Increased yield and selected properties of bacterial cellulose exposed to different modes of a rotating magnetic field

Rotating magnetic field (RMF) is an interesting alternative to conventional bacterial cellulose (BC) production methods. The BC synthesis processes may be affected by RMF, which facilitates the transfer of oxygen and nutrients from the media to the microbial cells. RMF may also directly influence the various physical and chemical properties of BC. The main aim of the present study was to evaluate the impact of the RMF on the BC in regard to its yield and material properties. The correlation between the efficiency of polymer production and the different time of exposure to the RMF was also analyzed to determine the conditions of lower energy consumption during the cellulose formation process. It was found that the Gluconacetobacter xylinus cultures exposed to the RMF for a half of the time of the entire cellulose production process (72 h), considering the results obtained in controls, synthesized BC more effectively than bacteria continuously exposed to the RMF for 144 h. Furthermore, the application of the RMF, regardless of the exposure mode, did not negatively affect the polymer material properties. It was concluded that the use of the RMF may provide a novel technique for altering cellulose biogenesis and may be used in multiple biotechnological applications.     

Efficient bioconversion from acid hydrolysate of waste oleaginous yeast biomass after microbial oil extraction to bacterial cellulose by Komagataeibacter xylinus

Biomass acid hydrolysate of oleaginous yeast Trichosporon cutaneum after microbial oil extraction was applied as substrate for bacterial cellulose (BC) production by Komagataeibacter xylinus (also named as Gluconacetobacter xylinus previously) for the first time. BC was synthesized in static culture for 10 days, and the maximum BC yield (2.9?g/L) was got at the 4th day of fermentation. Most carbon sources in the substrate (glucose, mannose, formic acid, acetic acid) can be utilized by K. xylinus. The highest chemical oxygen demand (COD) removal (40.7?±?3.0%) was obtained at the 6th day of fermentation, and then the COD increased possibly due to the degradation of BC. The highest BC yield on COD consumption was 38.7?±?4.0% (w/w), suggesting that this is one efficient bioconversion for BC production. The BC structure was affected little by the substrate by comparison with that generated in classical HS medium using field-emission scanning electron microscope (FE-SEM), Fourier transform infrared, and X-ray diffraction. Overall, this technology can both solve the issue of waste oleaginous yeast biomass and produce valuable biopolymer (BC).     

CRISPR/dCas9干扰bcsD基因表达调控细菌纤维素结构

木葡糖酸醋杆菌(Gluconacetobacter xylinus)是细菌纤维素的主要生产菌株。在该菌中,BcsD是纤维素合酶的亚基之一,参与细菌纤维素的组装过程。利用CRISPR/dCas9系统调控bcsD基因的表达量,获得了一系列bcsD基因表达量不同的木葡糖酸醋杆菌。通过分析细菌纤维素的结构特征发现,细菌纤维素的结晶度和孔隙率随着木葡糖酸醋杆菌中bcsD表达量的变化而发生改变。其中孔隙率的变化范围在59.95%–84.05%之间,结晶度的变化范围在74.26%–93.75%之间,而细菌纤维素的产量并未因bcsD的表达量变化而发生显著下降。结果表明,bcsD的表达量低于55.34%后,细菌纤维素的孔隙率显著上升,并且细菌纤维素的结晶度与bcsD的表达量呈正相关。最终,通过干扰bcsD基因的表达,实现了一步发酵木葡糖酸醋杆菌获得了产量稳定且结构不同的细菌纤维素。     

Viability and cellulose synthesizing ability of Gluconacetobacter xylinus cells under high-hydrostatic pressure

