Oxidative state in idiophase links reactive oxygen species (ROS) and lovastatin biosynthesis: Differences and similarities in submerged- and solid-state fermentations

The present work was focused on finding a relationship between reactive oxygen species (ROS) and lovastatin biosynthesis (secondary metabolism) in Aspergillus terreus. In addition, an effort was made to find differences in accumulation and control of ROS in submerged (SmF) and solid-state fermentation (SSF), which could help explain higher metabolite production in the latter. sod1 expression, ROS content, and redox balance kinetics were measured during SmF and SSF. Results showed that A. terreus sod1 gene (oxidative stress defence enzyme) was intensely expressed during rapid growth phase (trophophase) of lovastatin fermentations. This high expression decreased abruptly, just before the onset of production (idiophase). However, ROS measurements detected high concentrations only in idiophase, suggesting a link between ROS and lovastatin biosynthesis. Apparently sod1 down regulation promotes the rise of ROS during idiophase. This oxidative state in idiophase was further supported by a high redox balance observed in trophophase that changed to a low value in idiophase (around six-fold lower). The patterns of ROS accumulation, sod1 expression, and redox balance behaviour were similar in SmF and SSF. However, sod1 expression and ROS concentration (ten-fold), were higher in SmF. Our results indicate a link between ROS and lovastatin biosynthesis. Also, showed differences of physiology in SSF that yield lower but more steady ROS concentrations, which could be associated to higher lovastatin production.     

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.     

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.     

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     

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 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.     

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.     

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     

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.     

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.     

Mycophenolic acid production in solid-state fermentation using a packed-bed bioreactor

Mycophenolic acid (MPA) was produced from Penicillium brevicompactum by solid-state fermentation (SSF) using pearl barley, and submerged fermentation (SmF) using mannitol. It was found that SSF was superior to SmF in terms of MPA concentration (1219 mg/L vs. 60 mg/L after 144 h fermentation), and the product yields were 6.1 mg/g pearl barley for SSF and 1.2 mg/g mannitol for SmF. The volumetric productivities were 8.5 and 0.42 mg/L h for SSF and SmF, respectively.The optimum solid substrate of SSF for MPA production was pearl barley, producing 5470 mg/kg compared with wheat bran (1601 mg/kg), oat (3717 mg/kg) and rice (2597 mg/kg). The optimum moisture content, incubation time and inoculum concentrations were 70%, 144 h and 6%, respectively. Neither the addition of mannitol or (NH4)₂HPO₄ nor adjustment of media pH within the range of 3–7 significantly enhanced MPA production.MPA production by SSF using a packed-bed bioreactor was performed and an increased maximum production of MPA 6.9 mg/g was achieved at 168 h incubation time. The higher volumetric productivity and concentrations makes SSF an attractive alternative to SmF for MPA production.     

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.     

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.     

Phytase isozymes from Aspergillus niger NCIM 563 under solid state fermentation: Biochemical characterization and their correlation with submerged phytases

Aspergillus niger NCIM 563 produces dissimilar phytase isozymes under solid state and submerged fermentation conditions. Biochemical characterization and applications of phytase Phy III and Phy IV in SSF and their comparison with submerged fermentation Phy I and Phy III were studied. SSF phytases have a higher metabolic potential as compared to SmF. Phy I is tetramer and Phy II, III and IV are monomers. Phy I and IV have pH optima of 2.5 and Phy II and III have pH optima of 5.0 and 5.6, respectively. Phy I, III and IV exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. SSF phytase is less thermostable as compared to SmF phytase. Phy I and II show homology with other known phytases while Phy III and IV show no homology with SmF phytases and any other known phytases from the literature suggesting their unique nature. This is the first report about differences among phytase produced under SSF and SmF by A. niger and this study provides basis for explanation of the stability and catalytic differences observed for these enzymes. Exclusive biochemical characteristics and multilevel application of SSF native phytases determine their efficacy and is exceptional.     

Effect of fermentation system on the production and properties of tannase of Aspergillus niger van Tieghem MTCC 2425

The tannase-producing efficiency of liquid-surface fermentation (LSF) and solid-state fermentation (SSF) vis-à-vis submerged fermentation (SmF) was investigated in a strain of Aspergillus niger, besides finding out if there was a change in the activity pattern of tannase in these fermentation processes. The studies on the physicochemical properties were confined to intracellular tannase as only this form of enzyme was produced by A. niger in all three fermentation processes. In LSF and SmF, the maximum production of tannase was observed by 120 h, whereas in SSF its activity peaked at 96 h of growth. SSF had the maximum efficiency of enzyme production. Tannase produced by the SmF, LSF and SSF processes had similar properties except that the one produced during SSF had a broader pH stability of 4.5-6.5 and thermostability of 20 degrees-60 degrees C.     

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.