Design of a new rotating drum bioreactor for ligninolytic enzyme production by Phanerochaete chrysosporium grown on an inert support

doi:10.1016/S0032-9592(01)00233-3

Process Biochemistry

Volume 37, Issue 5, 20 December 2001, Pages 549-554

https://doi.org/10.1016/S0032-9592(01)00233-3 Get rights and content

Abstract

The production of ligninolytic enzymes by the white-rot fungus Phanerochaete chrysosporium BKM-F-1767 was studied in a new bioreactor configuration based on a standard rotating drum bioreactor. P. chrysosporium was grown on cubes of nylon sponge, and cultivation was carried out in batch. Two aeration levels: 0.5 and 1 vvm were tested. The latter led to activities about 3-fold higher than the former, achieving maximum manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 1350 and 364 U/l, respectively. Moreover, laccase activity was also detected, showing a highest activity of 56 U/l. In addition, the in vitro decolorisation of a model dye (Poly R-478) by the extracellular liquid obtained in the bioreactor was monitored in order to assess its ligninolytic ability. A percentage of Poly R-478 decolorisation of about 19% was achieved, after 15 min of dye incubation.

Introduction

The white-rot fungus Phanerochaete chrysosporium secretes, during its secondary metabolism, several lignin-degrading enzymes including lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase. The secondary metabolism in this fungus is triggered by nitrogen [1], carbon or sulphur [2] deprivation. In addition to lignin, the above-mentioned enzymes are also able to degrade a wide range of hazardous environmental pollutants [3], [4]. Their application to industrial processes (biobleaching, biopulping, decolorisation, etc.) on a large scale requires the production of high amount of enzyme at low cost. Thus, the design of a system permitting the continuous production of ligninolytic enzymes efficiently is required.

Solid-state fermentation (SSF) involves the growth of microorganisms on moist solid substrates in the absence or near absence of free liquid [5]. The physical nature of the medium is quite different from that in submerged fermentation. For example, a solid bed is more difficult to mix effectively than a liquid broth, and as a consequence, O₂ supply and heat removal can be restricted in SSF processes [6]. The kind of solid materials that can be employed in this type of cultivation are classified in two main categories: inert support and non-inert support. The former acts as an attachment place (e.g. plastic foams) whereas the latter also functions as a source of nutrients (e.g. crop wastes).

SSF has gained importance in recent years due to several advantages over submerged fermentation such as superior productivity, simpler techniques, reduced energy requirements, low wastewater output, and improved product recovery [7], [8], [9]. Nevertheless, bioreactor design aspects have not been received enough attention by researchers of SSF and the present state of the art does not indicate an ideal type of bioreactor for solid state processes.

In earlier reports, a high production of extracellular ligninolytic enzymes in semi-solid-state cultures has been achieved and the utility of such cultures has been demonstrated [10], [11], [12].

Among the diverse types of support tested for semi-solid-state processes in previous papers [11], [12], [13], nylon sponge was preferred in the present work, mainly due to its inert nature, which allowed study the efficiency of the bioreactor without interactions of a series of variables related to the composition of a non-inert support. Moreover, its physical features (high roughness, hydrophobic nature and high porosity), permit good attachment of the fungus to the carrier as well as efficient oxygen and nutrients diffusion into the reactor bed.

In previous work [14], [15], different bioreactor configurations were assayed to produce ligninolytic enzymes in semi-solid-state conditions. Those results showed that the choice of an adequate reactor configuration is essential operating in such conditions. Most of these configurations are a modification of conventional bioreactors. Hence, this paper focuses on the development of a new bioreactor configuration, which produces high ligninolytic activities, functioning in semi-solid-state conditions, for a long time period without operational problems.

The bioreactor configuration employed in the present work is very appropriate to operate with immobilised biomass. In order to apply this bioreactor to semi-solid-state processes the volume of the culture medium in contact with the microorganism has been reduced to conditions near to semi-solid-state.

Section snippets

Microorganism and growth medium

P. chrysosporium BKM-F-1767 (ATCC 24725) was grown on a medium prepared according to Tien and Kirk [16] with 10 g glucose/l as carbon source, and replacement of dimethylsuccinate with 20 mM acetate buffer (pH 4.5) [17]. The fungus was grown in 90 ml of this medium at 37 °C in complete darkness for 48 h. After this, the whole culture was homogenised in a blender for 1 min. This homogenate suspension was used to inoculate (10% v/v) the production medium for the preinoculum.

