Production of pectinase from deseeded sunflower head by Aspergillus niger in submerged and solid-state conditions

Studies were carried out on the production of pectinases using deseeded sunflower head by Aspergillus niger DMF 27 and DMF 45 in submerged fermentation (SmF) and solid-state fermentation (SSF). Higher titres of endo- and exo-pectinases were observed when medium was supplemented with carbon (4% glucose for SmF and 6% sucrose for SSF) and nitrogen (ammonium sulphate, 0.3% for both SmF and SSF) sources. Green gram husk proved to be relatively a better supplement to attain higher yield of endo-pectinase (11.7 U/g) and exo-pectinase (30.0 U/g) in solid-state conditions. Maximum production of endo-pectinase (19.8 U/g) and exo-pectinase (45.9 U/g) by DMF 45 were recorded in SSF when compared to endo-pectinase (18.9 U/ml) and exo-pectinase (30.3 U/ml) by DMF 27 in SmF under optimum process conditions.     

In vivo enzymatic digestion, in vitro xylanase digestion, metabolic analogues, surfactants and polyethylene glycol ameliorate laccase production from Ganoderma sp. kk-02

AIMS: The effect of in vivo enzymatic digestion (IVED), in vitro xylanase digestion (IVXD), metabolic analogues, surfactants and polyethylene glycol (PEG) on laccase production from Ganoderma sp. kk-02 was studied. METHODS AND RESULTS: An acidic laccase producing Ganoderma sp. kk-02 produced 16.0 U ml(-1) and 365.0 U g(-1) of laccase, when grown under submerged (SmF) and solid state (SSF) fermentation conditions, respectively. Modification of the substrate (wheat bran) molecular architecture by IVED and IVXD increased subsequent laccase production from Ganoderma sp. kk-02 by 1.31-fold (21.0 U ml(-1)) (SmF); 2.21-fold (810.0 U g(-1)) (SSF) and 1.10-fold (18.0 U ml(-1)) (SmF); 1.78-fold (650.0 U g(-1)) (SSF) when compared with untreated wheat bran. Further enhancement in laccase yield under SmF and SSF was obtained when IVED treated wheat bran was used in conjunction with amino acids [DL-tryptophan, 2.66-fold (56.0 U ml(-1)) SmF; 2.86-fold (2324.0 U g(-1)) SSF], vitamins [biotin, 1.71-fold (36.0 U ml(-1)) SmF; 3.06-fold (2483.0 U g(-1)) SSF], surfactants [Tween-40, 1.85-fold (39.0 U ml(-1)) SmF; 2.25-fold (1828.0 U g(-1)) SSF], and PEG [PEG 6000, 1.93-fold (40.0 U ml(-1)) SmF; 1.58-fold (1284.0 U g(-1)) SSF]. CONCLUSIONS: The IVED of substrate (wheat bran) facilitated hyper laccase production in presence of additives from Ganoderma sp. kk-02. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights a new methodology viz. IVED for concomitant and economic production of diverse enzymes using the same substrate. The hyper laccase levels obtained could improve the economic competitiveness of environmentally benign processes applied in varied industries. The work also provides an insight into the regulation of complex metabolic pathways governing the expression of extra cellular proteins from white-rot fungi.     

Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation

The kinetics of β-fructofuranosidase (Ffase) production by Aspergillus niger in submerged (SmF) and solid-state fermentation (SSF) systems was investigated. The maximum productivity of Ffase (81.8 U/l per h) was obtained in SSF for 72 h while it was 18.3 U/l per h in SmF for 120 h. The productivity of extra cellular Ffase produced in SSF was 5-fold higher than in SmF. Optimization of fermentation medium for Ffase production was carried out using De Meo's fractional factorial design with seven components such as (NH₄)₂SO₄, KH₂PO₄, FeSO₄, MgSO₄ · 7H₂O, sucrose, urea and yeast extract. The media designed for SmF after two steps of optimization supported the growth of A. niger and higher productivity of Ffase (58.3 U/l per h) than with the medium before optimization. The optimized medium of SmF when used in SSF, did not improve the Ffase productivity and therefore medium for SSF was optimized independent of SmF. After two optimization steps, the media was defined for SSF which supported the growth and high level of Ffase productivity (149.1 U/l per h) in SSF compared to the medium before optimization (81.8 U/l per h) and optimized medium for SmF (58.3 U/l per h). Our results suggested that the optimized media for SmF and SSF for the production of Ffase have to be different.     

Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose

Mutants of Penicillium janthinellum NCIM 1171 were evaluated for cellulase production using both submerged fermentation (SmF) and solid state fermentation (SSF). Mutant EU2D-21 gave highest yields of cellulases in both SmF and SSF. Hydrolysis of Avicel and cellulose were compared using SmF and SSF derived enzyme preparations obtained from EU2D-21. Surprisingly, the use of SSF derived preparation gave less hydrolysis compared to SmF derived enzymes. This may be due to inactivation of β-glucosidase at 50 °C in SSF derived enzyme preparations. SmF derived enzyme preparations contained both thermostable and thermosensitive β-glucosidases where as SSF derived enzyme preparations contained predominantly thermosensitive β-glucosidase. This is the first report on less thermostability of SSF derived β-glucosidase which is the main reason for getting less hydrolysis.     

Beta-fructofuranosidase production by 2-deoxyglucose resistant mutants of aspergillus niger in submerged and solid-state fermentation

Aspergillus niger produces extracellular beta-fructofuranosidase under submerged (SmF) and solid state fermentation (SSF) conditions. After UV mutagenesis of conidiospores of A. niger, 2-deoxyglucose (10 g/l) resistant mutants were isolated on Czapek's minimal medium containing glycerol as a carbon source and the mutants were examined for improved production of beta-fructofuranosidase in SmF and SSF conditions. One of such mutant DGRA-1 overproduced beta-fructofuranosidase in both SmF and SSF conditions. In SmF, the mutant DGRA-1 showed higher beta-fructofuranosidase productivity (110.8 U/l/hr) than the wild type (48.3 U/l/hr). While in SSF the same strain produced 322 U/l/hr of beta-fructofuranosidase, 2 times higher than that of wild type (154.2 U/l/hr). In SmF, both wild type and mutants produced relatively low level of beta-fructofuranosidase in medium containing sucrose with glucose than from the sucrose medium. However in SSF, the DGRA-1 mutant grown in sucrose and sucrose+ glucose did not show any difference with respect to beta-fructofuranosidase production. These results indicate that the catabolite repression of beta-fructofuranosidase synthesis is observed in SmF whereas in SSF such regulation was not prominent.     

Effect of different cultural conditions for phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> CFR 335 in submerged and solid-state fermentations

The present article deals with the studies on the effect of media ingredients, such as carbon, nitrogen, inorganic phosphates, surfactants, and metal salts, on phytase enzyme production by Aspergillus niger CFR 335 in submerged (SmF) and solid-state fermentations (SSF). The results obtained showed a 1.5-fold higher enzyme yield in the presence of sucrose in both SmF and SSF, while peptone was found to be a favorable nitrogen source for SmF. Sodium dihydrogen phosphate (NaH₂PO₄) favored 34% higher enzyme yield than the control, which was followed by 19% higher activity in potassium dihydrogen phosphate (KH₂PO₄) in SSF at 0.015% w/v. The addition of Tween-20 in SmF showed a maximum yield of 12.6 U/mL while, SDS suppressed the growth of the fungus. None of the surfactants favored the enzyme yield in SSF. Calcium chloride (CaCl₂) was extensively efficient in stimulating more than 55% higher phytase production in SmF at 0.01% v/v. In SSF, none of the metal salts stimulated phytase production.     

Biotechnological advantages of laboratory-scale solid-state fermentation with fungi

Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented. This is due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O₂, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology. This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF. In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.     

Production and Regulation of Lipase Activity from <Emphasis Type="Italic">Penicillium restrictum</Emphasis> in Submerged and Solid-State Fermentations

Different carbon (C) sources, mainly carbohydrates and lipids, have been screened for their capacity to support growth and lipase production by Penicillium restrictum in submerged fermentation (SmF) and in solid-state fermentation (SSF). Completely different physiological behaviors were observed after the addition of easily (oleic acid and glucose) and complex (olive oil and starch) assimilable C sources to the liquid and solid media. Maximal lipolytic activities (12.1 U/mL and 17.4 U/g) by P. restrictum were obtained with olive oil in SmF and in SSF, respectively. Biomass levels in SmF (12.2–14.1 mg/mL) and SSF (7.0–8.0 mg/g) did not varied greatly with the distinct C sources used. High lipase production (12.3 U/g) using glucose was only attained in SSF, perhaps due to the ability of this fermentation process to minimize catabolite repression.     

Applicability of Coriolopsis rigida for Biodegradation of Polycyclic Aromatic Hydrocarbons

Laccase was produced by Coriolopsis rigida using barley bran as substrate in solid-state fermentation (SSF) and also by submerged fermentation (SmF). The best results were obtained in SSF with twice the amount of laccase production. Laccase could be produced from repeated batch cultures of SSF over 30 days. The laccase degraded several polycyclic aromatic hydrocarbons (PAHs) in vivo and in vitro. The addition of an effective mediator, 1-hydroxybenzotriazol (50 µM), during in vitro treatment increased the degradation rate.     