The effect of pressure on viability and the synthesis of bacterial cellulose (BC) by Gluconacetobacter xylinus ATCC53582 were investigated. G. xylinus was statically cultivated in a pressurized vessel under 0.1, 30, 60, and 100 MPa at 25°C for 6 days. G. xylinus cells remained viable and retained cellulose producing ability under all the conditions tested, though the production of cellulose decreased with increasing the pressure. The BCs produced at each pressure condition were analyzed by field emission scanning electron microscopy (FE-SEM) and Fourier Transform Infrared (FT-IR). FE-SEM revealed that the widths of BC fibers produced under high pressure decreased as compared with those produced under the atmospheric pressure. By FT-IR, all the BCs were found to be of Cellulose type I, as the same as typical native cellulose. Our findings evidently showed that G. xylinus possessed a piezotolerant (barotolerant) feature adapting to 100 MPa without losing its BC producing ability. This was the first attempt in synthesizing BC with G. xylinus under elevated pressure of 100 MPa, which corresponded to the deep sea at 10,000 m.     

Performance of improved bacterial cellulose application in the production of functional paper

Aim: The purpose of this work was to study the feasibility of producing economic flame retardant bacterial cellulose (BC) and evaluating its behaviour in paper production. Methods and Results: This type of BC was prepared by Gluconacetobacter subsp. xylinus and substituting the glucose in the cultivation medium by glucose phosphate as a carbon source; as well as using corn steep liquor as a nitrogen source. The investigated processing technique did not dispose any toxic chemicals that pollute the surroundings or cause unacceptable effluents, making the process environmentally safe. The fire retardant behaviour of the investigated BC has been studied by non‐isothermal thermogravimetric analysis (TGA & DTGA). The activation energy of each degradation stage and the order of degradation were estimated using the Coats–Redfern equation and the least square method. Strength, optical properties, and thermogravimetric analysis of BC‐phosphate added paper sheets were also tested. Conclusions: The study confirmed that the use of glucose phosphate along with glucose was significant in the high yield production of phosphate containing bacterial cellulose (PCBC1); more so than the use of glucose phosphate alone (PCBC2). Incorporating 5% of the PCBC with wood pulp during paper sheet formation was found to significantly improve kaolin retention, strength, and fire resistance properties as compared to paper sheets produced from incorporating bacterial cellulose (BC). Significance and Impact of the Study: This modified BC is a valuable product for the preparation of specialized paper, in addition to its function as a fillers aid.     

Effects of 50 Hz rotating magnetic field on the viability of Escherichia coli and Staphylococcus aureus

This study presents results of research on the influence of rotating magnetic field (RMF) of the induction of 30?mT and the frequency of 50?Hz on the growth dynamics and cell metabolic activity of E. coli and S. aureus, depending on the exposure time. The studies showed that the RMF caused an increase in the growth and cell metabolic activity of all the analyzed bacterial strains, especially in the time interval t?=?30 to 150?min. However, it was also found that the optical density and cell metabolic activity after exposition to RMF were significantly higher in S. aureus cultures. In turn, the study of growth dynamics, revealed a rapid and a significant decrease in these values from t?=?90?min) in the case of E. coli samples. The obtained results prove that RMF (B?=?30?mT, f?=?50?Hz) has a stimulatory effect on the growth and metabolic activity of E. coli and S. aureus. Furthermore, taking into account the time of exposure, stronger influence of RMF on the viability was observed in S. aureus cultures, which may indicate that this effect depends on the shape of the exposed cells.     

Effects of 0.4 T rotating magnetic field exposure on density, strength, calcium and metabolism of rat thigh bones

