Fungal and enzymatic treatment of mature municipal landfill leachate.

The aim of our study was to evaluate biotreatability of mature municipal landfill leachate by using white rot fungus and its extracellular enzymes. Leachates were collected in one active and one closed regional municipal landfill. Both chosen landfills were operating for many years and the leachates generated there were polluted by organic and inorganic compounds. The white rot fungus Dichomitus squalens was able to grow in the mature leachate from the closed landfill and as it utilizes present organic matter as a source of carbon, the results were showing 60% of DOC and COD removal and decreased toxicity to the bacterium Aliivibrio fischeri. On the other hand, growth of the fungus was inhibited in the presence of the leachate from the active landfill. However, when the leachate was introduced to a crude enzyme filtrate containing extracellular ligninolytic enzymes, removal levels of COD and DOC reached 61% and 44%, respectively. Furthermore, the treatment led to detoxification of the leachate to the bacterium Aliivibrio fischeri and to reduction of toxicity (42%) to the plant Sinapis alba. Fungal and enzymatic treatment seems to be a promising biological approach for treatment of mature landfill leachates and their application should be further investigated.

Optimization of ligninolytic enzyme activity and production rate with Ceriporiopsis subvermispora for application in bioremediation by varying submerged media composition and growth immobilization support.

Response surface methodology (central composite design of experiments) was employed to simultaneously optimize enzyme production and productivities of two ligninolytic enzymes produced by Ceriporiopsis subvermispora. Concentrations of glucose, ammonium tartrate and Polysorbate 80 were varied to establish the optimal composition of liquid media (OLM), where the highest experimentally obtained activities and productivities were 41 U L(-1) and 16 U L(-1) day(-1) for laccase (Lac), and 193 U L(-1) and 80 U L(-1) day(-1) for manganese peroxidase (MnP). Considering culture growth in OLM on various types of immobilization support, the best results were obtained with 1 cm beech wood cubes (BWCM). Enzyme activities in culture filtrate were 152 U L(-1) for Lac and 58 U L(-1) for MnP, since the chemical composition of this immobilization material induced higher Lac activity. Lower enzyme activities were obtained with polyurethane foam. Culture filtrates of OLM and BWCM were applied for dye decolorization. Remazol Brilliant Blue R (RBBR) was decolorized faster and more efficiently than Copper(II)phthalocyanine (CuP) with BWCM (80% and 60%), since Lac played a crucial role. Decolorization of CuP was initially faster than that of RBBR, due to higher MnP activities in OLM. The extent of decolorization after 14 h was 60% for both dyes.

The use of the fungus Dichomitus squalens for degradation in rotating biological contactor conditions.

Biodegradation potential of Dichomitus squalens in biofilm cultures and rotating biological contactor (RBC) was investigated. The fungus formed thick biofilms on inert and lignocellulosic supports and exhibited stable activities of laccase and manganese peroxidase to reach 40-62 and 25-32% decolorization of anthraquinone Remazol Brilliant Blue R and heterocyclic phthalocyanine dyes, respectively. The decolorization ceased when glucose concentration dropped to 1 mmol l(-1). In RBC reactor, respective decolorizations of Remazol Brilliant Blue R and heterocyclic Methylene Blue and Azure B dyes (50 mg l(-1)) attained 99%, 93%, and 59% within 7, 40 and 200 h. The fungus exhibited tolerance to coliform and non-coliform bacteria on rich organic media, the inhibition occurred only on media containing tryptone and NaCl. The degradation efficiency in RBC reactor, capability to decolorize a wide range of dye structures and tolerance to bacterial stress make D. squalens an organism applicable to remediation of textile wastewaters.

Enhanced enzyme production with the pelleted form of D. squalens in laboratory bioreactors using added natural lignin inducer.

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.

Oxygen transfer in a laboratory stirred tank bioreactor during Mammalian cell culture cultivation.

The influence of power consumption on the volumetric mass transfer coefficient was studied in a 5 liter stirred tank bioreactor during cultivation of a recombinant Chinese Hamster Ovary (CHO) cell line, which requires low aeration and mixing intensity. Under these conditions and at high cell concentrations, oxygen mass transfer coefficient and consequently volumetric oxygen mass transfer rate was not sufficient for the oxygen requirements. This problem was successfully solved by introducing pure oxygen into the bioreactor and the oxygen mass balance was studied. For that purpose, respiration rate as a function of cell concentration, as well as volumetric oxygen mass transfer rate at different operating conditions were measured. Graphical presentation of the results show the process control possibilities and the role of mixing intensity necessary to ensure that there is sufficient oxygen supply for a given cell concentration and respiration rate.

Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR,PL promoters from phage lambda.

Constitutive and thermoinducible expression plasmids based on strong P(R),P(L) promoters from phage lambda were compared for production of TNF-alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF-alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2-3-fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible.