms as associated polymer viscoelastic systems. Supporting experimental results and a system of viscoelastic fluid equations with a linear Jeffreys viscoelastic stress-strain law are presented here. This system of equations is based on elements of associated polymer physics and is also consistent with presented and previous experimental results. A number of predictions can be made. One particularly interesting result is the prediction of an elastic relaxation time on the order of a few minutes-biofilm disturbances on shorter time scales produce an elastic response, biofilm disturbances on longer time scales result in viscous flow, i.e., nonreversible biofilm deformation. Although not previously recognized, evidence of this phenomenon is in fact present in recent experimental results.     

Multiplex FISH analysis of a six-species bacterial biofilm

Established procedures use different and seemingly incompatible experimental protocols for fluorescent in situ hybridization (FISH) with Gram-negative and Gram-positive bacteria. The aim of this study was to develop a procedure, based on FISH and confocal laser scanning microscopy (CLSM), for the analysis of the spatial organization of in vitro biofilms containing both Gram-negative and Gram-positive oral bacteria. Biofilms composed of the six oral species Actinomyces naeslundii, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, and Veillonella dispar were grown anaerobically for 64.5 h at 37 degrees C on hydroxyapatite disks preconditioned with saliva. Conditions for the simultaneous in situ hybridization of both Gram-negative and Gram-positive bacteria were sought by systematic variation of fixation and exposure to lysozyme. After fixation and permeabilization biofilms were labeled by FISH with 16S rRNA-targeted oligonucleotide probes ANA103 (for the detection of A. naeslundii), EUK116 (C. albicans), FUS664 (F. nucleatum), MIT447 and MIT588 (S. oralis), SOB174 (S. sobrinus), and VEI217 (V. dispar). Probes were used as 6-FAM, Cy3 or Cy5 conjugates, resulting in green, orange-red or deep-red fluorescence of target cells, respectively. Thus, with two independent triple-hybridizations with three probes carrying different fluorescence-tags, all six species could be visualized. Results show that the simultaneous investigation by FISH of complex biofilms composed of multiple bacterial species with differential Gram-staining properties is possible. In combination with the optical sectioning properties of CLSM the technique holds great promise for the analysis of spatial alterations in biofilm composition in response to environmental challenges.