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1.
Habib Driouch Andreas Roth Petra Dersch Christoph Wittmann 《Applied microbiology and biotechnology》2010,87(6):2011-2024
A comprehensive approach of bioprocess design at various levels was used to optimize microbial production of extracellular
fructofuranosidase, important as biocatalyst to derive fructooligosaccharides with broad application in food or pharmaceutical
industry. For production, the recombinant strain Aspergillus niger SKAn1015 was used, which expresses the fructofuranosidase encoding gene suc1 under control of a strong constitutive promoter. In a first screening towards an optimized medium, glucose, nitrate, Fe2+, and Mn2+ were identified as beneficial for production. A minimal medium with optimized concentration of these key nutrients, obtained
by central composite design experiments and quadratic modelling, provided a threefold increased fructofuranosidase activity
in the culture supernatant (400 U/mL) as compared to the originally described medium. Utilizing the optimized medium, the
process was then transferred from shake flask into a fed-batch-operated bioreactor. Hereby, the intended addition of talc
microparticles allowed engineering the morphology of A. niger into a highly active mycelial form, which strongly boosted production. Fructofuranosidase production was highly specific
as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The secreted enzyme activity of 2,800 U/mL,
corresponding to about 3 g/L of fructofuranosidase, achieved by the microparticle-enhanced fed-batch process, is tenfold higher
than that of any other process reported so far, so that the presented bioprocess strategy appears as a milestone towards future
industrial fructofuranosidase production. 相似文献
2.
Improved enzyme production by bio-pellets of Aspergillus niger: targeted morphology engineering using titanate microparticles 总被引:1,自引:0,他引:1
Driouch H Hänsch R Wucherpfennig T Krull R Wittmann C 《Biotechnology and bioengineering》2012,109(2):462-471
The present study describes the design of bio-pellet morphologies of the industrial working horse Aspergillus niger strains in submerged culture. The novel approach recruits the intended addition of titanate microparticles (TiSiO(4), 8 μm) to the growth medium. As tested for two recombinant strains producing fructofuranosidase and glucoamylase, the enzyme titer by the titanate-enhanced cultures in shake flasks was increased 3.7-fold to 150 U/mL (for fructofuranosidase) and 9.5-fold to 190 U/mL (for glucoamylase) as compared to the control. This could be successfully utilized for improved enzyme production in stirred tank reactors. Stimulated by the particles, the achieved final glucoamylase activity of 1,080 U/mL (fed-batch) and 320 U/mL (batch) was sevenfold higher as compared to the conventional processes. The major reason for the enhanced production was the close association between the titanate particles and the fungal cells. Already below 2.5 g/L the micromaterial was found inside the pellets, including single particles embedded as 50-150 μm particle aggregates in the center resulting in core shell pellets. With increasing titanate levels the pellet size decreased from 1,700 μm (control) to 300 μm. Fluorescence based resolution of GFP expression revealed that the large pellets of the control were only active in a 200 μm surface layer. This matches with the critical penetration depth for nutrients and oxygen typically observed for fungal pellets. The biomass within the titanate derived fungal pellets, however, was completely active. This was due a reduced thickness of the biomass layer via smaller pellets as well as the core shell structure. Moreover, also the created loose inner pellet structure enabled a higher mass transfer and penetration depths for up to 500 μm. The creation of core-shell pellets has not been achieved previously by the addition of microparticles, for example, made of talc or alumina. Due to this, the present work opens further possibilities to use microparticles for tailor-made morphology design of filamentous fungi, especially for pellet based processes which have a long and strong industrial relevance for industrial production. 相似文献
3.
Filamentous fungi such as Aspergillus?niger are important biocatalysts for industrial production of various enzymes as well as organic acids or antibiotics. In suspended culture these microorganisms exhibit a complex morphology which typically has a strong influence on their production properties. In this regard, we have recently shown that the addition of inorganic micro particles to the culture medium is a straightforward and elegant approach to precisely tame fungal morphology. For A.?niger a full range of morphological forms from pellets with different diameters to free mycelium could be adjusted by supplementation with talc powder. Aluminium oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition.?Exemplified for different recombinant A.?niger strains enzyme production could be strongly enhanced by the addition of microparticles. This was demonstrated for the production of fructofuranosidase, an important high-value biocatalyst for pre-biotic fructo-oligosaccharides, by recombinant A.?niger. In a microparticle enhanced fed-batch process, a highly productive mycelium could be achieved. The enzyme titre of 2800?U/mL finally reached was more then tenfold higher then that of any other process reported so far. Here we provide additional insights into the novel production process. This includes the confirmation of the highly selective production of the target enzyme fructofuranosidase using MALDI-TOF?MS analysis. Moreover, we show that the obtained enzyme suspension can be efficiently used with minimal pre-treatment for the biosynthesis of short chain fructooligosaccharides of the inulin type, such as 1-kestose and 1-nystose, prebiotics with substantial commercial interest. In particular, these compounds are highly attractive for human consumption, since they have been shown to reduce the risk of colon cancer. In summary, the use of microparticles opens a new avenue of engineering fungal morphology into the desired form for specific production processes. 相似文献
4.
