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.     

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.     

Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

Use of a low-cost methodology for biodetoxification of castor bean waste and lipase production

The castor bean (Ricinus communis) represents a potential candidate for biodiesel production. The Petrobras Research Center is developing a biodiesel production process from castor bean seeds, in which an unwanted byproduct named castor bean waste is produced. This extremely alkaline waste is toxic and allergenic and, as such, poses a significant environmental problem. Solid-state fermentation (SSF) of castor bean waste was carried out to achieve ricin detoxification, reduce allergenic potential and stimulate lipase production. The fungus, Penicillium simplicissimum, an excellent lipase producer, was able to grow and produce lipase enzyme. After an optimization process, the maximum lipase activity achieved was 44.8 U/g. Moreover, the fungus P. simplicissimum was able to reduce the ricin content to non-detectable levels in addition to diminishing castor bean waste allergenic potential by approximately 16%. In this way, SSF of castor bean waste by P. simplicissimum may increase the utility of the waste by promoting enzyme production and eliminating the principal toxic element, ricin.     

固液态发酵中橄榄油对Rhizopus chinensis全细胞脂肪酶的影响

主要对华根霉全细胞脂肪酶固态和液态两种发酵过程进行比较,并着重探讨不同培养方式下橄榄油对其合成活力和水解活力的影响。结果表明：液态培养较有利于菌体生长,对脂肪酶的生产也有一定的促进作用。橄榄油的加入不仅有利于菌体生长、提高脂肪酶水解活力,更可使脂肪酶的合成活力显著增加,液态发酵下的效果更为明显。橄榄油在整个发酵过程中可能既作为碳源又是脂肪酶的诱导物。另外,全细胞脂肪酶的水解活力和合成活力在固液态发酵条件下均存在不对应性,表明华根霉可能产性质不同的脂肪酶同功酶。     

Production,partial characterization,and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus <Emphasis Type="Italic">Myceliophthora</Emphasis> sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.     

Effects of different carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentations

A study was made to compare the production of pectinase by Aspergillus niger CH4 in solid-state (SSF) and submerged (SmF) fermentations. Production of endo- (endo-p) and exo-pectinase (exo-p) by SSF was not reduced when glucose, sucrose or galacturonic acid (up to 10%) were added to a culture medium containing pectin. Moreover, both activities increased when concentrations of the carbon sources were also increased. In SmF, these activities were strongly decreased when glucose or sucrose (3%) was added to culture medium containing pectin. The addition of galacturonic acid affected endo-p activity production to a lesser extend than exo-p. Final endo-p and exo-p activities in SSF were three and 11 times higher, respectively, than those obtained in SmF. The overall productivities of SSF were 18.8 and 4.9 times higher for endo-p and exo-p, respectively, than those in SmF. These results indicate that regulatory phenomena, such as induction-repression or activation-inhibition, related to pectinase synthesis by A. niger CH4 are different in the two types of fermentation. Correspondence to: E. Favela-Torres     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

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.     

Production of phytase by Mucor racemosus in solid-state fermentation

Phytase production was studied by three Mucor and eight Rhizopus strains by solid-state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosus NRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH(4))(2)SO(4), phytase production in solid-state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett-Burman and central composite experimental designs. Using the optimized medium phytase, alpha-amylase and lipase production of Mucor racemosus NRRL 1994 was compared in solid-state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food-grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.     

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.     

Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid

Tannase production by Aspergillus niger Aa-20 was studied in submerged (SmF) and solid-state (SSF) fermentation systems with different tannic acid and glucose concentrations. Tannase activity and productivity were at least 2.5 times higher in SSF than in SmF. Addition of high tannic acid concentrations increased total tannase activity in SSF, while in SmF it was decreased. In SmF, total tannase activity increased from 0.57 to 1.03 IU/mL, when the initial glucose concentration increased from 6.25 to 25 g/L, but a strong catabolite repression of tannase synthesis was observed in SmF when an initial glucose concentration of 50 g/L was used. In SSF, maximal values of total tannase activity decreased from 7.79 to 2.51 IU when the initial glucose concentration was increased from 6.25 to 200 g/L. Kinetic results on tannase production indicate that low tannase activity titers in SmF could be associated to an enzyme degradation process which is not present in SSF. Tannase titers produced by A. niger Aa-20 are fermentation system-dependent, favoring SSF over SmF. Journal of Industrial Microbiology & Biotechnology (2001) 26, 296–302. Received 07 July 2000/ Accepted in revised form 15 February 2001     

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.     

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.     