Simultaneous saccharification and fermentation of lignocellulosic residues pretreated with phosphoric acid–acetone for bioethanol production

Bermudagrass, reed and rapeseed were pretreated with phosphoric acid–acetone and used for ethanol production by means of simultaneous saccharification and fermentation (SSF) with a batch and fed-batch mode. When the batch SSF experiments were conducted in a 3% low effective cellulose, about 16 g/L of ethanol were obtained after 96 h of fermentation. When batch SSF experiments were conducted with a higher cellulose content (10% effective cellulose for reed and bermudagrass and 5% for rapeseed), higher ethanol concentrations and yields (of more than 93%) were obtained. The fed-batch SSF strategy was adopted to increase the ethanol concentration further. When a higher water-insoluble solid (up to 36%) was applied, the ethanol concentration reached 56 g/L of an inhibitory concentration of the yeast strain used in this study at 38 °C. The results show that the pretreated materials can be used as good feedstocks for bioethanol production, and that the phosphoric acid–acetone pretreatment can effectively yield a higher ethanol concentration.     

Gibberellic acid by solid state fermentation: Consistent and improved yields

Five strains each of Gibberella fujikuroi and Fusarium monoliforme were screened to select G. fujikuroi P-3, a strain capable of giving consistent production of gibberellic acid (GA(3)) by solid state fermentation (SSF). The comparative production of GA(3) by SSF and submerged fermentation (SmF) indicated better productivity with the former technique. The accumulation of GA(3) was 1.626 times higher in the case of SSF. On the basis of available carbohydrates in the media, the percent conversions were 0.096 and 0.156 in SmF and SSF, respectively. The use of coarse wheat bran of the particle size of 0.3-0.4 cm resulted in an increase of 2.5 times in the yield of GA(3). The enrichment of commercial wheat bran with soluble starch gave enhanced accumulation to an extent of 3.5 times. The relation between GA(3) production and cell growth in SSF was similar to that encountered in SmF. The consistent and improved yields to a tune of 1.22 g GA(3) per kilogram dry moldy bran (DMB) establish the potential and feasibility of SSF for the production of GA(3) by G. fujikuroi P-3. On preliminary cost analysis, a net savings of about 60% and 50% on fermentation medium cost and the expenditure on down-stream processing, respectively, as compared to the presently employed SmF technique was evident.     

Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

Improved polygalacturonase production from Bacillus sp. MG-cp-2 under submerged (SmF) and solid state (SSF) fermentation

AIMS: To investigate the effect of amino acids, vitamins and surfactants on polygalacturonase production from Bacillus sp. MG-cp-2 under submerged (SmF) and solid state fermentation (SSF). METHODS AND RESULTS: Bacillus sp. MG-cp-2 was isolated from the outer covering of the seeds of Celastrus paniculatus. Out of the various surfactants, amino acids and vitamins, Tween-60, DL-serine and folic acid maximally enhanced polygalacturonase production by 2.7-fold (240.0 U x ml(-1)), 4.0-fold (360.0 U x ml(-1)) and 3.8-fold (342.0 U x ml(-1)) respectively, under submerged fermentation (SmF). In solid state fermentation (SSF), Tween-80, pyridoxine and DL-ornithine monohydrochloride induced highest enzyme production up to 1.73-fold (6956.5 U x g(-1)), 5.3-fold (21224.4 U x g(-1)) and 5.74-fold (23076.9 U x g(-1)), respectively. CONCLUSION: Amino acids and their analogues, vitamins and surfactants effect significantly polygalacturonase production by Bacillus sp. MG-cp-2 when grown under submerged (SmF) and solid state fermentation (SSF) conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides useful information about regulation of polygalacturonase biosynthesis in Bacillus sp. MG-cp-2, which appears to be an interplay of nutritional and physical factors. Alkaline polygalacturonase from Bacillus sp. MG-cp-2 will be extremely useful in the treatment of alkaline pectic waste waters from vegetable and fruit processing industries and in degumming of bast fibres.     

Pectinase production by a Brazilian thermophilic fungus <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> N31 in solid-state and submerged fermentation

Thermophilic organisms produce thermostable enzymes, which have a number of applications, justifying the interest in the isolation of new thermophilic strains and study of their enzymes. Thirty-four thermophilic and thermotolerant fungal strains were isolated from soil, organic compost, and an industrial waste pile based on their ability to grow at 45°C and in a liquid medium containing pectin as the only carbon source. Among these fungi, 50% were identified at the genus level as Thermomyces, Aspergillus, Monascus, Chaetomium, Neosartoria, Scopulariopsis, and Thermomucor. All isolated strains produced pectinase during solid-state fermentation (SSF). The highest polygalacturonase (PG) activity was obtained in the culture medium of thermophilic strain N31 identified as Thermomucor indicae-seudaticae. Under SSF conditions on media containing a mixture of wheat bran and orange bagasse (1 : 1) at 70% of initial moisture, this fungus produced the maximum of 120 U/ml of exo-PG, while in submerged fermentation (SmF) it produced 13.6 U/ml. The crude PG from SmF was more thermostable than that from SSF and exhibited higher stability in acidic pH.     

