Production of antifungal antibiotic,iturin in a solid state fermentation byBacillus subtilis NB22 using wheat bran as a substrate

Summary Production of a family of lipopeptide antibiotic, iturin byB. subtilis NB22, in solid state fermentation (SSF) of wheat bran (WB) was investigated. The amount of iturin produced per unit weight of wet substrate was 5–6 times more than that in the submerged fermentation (SMF). SSF enabled to produce a homologue of iturin with strong antibiotic activity in a larger fraction compared with the SMF.     

Hydrolysis and acidogenic fermentation of gelatin under anaerobic conditions in a laboratory scale upflow reactor

Summary Gelatin was dissolved in a mineral salts medium for growth under carbon limitation and fed to a mixed population of bacteria in a lab-scale upflow reactor for hydrolysis and acidogenic fermentation under anaerobic conditions, at pH=7 and 30°C.With the shortest applied liquid retention time (30 min), 84% of the protein was hydrolysed. The majority (85%) of the hydrolysed protein was fermented. The ammonia concentration in the reactor was about 1,400 mg N·l^-1.The fermentation products were mainly acetate, propionate, and valerate. Iso-butyrate, butyrate, and iso-valerate were formed to a limited extent. Gas production was very low and consisted of carbon dioxide and methane. The product composition was independent of the retention time applied. The sludge formed was slimy. No granules were formed, however a hold-up factor of 46 could be obtained.     

Durable continuous fermentation of a model mixture of d(+)-glucose and l(−)-sodium monoglutamate under non-axenic conditions

The possible durability of non-axenic fermentation profiles is of major importance in controlled environmental biotechnology. The durability of the non-axenic anaerobic fermentation profile of a model mixture of d(+)-glucose and l(–)-sodium monoglutamate was documented in a fabricated bioreactor for a period of more than 6 months. A cyclic scheme of reactor loading induced a cyclic, reproducible and durable fermentation profile. Equimolar amounts of n-butyric acid as well as acetic acid, and of H₂ and CO₂ were observed as steady metabolites. Correspondence to: E.-J. Nyns     

Morphology and laccase production of white-rot fungi grown on wheat bran flakes under semi-solid-state fermentation conditions

In this paper, we studied the laccase production and the growth morphology of different white-rot fungi, i.e. Pleurotus ostreatus, Trametes pubescens, Cerrena unicolor and Trametes versicolor, cultured under semi-solid-state fermentation conditions using wheat bran flakes as a natural low-cost support substrate. Trametes versicolor exhibited the highest laccase activity per gram of total dry matter, followed by P. ostreatus (63.5 and 58.2Ug(-1) , respectively). In addition, they showed a time profile of laccase production that was quite similar. Growth morphology was studied using environmental microscopic images and analyzed by discrete Fourier transformation-based software to determine the mean diameter of the hyphae, the number of hypha layers and the global micromorphology. The four strains exhibited different micromorphologies of growth. Pleurotus ostreatus presented narrow hyphae, which formed many thick clumps, T. pubescens and T. versicolor showed clumps of different sizes and C. unicolor showed thick hyphae that formed larger clumps, but in less amounts.     

Chitinase production by Beauveria felinaRD 101: optimization of parameters under solid substrate fermentation conditions

Major parameters affecting the production of chitinase by Beauveria felinaRD 101 under solid substrate fermentation conditions have been optimized. Wheat bran moistened with 100 MS-HCl medium adjusted to pH 5.0, inoculated with 1 × 10¹⁰ conidia g^–1 initial dry bran and incubated at 28 °C for 6 days produced maximum chitinase activity of 6.34 U g^–1 initial dry substrate.     

Influence of solid substrate fermentation on the chemical composition of chickpea

A basic procedure was developed to produce a fermented product by solid substrate fermentation using Rhyzopus oligosporus and chickpea as substrate. Water activity was kept at 0.92 throughout the process. Fermentation increased total, ‘true’ and soluble proteins, soluble solids and soluble carbohydrates, and decreased fiber content and pH. About 12% of solids were lost during 72 h of fermentation. The content of most fatty acids was enhanced by fermentation, whereas peroxide value and tannins declined. The color of the fermented product was not deteriorated after 72 h of fermentation. Scanning electron microscopy studies of microbial growth on the substrate showed penetration of the fungus hyphae and degradation effects on the chickpea cotyledon cells.     