Carrier

The bioreactor was

Results and discussion

Several bioreactor configurations have been employed to obtain ligninolytic enzymes not only in submerged but also in immobilised conditions [25], [26], [27], [28]. In contrast there are few studies on the production of such enzymes in bioreactors operating in semi-solid-state conditions [6], [29]. In the present report, a new bioreactor design, based on conventional rotating drum bioreactors, was tested for the production of ligninolytic enzymes in semi-solid-state conditions.

Conclusions

In view of the results attained, it can be concluded that the bioreactor configuration developed in the present paper is appropriate for the production of ligninolytic enzymes in semi-solid-state conditions. Moreover, it allowed the continuous production of LiP at high activity levels for long times without operational problems.

Acknowledgements

This research was financed by Xunta (PGIDT00PXI30118PR).

References (33)

J.S. Knapp et al.
Decolorization of dyes by wood-rotting Basidiomycete fungi
Enzyme Microbial. Biotechnol.
(1995)
J. Swamy et al.
The evaluation of white rot fungi in the decoloration of textile dyes
Enzyme Microbial. Biotechnol.
(1999)
A. Pandey
Recent process developments in solid-state fermentation
Process Biochem.
(1992)
R.P. Tengerdy
Solid substrate fermentation
Trends Biotechnol.
(1985)
M. Tien et al.
Lignin peroxidase of Phanerochate chrysosporium
Methods Enzymol.
(1988)
M. Kuwahara et al.
Separation and characterization of two extracellular H₂O₂-dependent oxidases from ligninolytic cultures of Phanerochaete chrysosporium
FEBS Lett.
(1984)
M.L. Niku-Paavola et al.
Detection of white-rot fungi by a non-toxic stain
Mycol. Res.
(1990)
F. Bosco et al.
Experimental identification of a scalable reactor configuration for lignin peroxidase production by Phanerochaete chrysosporium
J. Biotechnol.
(1996)
M.T. Moreira et al.
Strategies for the continuous production of ligninolytic enzymes in fixed and fluidised bed bioreactors
J. Biotechnol.
(1998)
P. Keyser et al.
Ligninolytic enzyme system of Phanerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation
J. Bacteriol.
(1978)

T.W. Jeffries et al.

Nutritional regulation of lignin degradation by Phanerochaete chrysosporium

Appl. Environ. Microbiol.

(1981)

A.J. Marsh et al.

O₂ uptake during solid-state fermentation in a rotating drum bioreactor

Biotechnol. Lett.

(1998)

C.W. Hesseltine

Solid state fermentation. Part I

Process Biochem.

(1977)

C.W. Hesseltine

Solid state fermentation: an overview

Int. Biodeter.

(1987)

S. Rodrı́guez et al.

Laccase production in semi solid state cultures of Phanerochaete chrysosporium

Biotechnol. Lett.

(1997)

S. Rodrı́guez Couto et al.

Effect of the different parts of the corncob employed as a carrier on ligninolytic activity in solid state cultures by Phanerochaete chrysosporium

Bioproc. Eng.

(1998)

Cited by (63)