The current study investigated the effects of 0.4 T rotary non-uniform magnetic field (RMF) exposure on bone density in ovariectomized (OVX) rats. Results showed that many bone indexes are significantly elevated after RMF exposure compared to the control OVX group and confirmed mechanistic evidence that strong magnetic field (MF) exposure could effectively increase bone density and might be used to treat osteoporosis. Synergy of daily RMF exposure (30 min a day for 30 days using an 8 Hz rotary 0.4 T MF) with calcium supplement tended to increase the indexes of thigh bone density, energy absorption, maximum load, maximum flexibility, and elastic deformation as compared to those of untreated OVX control group. Results also revealed that the indexes of alkaline phosphatase (ALP), serum phosphate, and serum calcium were higher in rats exposed to RMF with calcium than in the untreated OVX control group. Changes in bone mineral density (BMD) and bone mineral content (BMC) were observed in rats for three months including the first month RMF exposure. Bone density in rats exposed each day for 60 min increased during 1-month exposure and continued to increase during the post-exposure period. Furthermore, bone density and calcium content in rats exposed for 90 min daily decreased initially in the exposure month; however, ratio of increase was well above the control values by the end of the post-exposure period suggesting possible window and delayed effects. The study indicated that RMF exposure to both male and OVX female rats for 120 min a day over 15 day period should effectively promote increase of bone calcium contents (BCC) and bone-specific alkaline phosphatase (BAP) in rats thigh bone as well as a corresponding decrease in deoxypyridinoline crosslinks (DPD).     

Efficient Using Durian Shell Hydrolysate as Low-Cost Substrate for Bacterial Cellulose Production by <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>

Durian is one important tropical fruit with high nutritional value, but its shell is usually useless and considered as waste. To explore the efficient and high-value utilization of this agricultural and food waste, in this study, durian shell was simply hydrolyzed by dilute sulfuric acid, and the durian shell hydrolysate after detoxification was used for bacterial cellulose (BC) production by Gluconacetobacter xylinus for the first time. BC was synthesized in static culture for 10 days and the highest BC yield (2.67 g/L) was obtained at the 8th day. The typical carbon sources in the substrate including glucose, xylose, formic acid, acetic acid, etc. can be utilized by G. xylinus. The highest chemical oxygen demand (COD) removal (16.40%) was obtained at the 8th day. The highest BC yield on COD consumption and the highest BC yield on sugar consumption were 93.51% and 22.98% (w/w), respectively, suggesting this is one efficient bioconversion for BC production. Durian shell hydrolysate showed small influence on the BC structure by comparison with the structure of BC generated in traditional Hestrin–Schramm medium detected by FE-SEM, FTIR, and XRD. Overall, this technology can both solve the issue of waste durian shell and produce valuable bio-polymer (BC).     

Recent advances in bacterial cellulose production

Bacterial cellulose (BC), which is produced by some bacteria, has unique structural, functional, physical and chemical properties. Thus, the mass production of BC for industrial application has recently attracted considerable attention. To enhance BC production, two aspects have been considered, namely, the engineering and genetic viewpoints. The former includes the reactor design, nutrient selection, process control and optimization; and the latter the cloning of the BC synthesis gene, and the genetic modification of the speculated genes for higher BC production. In this review, recent advances in BC production from the two viewpoints mentioned above are described, mainly using the bacteriumGluconacetobacter xylinus.     

Illustration of the development of bacterial cellulose bundles/ribbons by Gluconacetobacter xylinus via atomic force microscopy

The development of bacterial cellulose (BC) fibrils biosynthesized by Gluconacetobacter xylinus was investigated using atomic force microscopy (AFM). After various incubation times at 30 °C, both the length of BC fibrils and their average diameters increased significantly. After the first 2-h incubation, not only single BC microfibrils with an average diameter of 5.8?±?0.7 nm were biosynthesized but single microfibrils also began to bind with each other forming bundles. After longer incubation times of 6 h, 16 h, and 48 h, only BC bundles and ribbons or even only ribbons were detectable. The development of BC fibrils and the formation of BC bundles/ribbons along with the biosynthesis time were illustrated using AFM. Furthermore, single BC fibrils were twisted in a right-handed manner. The twisting of BC fibrils possibly promoted the formation of bigger ribbons.     