Addition of aluminum oxide microparticles to Trichoderma viride My preculture enhances cellulase production and influences fungal morphology
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Miaoyin Dong Shuyang Wang Fuqiang Xu Qiaoqiao Li Wenjian Li 《Engineering in Life Science》2018,18(6):353-358
5.
Darah Ibrahim Haritharan Weloosamy Sheh-Hong Lim 《World journal of biological chemistry》2015,6(3):265-271
AIM: To investigate the impact of agitation speed on pectinase production and morphological changing of Aspergillus niger(A. niger) HFD5A-1 in submerged fermentation. METHODS: A. niger HFM5A-1 was isolated from a rotted pomelo. The inoculum preparation was performed by adding 5.0 m L of sterile distilled water containing 0.1% Tween 80 to a sporulated culture. Cultivation was carried out with inoculated 1 × 107 spores/m L suspension and incubated at 30 ℃ with different agitation speed for 6 d. The samples were withdrawn after 6 d cultivation time and were assayed for pectinase activity and fungal growth determination. The culture broth was filtered through filter paper(Whatman No. 1, London) to separate the fungal mycelium. The cell-free culture filtrate containing the crude enzyme was then assayed for pectinase activity. The biomass was dried at 80 ℃ until constant weight. The fungal cell dry weight was then expressed as g/L. The 6 d old fungal mycelia were harvested from various agitation speed, 0, 50, 100, 150, 200 and 250 rpm. The morphological changing of samples was then viewed under the light microscope and scanning electron microscope.RESULTS: In the present study, agitation speed was found to influence pectinase production in a batch cultivation system. However, higher agitation speeds than the optimal speed(150 rpm) reduced pectinase production which due to shear forces and also collision among the suspended fungal cells in the cultivation medium. Enzyme activity increased with the increasing of agitation speed up to 150 rpm, where it achieved its maximal pectinase activity of 1.559 U/m L. There were significant different(Duncan, P 0.05) of the pectinase production with the agitation speed at static, 50, 100, 200 and 250 rpm. At the static condition, a well growth mycelial mat was observed on the surface of the cultivation medium and sporulation occurred all over the fungal mycelial mat. However with the increased in agitation speed, the mycelial mat turned slowly to become a single circular pellet. Thus, it was found that agitation speed affected the morphological characteristics of the fungal hyphae/mycelia of A. niger HFD5A-1 by altering their external as well as internal cell structures.CONCLUSION: Exposure to higher shear stress with an increasing agitation speed could result in lower biomass yields as well as pectinase production by A. niger HFD5A-1. 相似文献
6.
Microscale bioprocessing techniques are rapidly emerging as a means to increase the speed of bioprocess design and to reduce material consumption. However, there is still a lack of suitable parallelized techniques to investigate the industrially important group of filamentous bacteria and fungi. Cultivation of filamentous organisms in shake flasks is still the favored technique for comparing and optimizing cultivation conditions of production strains at mL‐scale. In this paper, the application of a microtiter plate‐based cultivation system in combination with the filamentous fungus Aspergillus niger was investigated. A protocol for reproducible cultivation was developed and evaluated. Productivity of A. niger concerning the rose‐like aroma compound 2‐phenylethanol showed low standard deviations while regular and consistent morphologies appeared in the parallelized system. Furthermore, the effect of addition of microparticles on the morphology was investigated. The results can be used to accelerate the process development with A. niger and other filamentous organisms. 相似文献
7.