The removal of diuron from agricultural wastewaters by Trametes versicolor immobilized on pinewood in simple channel reactors
2020, Science of the Total Environment
Citation Excerpt :
In addition, the output pumps were discarded, and the effluent left the reactor by overflow. Fungal RDBs have been previously used for enzyme production (Colla et al., 2017; Domínguez et al., 2001) and biological desulfurization (Şener et al., 2018), and a similar rotating drum biological contactor of white-rot fungus has been applied for decolorization (Šíma et al., 2016). Recently, rotating biological contactors containing immobilized T. versicolor were implemented to remove pharmaceutical micropollutants from urban wastewaters (Cruz del Álamo et al., 2020).
The presence of pesticides in agricultural wastewater entails harmful risks to both the environment and public health. In this study, two channel-type bioreactors with Trametes versicolor immobilized on pinewood chips were evaluated in terms of the removal efficiency of diuron from agricultural wastewater under non-sterile conditions. First, both single and successive sorption processes of diuron on pinewood chips were evaluated. The Freundlich model showed the best correlation in the sorption isotherm study (R² = 0.993; Δq = 5.245), but according to repeated sorption experiments, the Langmuir model (R² = 0.993; Δq = 5.757) was considered more representative. Equilibrium was reached after approximately 48 h, and the Elovich kinetic model gave the best fit with the experimental data. A packed-bed channel bioreactor (PBCB) was found to be a remarkable alternative able to remove up to 94% diuron from agricultural wastewater during 35 d. However, periodic manual mixing was required to guarantee an aerobic process, and a rotating drum bioreactor (RDB) was subsequently proposed as an enhanced version. The RDB removed up to 61% diuron during 16 d using almost 7 times lower wood dose (152 g wood·L⁻¹) than in the PBCB (1000 g wood·L⁻¹).
Fungal bioremediation of toxic textile dye effluents
2020, Fungi Bio-prospects in Sustainable Agriculture, Environment and Nano-technology: Volume 2: Extremophilic Fungi and Myco-mediated Environmental Management
Dyes used in the textile industry for imparting color to clothes are designed to be resistant to sweat, light and microbial attack. A significant portion of the dyes that are produced for the textile industry is discharged into the process water without any pretreatment. Thus, synthetic dyes used in dyeing of textiles are major contaminants of the aquatic environment. Fungi, especially white rot fungi (WRF), which produce extracellular lignin modifying enzymes have been found to be capable of degrading toxic synthetic dyes. Some of the mechanisms fungi use to detoxify dyes include biosorption, decolourisation and degradation or metabolism. Apart from WRF, fungi of the genera Aspergillus, Fusarium, Trichoderma, and Penicillium have been reported as good candidates for treating dye effluents. Several fungal bioreactors have been developed for the treatment of synthetic dyes and dye effluents.
Potential of fungal laccase in decolorization of synthetic dyes
2019, Microbial Wastewater Treatment
The continuous discharge of effluents containing synthetic dyes and nonessential heavy metals at a continually increasing rate, even beyond permissible level from various industries into adjoining aquatic environment, might result in accumulation and subsequent magnification to dangerous level, thus affecting marine life. Recent research have used enzymes to remove phenolic compounds and synthetic dyes from contaminated wastewater. Oxidoreductive enzymes, such as laccases, have the potential of degrading a wide range of aromatic pollutants and their substrates present in a shallow concentration in the contaminated sites. The use of fungal laccases in the decolorization of some recalcitrant textile and nontextile dyes need redox mediators for effective treatment of wastewater; hence, they will prove to be a cheaper alternative for the removal of aromatic pollutants from wastewater. The present review focuses on recent developments in the treatment of specific pollutants and synthetic dyes using fungal laccases.
Improved methane removal in exhaust gas from biogas upgrading process using immobilized methane-oxidizing bacteria
2018, Bioresource Technology
Citation Excerpt :
Sponge, as a loose porous material with polyurethane composition, has been used as the microbial immobilization support in several studies. Due to the features of porosity, lightweight, inertia and desirable hydroscopicity, the sponge could favor the microbial adhesion meanwhile not interfere with cell properties (de Oliveira Delani et al., 2012; Domínguez et al., 2001). In this work, the sponge possessed a bulk of pores with pore size of 200–300 μm, which formed by rough fibers with the width of 70–80 μm (Fig. S2(a)), MIP analysis also showed that the sponge has a great number of large pores, which had a Vmac of 5.4380 cm3 g−1.
Methane in exhaust gas from biogas upgrading process, which is a greenhouse gas, could cause global warming. The biofilter with immobilized methane-oxidizing bacteria (MOB) is a promising approach for methane removal, and the selections of inoculated MOB culture and support material are vital for the biofilter. In this work, five MOB consortia were enriched at different methane concentrations. The MOB-20 consortium enriched at the methane concentration of 20.0% (v/v) was then immobilized on sponge and two particle sizes of volcanic rock in biofilters to remove methane in exhaust gas from biogas upgrading process. Results showed that the immobilized MOB performed more admirable methane removal capacity than suspended cells. The immobilized MOB on sponge reached the highest methane removal efficiency (RE) of 35%. The rough surface, preferable hydroscopicity, appropriate pore size and particle size of support material might favor the MOB immobilization and accordingly methane removal.
Sweet sorghum-a promising alternative feedstock for biofuel production
2018, Renewable and Sustainable Energy Reviews
The key principle in resource management is sustainability which consists of operational robustness, attenuation of environmental footprint and socio-economic considerations. Dependence on fossil fuels is unviable due to their continuous depletion all over the world and also the inexperienced greenhouse gas emissions related to their utilization. Therefore, the continuous initiatives geared towards developing various renewable and probably carbon neutral biofuels as energy resources are being taken up. Alternate energy resources such as 1st generation biofuels derived from terrestrial crops like sugarcane, sugar beet, corn and wheat place a colossal stress on global food markets, but this potential food versus fuel conflict is palliated by using sweet sorghum as a bioenergy crop. It can be processed into both biofuel and valuable co-products, thus meeting the various requirements of food, fuel and fodder.
This paper mainly reviewed the technologies for bioethanol production from sweet sorghum, focussing on its potential benefits as feedstock for ethanol production over other substrates and recent advancements to enhance ethanol yield. It also reviewed the advances in pretreatment along with the novel process of ethanol production from sweet sorghum stalks, biogas production from sweet sorghum and environmental cum socio-economic aspects. No doubt, there are outstanding issues related to ethanol production and yield, still sweet sorghum derived bioethanol could progressively substitute a significant proportion of the fossil fuels required to meet the growing energy demand.
Decolorization of Reactive Orange 16 in Rotating Drum Biological Contactor
2016, Journal of Environmental Chemical Engineering
A laboratory scale Rotating Drum Biological Contactor (RDBC; drum length × diameter: 255 × 130 mm) filled by randomly arranged particulate carriers with mycelium of the white rot fungus Irpex lacteus was tested for Reactive Orange 16 (RO16) decolorization. Five mycelium carriers were pre-screened for fungus immobilization and the dye degradation in static cultures. The best mycelium growth was observed with the straw, wooden shavings and polyether foam cylinders. Considerable dye adsorption (up to 30%) was observed with natural carriers; very low adsorption was observed with carrier particles made of plastics. Natural mycelium carriers with attached I. lacteus yielded the highest RO16 decolorization efficiency (50–90%). The straw, wooden shavings and polyether foam particles were tested in the RDBC in batch dye degradation experiments and the straw was also tested in continuous experiments. The straw yielded the fastest and stable dye decolorization in the subsequent batches. With the straw, wooden shavings and polyether foam particles RO16 decolorization higher than 90% was achieved within 48 h in batch experiments and better than 80% decolorization was attained at liquid mean residence time of 33.3 h in continuous experiments. Dye degradation rate of 3.5–4.0 g m⁻³ h⁻¹ was achieved in the batch RDBC and the rate of 1.1–1.5 g m⁻³ h⁻¹ in the continuously operated reactor.