Moderate-Intensity Rotating Magnetic Fields Do Not Affect Bone Quality and Bone Remodeling in Hindlimb Suspended Rats

Abundant evidence has substantiated the positive effects of pulsed electromagnetic fields (PEMF) and static magnetic fields (SMF) on inhibiting osteopenia and promoting fracture healing. However, the osteogenic potential of rotating magnetic fields (RMF), another common electromagnetic application modality, remains poorly characterized thus far, although numerous commercial RMF treatment devices have been available on the market. Herein the impacts of RMF on osteoporotic bone microarchitecture, bone strength and bone metabolism were systematically investigated in hindlimb-unloaded (HU) rats. Thirty two 3-month-old male Sprague-Dawley rats were randomly assigned to the Control (n = 10), HU (n = 10) and HU with RMF exposure (HU+RMF, n = 12) groups. Rats in the HU+RMF group were subjected to daily 2-hour exposure to moderate-intensity RMF (ranging from 0.60 T to 0.38 T) at 7 Hz for 4 weeks. HU caused significant decreases in body mass and soleus muscle mass of rats, which were not obviously altered by RMF. Three-point bending test showed that the mechanical properties of femurs in HU rats, including maximum load, stiffness, energy absorption and elastic modulus were not markedly affected by RMF. µCT analysis demonstrated that 4-week RMF did not significantly prevent HU-induced deterioration of femoral trabecular and cortical bone microarchitecture. Serum biochemical analysis showed that RMF did not significantly change HU-induced decrease in serum bone formation markers and increase in bone resorption markers. Bone histomorphometric analysis further confirmed that RMF showed no impacts on bone remodeling in HU rats, as evidenced by unchanged mineral apposition rate, bone formation rate, osteoblast numbers and osteoclast numbers in cancellous bone. Together, our findings reveal that RMF do not significantly affect bone microstructure, bone mechanical strength and bone remodeling in HU-induced disuse osteoporotic rats. Our study indicates potentially obvious waveform-dependent effects of electromagnetic fields-stimulated osteogenesis, suggesting that RMF, at least in the present form, might not be an optimal modality for inhibiting disuse osteopenia/osteoporosis.     

Bacterial cellulose synthesized with apple pomace enhanced by ionic liquid pretreatment

Abstract

Apple pomace was explored as alternative feedstock for producing bacterial cellulose (BC) by Gluconacetobacter xylinus following a cellulase saccharification performed after pretreatment of 1-allyl-3-methylimidazolium chloride ([AMIM]Cl). The dissolving process of apple pomace cellulose was observed by polarized light microscopy (PLM). As FT-IR and XRD results demonstrated, the IL pretreatment proved to be a physical process and no changes in the crystalline structure occurred during the pretreatment. However, the SEM result showed that more fissures and breakages appeared on the surface of pomace microfibers after IL-pretreating, which increased the contact area with cellulase and improved the enzymatic hydrolysis efficiency. An enhancing effect on the BC yield has been observed, 27% higher yield of BC obtained from hydrolysate as compared to sucrose-based medium indicates efficiency of IL-treated apple pomace to serve as high quality feedstock in BC production.     

Cellulose synthesis by Komagataeibacter rhaeticus strain P 1463 isolated from Kombucha

Isolate B17 from Kombucha was estimated to be an efficient producer of bacterial cellulose (BC). The isolate was deposited under the number P 1463 and identified as Komagataeibacter rhaeticus by comparing a generated amplified fragment length polymorphism (AFLP™) DNA fingerprint against a reference database. Static cultivation of the K. rhaeticus strain P 1463 in Hestrin and Schramm (HS) medium resulted in 4.40 ± 0.22 g/L BC being produced, corresponding to a BC yield from glucose of 25.30 ± 1.78 %, when the inoculum was made with a modified HS medium containing 10 g/L glucose. Fermentations for 5 days using media containing apple juice with analogous carbon source concentrations resulted in 4.77 ± 0.24 g/L BC being synthesised, corresponding to a yield from the consumed sugars (glucose, fructose and sucrose) of 37.00 ± 2.61 %. The capacity of K. rhaeticus strain P 1463 to synthesise BC was found to be much higher than that of two reference strains for cellulose production, Komagataeibacter xylinus DSM 46604 and Komagataeibacter hansenii DSM 5602^T, and was also considerably higher than that of K. hansenii strain B22, isolated from another Kombucha sample. The BC synthesised by K. rhaeticus strain P 1463 after 40 days of cultivation in HS medium with additional glucose supplemented to the cell culture during cultivation was shown to have a degree of polymerization of 3300.0 ± 122.1 glucose units, a tensile strength of 65.50 ± 3.27 MPa and a length at break of 16.50 ± 0.83 km. For the other strains, these properties did not exceed 25.60 ± 1.28 MPa and 15.20 ± 0.76 km.