Diogo Robl Priscila da Silva Delabona Patrícia dos Santos Costa Deise Juliana da Silva Lima Sarita Candida Rabelo Ida Chapaval Pimentel 《Biocatalysis and Biotransformation》2015,33(3):175-187
Fungal xylanases have been widely studied and various production methods have been proposed using submerged and solid-state fermentation. This class of enzyme is used to supplement cellulolytic enzyme cocktails in order to enhance the enzymatic hydrolysis of plant cell walls. The present work investigates the production of xylanase and other accessory enzymes by a recently isolated endophytic Aspergillus niger DR02 strain, using the pentose-rich liquor from hydrothermal pretreatment of sugarcane bagasse as carbon source. Batch and fed-batch submerged cultivation approaches were developed in order to minimize the toxicity of the liquor and increase enzyme production. Maximum xylanase activities obtained were 458.1 U/mL for constant fed-batch, 428.1 U/mL for exponential fed-batch, and 264.37 U/mL for pulsed fed-batch modes. The results indicated that carbon-limited fed-batch cultivation can reduce fungal catabolite repression, as well as overcome possible negative effects of toxic compounds present in the pentose-rich liquor. Enzymatic panel and mass spectrometric analyses of the fed-batch A. niger secretome showed high levels of xylanolytic enzymes (GH10, GH11, and GH62 Cazy families), together with cellobiohydrolase (G6 and GH7), β-glucosidase, β-xylosidase (GH3), and feruloyl esterase (CE1) accessory enzyme activities. The yields of glucose and xylose from enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse increased by 43.7 and 65.3%, respectively, when a commercial cellulase preparation was supplemented with the A. niger DR02 constant fed-batch enzyme complex. 相似文献
8.
Ercan Yatmaz Ercan Karahalil Mustafa Germec Merve Ilgin İrfan Turhan 《Bioprocess and biosystems engineering》2016,39(9):1391-1399
β-mannanase was produced mainly by Aspergillus species and can degrade the β-1,4-mannose linkages of galactomannans. This study was undertaken to enhance mannanase production using talcum and aluminum oxide as the microparticles, which control cell morphology of recombinant Aspergillus sojae in glucose and carob extract medium. Both microparticles improved fungal growth in glucose and carob pod extract medium. Aluminum oxide (1 g/L) was the best agent for glucose medium which resulted in 514.0 U/ml. However, the highest mannanase activity was found as 568.7 U/ml with 5 g/L of talcum in carob extract medium. Increase in microparticle concentration resulted in decreasing the pellet size diameter. Furthermore, more than 10 g/L of talcum addition changed the filamentous fungi growth type from pellet to pellet/mycelium mixture. Results showed that right type and concentration of microparticle in fermentation media improved the mannanase activity and production rate by controlling the growth morphology. 相似文献
9.
10.
Adding talc microparticles to Aspergillus terreus ATCC 20542 preculture decreases fungal pellet size and improves lovastatin production
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Changing fungal morphology with the use of morphological engineering techniques leads to improving the production of metabolites by filamentous fungi in the submerged culture. Adding mineral microparticles is one such simple method to change fungal pellet size. Here, it was studied for a lovastatin producer, Aspergillus terreus ATCC 20542. The experiments were conducted in shake flasks and 10 μm talc microparticles were added to the preculture. Intrapellet oxygen concentration profiles were determined by an oxygen microprobe. Talc microparticles caused a decrease of A. terreus pellets diameter from about 2000 to 900 μm, dependent on their concentration in the preculture. Smaller pellets produced more lovastatin, whose titre exceeded then 120 mg L?1, utilising more lactose. The decrease in pellet size resulted in changes of oxygen concentration profiles in the pellets. The estimated critical pellet diameter, at which the non‐oxygenated zone was observed in the centre of the pellets, was 1700 μm. Smaller pellets were fully penetrated by oxygen. To conclude, facilitated diffusion of oxygen into the pellets of smaller diameter and their less dense structure made lactose utilisation by A. terreus more efficient, which ultimately increased lovastatin production in the runs with talc microparticles added, compared to the control runs. 相似文献
11.
Mutants ofA. niger K 69/26, prepared by multistep mutagenesis (UV, MNNG, heating) have been screened for pectinase activities. Mutants with
altered levels of certain pectinases, such as endo- and exopolygalacturonase (PG vis, red), pectinesterase (PE) and pectinlyase
(PL), were isolated. The enzyme activities of the best mutants M 1348/126 were increased 2–3-fold compared to the parent strain
after a 6-d cultivation of filamentous mycelium on a shaker. Further mutagenesis of mutants with decreased pectinase activities
(e.g. Se3) produced revertants. PG (vis) synthesis of revertant Se5 was increased 1.7 times compared to the control strain K 69/26.
Independent of these increased rates, the general level of pectinase activities synthesized by the filamentous mycelium ofA. niger mutants amounts to about 10–20% compared with those produced by aggregated mycelium. It appears that the enzyme synthesis
related to mycelium structure is independent of the mechanism which regulates the level of pectinase synthesis within a specific
morphological structure. 相似文献
12.