View all citing articles on Scopus

View full text

Design of a new rotating drum bioreactor for ligninolytic enzyme production by Phanerochaete chrysosporium grown on an inert support

Abstract

Introduction

Section snippets

Microorganism and growth medium

Carrier

Results and discussion

Conclusions

Acknowledgements

Enzyme Microbial. Biotechnol.

Enzyme Microbial. Biotechnol.

Process Biochem.

Trends Biotechnol.

Methods Enzymol.

FEBS Lett.

Mycol. Res.

J. Biotechnol.

J. Biotechnol.

Ligninolytic enzyme system of Phanerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation

J. Bacteriol.

Nutritional regulation of lignin degradation by Phanerochaete chrysosporium

Appl. Environ. Microbiol.

O2 uptake during solid-state fermentation in a rotating drum bioreactor

Biotechnol. Lett.

Solid state fermentation. Part I

Process Biochem.

Solid state fermentation: an overview

Int. Biodeter.

Laccase production in semi solid state cultures of Phanerochaete chrysosporium

Biotechnol. Lett.

Effect of the different parts of the corncob employed as a carrier on ligninolytic activity in solid state cultures by Phanerochaete chrysosporium

Bioproc. Eng.

O₂ uptake during solid-state fermentation in a rotating drum bioreactor