     

Aluminium effects on mechanical properties of cell wall analogues

Aluminium (Al) toxicity adversely impacts plant productivity in acid soils by restricting root growth and although several mechanisms are involved the physiological basis of decreased root elongation remains unclear. Understanding the primary mechanisms of Al rhizotoxicity is hindered due to the rapid effects of soluble Al on root growth and the close proximity of many cellular components within the cell wall, plasma membrane, cytosol and nucleus with which Al may react. To overcome some of these difficulties, we report on a novel method for investigating Al interactions with Komagataeibacter xylinus bacterial cellulose (BC)‐pectin composites as cell wall analogues. The growth of K. xylinus in the presence of various plant cell wall polysaccharides, such as pectin, has provided a unique in vitro model system with which to investigate the interactions of Al with plant cell wall polysaccharides. The BC‐pectin composites reacted in a similar way with Al as do plant cell walls, providing insights into the effects of Al on the mechanical properties of the BC‐pectin composites as cell wall analogues. Our findings indicated that there were no significant effects of Al (4–160 μM) on the tensile stress, tensile strain or Young's modulus of the composites. This finding was consistent with cellulose, not pectin, being the major load bearing component in BC‐pectin composites, as is also the case in plant cell walls.     

Determination of live and dead Komagataeibacter xylinus cells and first attempt at precise control of inoculation in nanocellulose production

The timely enumeration of cells of nanocellulose-producing bacteria is challenging due to their unique growth properties. To better understand the metabolism of the bacteria and better control the concentration of living cells during cultivation, a prompt cell counting technology is crucial and urgently required. In this work, two fluorescent dyes, the asymmetrical anthocyanidin dye SYBR Green I (SG) and propidium iodide (PI), were first combined for Komagataeibacter xylinus species to determine live/dead bacterial cells quantitatively and promptly. The number of live and dead K. xylinus cells determined using an epifluorescence microscope corresponded well to the results obtained using a fluorescence microplate reader. The R² values were 0.9986 and 0.9920, respectively, and were similar to those obtained with the LIVE/DEAD^® BacLight^TM commercial kit. SG/PI double-staining showed proper efficiency in distinguishing live/dead cells for the K. xylinus strain (R² = 0.9898). The technology was applied to standardize four different K. xylinus strains, and the initial cell concentration of the strains was precisely controlled (no significant difference among the strains, P> 0.05). The cellulose yield per live cell was calculated, and significant differences (P < 0.05) were found among the four strains in the following order: DHU-ATCC-1> DHU-ZCY-1> DHU-ZGD-1> ATCC 23770. The study shows (i) the application of the SG/PI staining to standardizing inocula for bacterial cellulose production so that a more accurate comparison can be made between different strains, and (ii) the lower cost of using SG rather than the SYTO 9 of the commercially available LIVE/DEAD^® BacLight^TM kit.     