Novel Characteristics of Bifidobacterium bifidum in Solid State Fermentation System

Summary The characteristics of Bifidobacterium bifidum grown in solid state fermentation (SSF) system (water content of media 54.5 and 68.8%) was compared with the submerged fermentation (SmF) system (water content of medium: 89.8%). Besides lactic acid (lactate) and acetic acid (acetate), the bacterium was able to secrete propionic acid (propionate) and butyric acid (butyrate) under SSF conditions. However, it only produced lactate and acetate under SmF conditions. The ratio of lactate to acetate was 1.26–1.62:1 in SSF but it was 1:2 in SmF. A higher content of C16:0 and C18:1 as well as a lower content of C18:0 cell membrane fatty acids were observed in SSF than in SmF. There was a lower growth rate, a lower viable count and a longer logarithmic growth phase for B. bifidum cultivated in SSF than in SmF.     

Fed-batch cultivation for the production of clavulanic acid by an immobilized Streptomyces clavuligerus mutant

In order to obtain high productivity of clavulanic acid, a newly-introduced carrier, polyurethane pellet (PUP) Z97-020 was used for the immobilization process. In a stirred-tank bioreactor, batch cultivation by Streptomyces clavuligerus KK immobilized on PUP Z97-020 gave about 3100 mg of clavulanic acid per litre, representing an increase of 200% in productivity compared with that by fed-batch cultivation of free cells (1500 mg/l). However, the clavulanic acid produced rapidly decomposed due to the pH change during batch cultivation. Fed-batch cultivation by immobilized S. clavuligerus KK gave an excellent level of clavulanic acid up to 3250 mg/l, a productivity increase of 220% compared with that by fed-batch cultivation of free cells. These results suggest that immobilization with PUP Z97-020 is a more effective process for the production of clavulanic acid and that the maintenance of pH by fed-batch cultivation with glycerol as a limiting substrate prevents the clavulanic acid from decomposing during the fermentation.     

Biodegradation of hexadecane in liquid and solid-state fermentations by Aspergillus niger

The biodegradation and mineralisation of hexadecane (HXD) by Aspergillus niger were studied in SmF and Solid-state fermentation (SSF). HXD concentrations ranging from 45 to 180 g/l (SSF) and from 20 to 80 g/l (SmF) were tested. HXD consumption was three times higher and fungal growth was up to 30 times faster in SSF than in SmF. The maximum HXD consumption in SmF was 62% (18% mineralised) and in SSF 100% (52% mineralised) for initial HXD concentrations of 20 and 45 g/l, respectively. The respiratory quotient in SmF increased (from 0.85 to 1.08) with increase in HXD concentration, while it was independent (approximately 0.68) of the initial HXD concentration in SSF. These results showed that the consumption rate and biodegradation efficiency for HXD were higher in SSF than in SmF.     

Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)-hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6g/l via fed-batch fermentation.     

Effect of nonionic surfactants on Rhizopus homothallicus lipase activity: a comparative kinetic study

Based on amino-terminal sequencing and mass spectrometry data on the Rhizopus homothallicus lipase extracted using solid (SSF) and submerged state fermentation (SmF) methods, we previously established that the two enzymes were identical. Differences were observed, however, in terms of the specific activity of these lipases and their inhibition by diethyl p-nitrophenyl phosphate (E600). The specific activity of the SSF lipase (10,700 μmol/min/mg) was found to be 1.2-fold that of SmF lipase (8600 μmol/min/mg). These differences might be the result of residual Triton X-100 molecules interacting with the SSF lipase. To check this hypothesis, the SmF lipase was incubated with submicellar concentrations of Triton X-100. The specific activity of the lipase increased after this treatment, reaching similar values to those measured with the SSF lipase. Preincubating SSF and SmF lipases with E600 at a molar excess of 100 for 1 h resulted in 80% and 60% enzyme inhibition levels, respectively. When the SmF lipase was preincubated with Triton X-100 for 1 h at a concentration 100 times lower than the Trition X-100 critical micellar concentration, the inhibition of the lipase by E600 increased from 60% to 80%. These results suggest that residual detergent monomers interacting with the enzyme may after the kinetic properties of the Rh. homothallicus lipase.