The ability of immobilized cell cultures of Aspergillus niger FETL FT3 to produce extracellular tannase was investigated. The production of enzyme was increased by entrapping the fungus
in scouring mesh cubes compared to free cells. Using optimized parameters of six scouring mesh cubes and inoculum size of
1 × 106 spores/mL, the tannase production of 3.98 U/mL was obtained from the immobilized cells compared to free cells (2.81 U/mL).
It was about 41.64% increment. The immobilized cultures exhibited significant tannase production stability of two repeated
runs. 相似文献
13.
White-rot fungi are extensively used in various submerged biotechnology processes to produce ligninolytic enzymes. Transfer
of the process from the laboratory to the industrial level requires optimization of the cultivation conditions on the laboratory
scale. An interesting area of optimization is pellet growth since this morphological form solves problems such as the decreased
oxygen concentration, limited heat, and nutrient transport, which usually occur in dispersed mycelium cultures. Many submerged
fermentations with basidiomycetes in pellet form were done with Phanerochaete, Trametes, and Bjerkandera species, among others. In our study, another promising basidiomycete, D. squalens, was used for ligninolytic enzyme production. With the addition of wood particles (sawdust) as a natural inducer and optimization
of mixing and aeration conditions in laboratory stirred tank (STR) and bubble column (BCR) reactors on pellet growth and morphology,
the secretion of laccase and the manganese-dependent peroxidase into the medium was substantially enhanced. The maximum mean
pellet radius was achieved after 10 days in the BCR (5.1 mm) where pellets were fluffy and 5 days in the STR (3.5 mm) where
they were round and smooth. The maximum Lac activity (1,882 U l−1) was obtained after 12 days in the STR, while maximum MnP activity (449.8 U l−1) occurred after 18 days in the BCR. The pellet size and morphology depended on the agitation and aeration conditions and
consequently influenced a particular enzyme synthesis. The enzyme activities were high and comparable with the activities
found for other investigations in reactors with basidiomycetes in the form of pellets. 相似文献
14.
Papagianni M Joshi N Moo-Young M 《Journal of industrial microbiology & biotechnology》2002,29(5):259-263
The effects of cell immobilization on the secretion of extracellular proteases and glucoamylase production by Aspergillus niger were investigated under a variety of immobilization techniques and culture conditions. Immobilization was achieved by means
of cell attachment on metal surfaces or spore entrapment and subsequent growth on porous Celite beads. Free-suspension cultures
were compared with immobilized mycelium under culture conditions that included growth in shake flasks and an airlift bioreactor.
Cell attachment on metal surfaces minimized the secretion of proteases while enhancing glucoamylase production by the fungus.
Growth on Celite beads in shake-flask cultures reduced the specific activity of the secreted proteases from 128 to 61 U g−1, while glucoamylase specific activity increased from 205 to 350 U g−1. The effect was more pronounced in bioreactor cultures. A reduction of six orders of magnitude in protease specific activities
was observed when the fungus grew immobilized on a rolled metal screen, which served as the draft tube of an airlift bioreactor.
Received 29 October 2001/ Accepted in revised form 14 June 2002 相似文献
15.
The authors studied the effect of the various components of synthetic nutrient medium on glucose oxidase production in submerged cultivation ofAspergillus niger. It was found that the optimal glucose concentration was 3.5–6%. The only suitable source of nitrogen was nitrate nitrogen. If the medium contained ammonia nitrogen, glucose oxidase was not formed. The addition of citric acid to the medium very effectively stimulated theQ O 2 of the mycelium. Calcium added in the form of calcium nitrate had the same effect. A decrease in the Mg2+ ion concentration raised the activity of the enzyme, while inhibiting growth of the mycelium. If the initial pH was less than 4, glucose oxidase production was inhibited and did not start until the pH rose in the course of fermentation. Differences in the initial pH affected not only production of the enzyme, but also the formation of acids and the morphological appearance of the submerged mycelium. On the basis of the findings the synthetic medium for submerged cultivation ofAspergillus niger was modified, resulting in a 50–100% increase in glucose oxidase production as compared with the original medium. 相似文献
16.