Effects of 50‐Hz magnetic field exposure on hormone secretion and apoptosis‐related gene expression in human first trimester villous trophoblasts in vitro

Evidence from epidemiological and animal studies showed that exposure to extremely low frequency magnetic fields (ELF‐MF) could produce deleterious effects on reproduction. In order to investigate the possible mechanism of MF exposure on reproductive effects, first trimester human chorionic villi at 8–10 weeks' gestation were obtained, and trophoblasts were isolated, cultured, and exposed to a 50‐Hz MF for different durations. The human chorionic gonadotropin (hCG) and progesterone in the culture medium was measured by electrochemiluminescence immunoassay. The mRNA levels of apoptosis‐related genes bcl‐2, bax, caspase‐3, p53, and fas in trophoblasts were analyzed using real‐time RT‐PCR. The results showed that exposure of trophoblasts to MF at 0.2 mT for 72 h did not affect secretion of hCG and progesterone from these cells. There was also no significant change in secretion of these hormones when trophoblasts were exposed to a 0.4 mT MF for 48 h. However, MF significantly inhibited hCG and progesterone secretion of trophoblasts after exposure for 72 h at 0.4 mT. Results of apoptosis‐related gene expression analysis showed that, within 72 h of exposure at 0.4 mT, there was no significant difference between MF exposure and control on the expression pattern of each gene. Based on results of the present experiment, it is suggested that exposure to MF for a longer duration (72 h) could inhibit secretion of hCG and progesterone by human first trimester villous trophoblasts, however, the effect might not be related to trophoblast apoptosis. Bioelectromagnetics 31:566–572, 2010. © 2010 Wiley‐Liss, Inc.     

Mediterranean flour moth Ephestia kuehniella eggs and larvae exposed to a static magnetic field and preference by Trichogramma embryophagum

This study investigated the effect of strong magnetic fields as insecticidal activity on Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) larvae and eggs at different stages of development and their preference by the egg parasitoid, Trichogramma embryophagum Hartig (Hymenoptera: Trichogrammatidae). Eggs ranging in age from 24-h to 48-h and 72-h-old and larvae (1 to 2 days) were exposed to 1.4 Tesla (T) magnetic fields from a DC power supply at 50 Hz for different time periods (3, 6, 12, 24, 48 and 72 h). Twelve hours of exposure at 1.4 T was toxic to 24-h-old eggs of E. kuehniella. The 72-h-old host eggs treated with 1.4 T for 6–72 h were not significantly preferred by T. embryophagum. The magnetic field was toxic to 24-h-old eggs of E. kuehniella exposed to 1.4 T for 12. The treatment of magnetic fields on the 72-h-old host egg with 1.4 T at 6–72 h was not significantly preferred by T. embryophagum. Magnetization of 24-h-old eggs of E. kuehniella for 3 h could be effectively used with T. embryophagum as sterilised host eggs. These eggs were markedly preferred by T. embryophagum. The LT₅₀ and LT₉₉ values of magnetic fields at different egg stages of E. kuehniella, and larvae were measured. A level of 1.4 T at 72 h completely prevented the development of the larvae. There was no significant effect on larval survival at 1.4 T at 48 and 72 h. Increasing magnetic fields exposure times for eggs that were 24-h, 48-h and 72-h-old prevented larval emergence and increased their mortality rate. Consequently, magnetic fields could be used in controlling stored-product pest eggs and larvae of E. kuehniella.     

Submerged Cultivation of a Fungal Mutant Strain Humicola Lutea 120-5 Producing Acid Proteinases

The optimal parameters for the cultivation in 10-l fermenters of a mutant strain Humicola lutea 120-5 were established:temperature 30°C, inoculum size 6%, inoculum age 24 h, aeration rate 0,6 vol/vol · min, medium agitation 620 rpm and cultivation time 72 h. A maximal proteolytic activity of 2000 µg tyrosine liberated from 2%casein ml^?1 culture filtrate min^?1 at pH 3.0 and 40°C was obtained under the fixed conditions. α-Amylase biosynthesis during the cultivation of H. lutea 120-5 was observed but it was insignificant to the 72nd h. It is demonstrated that starch can be used as alternative to glucose carbon source. It is proved that the mutant strain H.lutea 120-5 produced two acid proteinases.     

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.