Mixed substrate solid state fermentation for production and extraction of lipase from Aspergillus niger MTCC 2594

A novel mixed substrate solid-state fermentation (SSF) process has been developed for Aspergillus niger MTCC 2594 using wheat bran (WB) and gingelly oil cake (GOC) and the results showed that addition of GOC to WB (WB : GOC, 3 : 1, w/w) increased the lipase activity by 36.0% and the activity was 384.3+/-4.5 U/g dry substrate at 30 degrees C and 72 h. Scale up of lipase production to 100 g and 1 kg tray-level batch fermentation resulted in 95.0% and 84.0% of enzyme activities respectively at 72 h. A three-stage multiple contact counter-current extraction yielded 97% enzyme recovery with a contact time of 60 min. However, extraction by simple percolation and plug-flow methods resulted in decreased enzyme recoveries. The mixed substrate SSF process has resulted in a significant increase in specific activity (58.9%) when compared to a submerged fermentation (SmF) system. Furthermore, an efficient process of extraction has been standardized with this process. Use of GOC along with WB as potential raw materials for enzyme production could be of great commercial significance. This is the first report on the production and extraction of lipase from Aspergillus niger using mixed solid substrates, WB and GOC, which are potential raw materials for the production of enzymes and other value-added products.     

Improvement of penicillin yields in solid-state and submerged fermentation of Penicillium chrysogenum by amplification of the penicillin biosynthetic gene cluster

Penicillium chrysogenum low and high penicillin producing strains were transformed with a cosmid containing the whole penicillin biosynthetic gene cluster. The cosmid library was constructed in a newly developed cosmid vector, IztapaCos, which allows cloning and direct introduction of large DNA fragments in fungal recipients using phleomycin resistance as selection marker. The effect of increased gene dosage on penicillin production was evaluated both in submerged (SmF) and solid-state fermentation (SSF). Transformants from the low-producing strain Wis 54-1255, showed a 67.3 and 28.3% increased penicillin titer in SSF and SmF, respectively. In transformants from the high-producing strain P2-32 the increase was 92.9 and 158.4% respectively. Strain P2-32 already contains originally about 14 copies of the penicillin biosynthetic cluster, which shows that the strategy of increasing the gene dosage is still valid for high copy-number strains. The different behavior of the two strains in each type of culture is discussed, along with the practical implications for industrial penicillin production.     

Adding value to a toxic residue from the biodiesel industry: production of two distinct pool of lipases from <Emphasis Type="Italic">Penicillium simplicissimum</Emphasis> in castor bean waste

In countries with a strong agricultural base, such as Brazil, the generation of solid residues is very high. In some cases, these wastes present no utility due to their toxic and allergenic compounds, and so are an environmental concern. The castor bean (Ricinus communis) is a promising candidate for biodiesel production. From the biodiesel production process developed in the Petrobras Research Center using castor bean seeds, a toxic and alkaline waste is produced. The use of agroindustrial wastes in solid-state fermentation (SSF) is a very interesting alternative for obtaining enzymes at low cost. Therefore, in this work, castor bean waste was used, without any treatment, as a culture medium for fungal growth and lipase production. The fungus Penicillium simplicissimum was able to grow and produce an enzyme in this waste. In order to maximize the enzyme production, two sequential designs–Plackett-Burman (variable screening) followed by central composite rotatable design (CCRD)—were carried out, attaining a considerable increase in lipase production, reaching an activity of 155.0 U/g after 96 h of fermentation. The use of experimental design strategy was efficient, leading to an increase of 340% in the lipase production. Zymography showed the presence of different lipases in the crude extract. The partial characterization of such extract showed the occurrence of two lipase pools with distinct characteristics of pH and temperature of action: one group with optimal action at pH 6.5 and 45°C and another one at pH 9.0 and 25°C. These results demonstrate how to add value to a toxic and worthless residue through the production of lipases with distinct characteristics. This pool of enzymes, produced through a low cost methodology, can be applied in different areas of biotechnology.     

Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study

Exopectinase production by Aspergillus niger was compared in submerged fermentation (SmF) and solid-state fermentation (SSF). SSF was carried out using polyurethane foam (PUF) as the solid support. The purpose was to study the effect of sucrose addition (0 or 40 g/l) and water activity level (A _w=0.99 or 0.96) on the level of enzyme activity induced by 15 g/l of pectin. Mycelial growth, as well as extracellular protease production, was also monitored. Sucrose addition in SmF resulted in catabolite repression of exopectinase activity. However, in SSF, an enhancement of enzyme activity was observed. Protease levels were minimal in SSF experiments with sucrose and maximal in SmF without sucrose. Exopectinase yields (IU/g X) were negligible in SmF with sucrose. The high levels of exopectinase with sucrose and high A _w in SSF can be explained by a much higher level of biomass production without catabolite repression and with lower protease contamination. Journal of Industrial Microbiology & Biotechnology (2001) 26, 271–275. Received 05 July 2000/ Accepted in revised form 27 January 2001