Hua‐Wei Zeng Yu‐Jie Cai Xiang‐Ru Liao Feng Zhang Yu‐Lin Li Xiang‐Kang Zeng Da‐Bing Zhang 《Engineering in Life Science》2011,11(1):37-43
A high‐catalase‐producing strain, which was isolated from sludge containing hydrogen peroxide, was identified as Serratia marcescens SYBC08 by 16S rDNA sequence analysis. Serratia spp. was reported as non‐spore‐forming bacterium (except S. marcescens spp. sakuensis), but in our study electron microscopic observation revealed that the strain did produce spores. The content of the main fatty acid C16:0 (14.8%) was significantly different from that of S. marcescens spp. sakuensis (33.2%) and S. marcescens spp. marcescens DSM 30121T (34.8%), and the biochemical characteristics were not identical to those of S. marcescens spp. sakuensis. We speculate that the relatively high catalase activity and the spore structures may enable the strain to survive in a hydrogen peroxide environment. The most suitable carbon and nitrogen sources for the catalase production by S. marcescens SYBC08 were citric acid and corn steep liquor powder. A strategy of carbon metabolism regulation to enhance the catalase production was exploited. In the 7‐L fermenter, catalase production (20 353 U/mL) obtained in the presence of glucose and citric acid was 1.68‐ and 1.31‐fold higher than that obtained in the presence of glucose or citric acid, at equimolar carbon concentration. This production yield was much higher than that of many catalase‐producing strains, but only slightly lower than the production by Micrococcus luteus (34 601 U/mL). The results suggest that the new spore‐forming S. marcescens SYBC08 is a potential candidate for the production of catalase. 相似文献
17.
Morozova E. V. Baranova M. V. Kozlov V. P. Tereshina V. M. Memorskaya A. S. Feofilova E. P. 《Microbiology》2001,70(5):527-534
Aspergillus nigerconidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice-distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. nigerconidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45°C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. nigerconidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase. 相似文献
18.
Physiological description of multivariate interdependencies between process parameters,morphology and physiology during fed‐batch penicillin production
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Optimization of productivity and economics of industrial bioprocesses requires characterization of interdependencies between process parameters and process performance. In the case of penicillin production, as in other processes, process performance is often closely interlinked with the physiology and morphology of the organism used for production. This study presents a systematic approach to efficiently characterize the physiological effects of multivariate interdependencies between bioprocess design parameters (spore inoculum concentration, pO2 control level and substrate feed rate), morphology, and physiology. Method development and application was performed using the industrial model process of penicillin production. Applying traditional, statistical bioprocess analysis, multivariate correlations of raw bioprocess design parameters (high spore inoculum concentration, low pO2 control as well as reduced glucose feeding) and pellet morphology were identified. A major drawback of raw design parameter correlation models; however, is the lack of transferability across different process scales and regimes. In this context, morphological and physiological bioprocess modeling based on scalable physiological parameters is introduced. In this study, raw parameter effects on pellet morphology were efficiently summarized by the physiological parameter of the biomass yield per substrate. Finally, for the first time to our knowledge, the specific growth rate per spore was described as time‐independent determinant for switching from pellet to disperse growth during penicillin production and thus introduced as a novel, scalable key process parameter for pellet morphology and process performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:689–699, 2014 相似文献
19.
Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification
reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained
using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations
than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl
acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity.
Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification.
The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion
efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High
ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions. 相似文献
20.
Rao ChS Sathish T Mahalaxmi M Laxmi GS Rao RS Prakasham RS 《Journal of applied microbiology》2008,104(3):889-898
Aim: Modelling and optimization of fermentation factors and evaluation for enhanced alkaline protease production by Bacillus circulans. Methods and Results: A hybrid system of feed‐forward neural network (FFNN) and genetic algorithm (GA) was used to optimize the fermentation conditions to enhance the alkaline protease production by B. circulans. Different microbial metabolism regulating fermentation factors (incubation temperature, medium pH, inoculum level, medium volume, carbon and nitrogen sources) were used to construct a ‘6‐13‐1’ topology of the FFNN for identifying the nonlinear relationship between fermentation factors and enzyme yield. FFNN predicted values were further optimized for alkaline protease production using GA. The overall mean absolute predictive error and the mean square errors were observed to be 0·0048, 27·9, 0·001128 and 22·45 U ml?1 for training and testing, respectively. The goodness of the neural network prediction (coefficient of R2) was found to be 0·9993. Conclusions: Four different optimum fermentation conditions revealed maximum enzyme production out of 500 simulated data. Concentration‐dependent carbon and nitrogen sources, showed major impact on bacterial metabolism mediated alkaline protease production. Improved enzyme yield could be achieved by this microbial strain in wide nutrient concentration range and each selected factor concentration depends on rest of the factors concentration. The usage of FFNN–GA hybrid methodology has resulted in a significant improvement (>2·5‐fold) in the alkaline protease yield. Significance and Impact of the Study: The present study helps to optimize enzyme production and its regulation pattern by combinatorial influence of different fermentation factors. Further, the information obtained in this study signifies its importance during scale‐up studies. 相似文献