Performance of the anaerobic baffled reactor under steady-state and shock loading conditions

The anaerobic baffled reactor (ABR) contains a granulated, mixed anaerobic culture segregated into compartments. Operation of four reactors under a range of hydraulic retention times showed that this novel reactor design offers highly efficient performance in the conversion of carbon in the feed stream to methane and carbon dioxide. The design parameter varied was the number of compartments. COD removal at 20 h retention time was routinely over 95% in all reactors, with low washout of biomass. Very high specific reaction rates were achievable (although with a loss of efficiency) at low biomass concentrations and high loading rates. In order to optimize volumetric reaction rates, a tradeoff has to be made between high biomass concentration, granule size, and the resulting mass transfer limitations. Formate is shown to be an important intermediate in the process under conditions of high loading.     

Hyper production of alkali stable xylanase in lesser duration by Bacillus pumilus SV-85S using wheat bran under solid state fermentation

High level production of an extracellular cellulase-poor alkali stable xylanase has been conceded from newly isolated Bacillus pumilus SV-85S under solid state fermentation using wheat bran as a substrate. Optimization of the fermentation conditions enhanced the enzyme production to 73,000 ± 1,000 IU/g dry substrate, which was 13.8-fold higher than unoptimized conditions (5,300 IU/g). The enzyme titre was highest after 48 h of incubation at 30°C with 1:3 ratios of substrate to moistening agent using wheat bran as a carbon source. The enzyme could be produced in significant levels by using either tap water or distilled water alone as a moistening agent. An elevated production of xylanase by B. pumilus SV-85S in the presence of wheat bran, a cheap and easily available agro-residue, in shorter duration would apparently reduce the enzyme cost substantially. The enzyme was completely stable over a broad pH (5-11) range and retained 52% of its activity at a temperature of 70°C for 30 min. The desired characteristics of this enzyme together with economic production would be important for its application in paper and pulp industry.     

Influence of transitional states on the microbial ecology of anaerobic digesters treating solid wastes

A better understanding of the microbial ecology of anaerobic processes during transitional states is important to achieve a long-term efficient reactor operation. Five wastes (pig manure, biodiesel residues, ethanol stillage, molasses residues, and fish canning waste) were treated in five anaerobic reactors under the same operational conditions. The influence of the type of substrate and the effect of modifying feeding composition on the microbial community structure was evaluated. The highest biomethanation efficiency was observed in reactors fed with fish canning waste, which also presented the highest active archaeal population and the most diverse microbial communities. Only two Bacteria populations could be directly related to a particular substrate: Ilyobacter with biodiesel residues and Trichococcus with molasses residues. Results showed that the time to achieve steady-state performance after these transitional states was not dependent on the substrate treated. But reactors needed more time to handle the stress conditions derived from the start-up compared to the adaptation to a new feeding. Cluster analyses showed that the type of substrate had a clear influence on the microbiology of the reactors, and that segregation was related to the reactors performance. Finally, we conclude that the previous inoculum history treating solid waste and higher values of active Archaea population are important factors to face a successful change in substrate not entailing stability failure.     

Optimization of some parameters of solid state fermentation of wheat bran for protease production by a local strain of Rhizopus oryzae

Some parameters of the production of an alkaline protease by Rhizopus oryzae in the solid state fermentation of wheat bran were optimized. Using the optimum parameters of an inoculum age of 7 days, an incubation time of 9 days, an amount of CZAPEK ‐DOX (liquid medium) of 6 ml/g bran and an incubation temperature of 33°C, an activity of 50 U/g bran was achieved. The initial pH of the CZAPEK ‐DOX medium had little effect. Re‐incubation of mouldy bran with only fresh CZAPEK ‐DOX yielded 3 times total activity compared to single‐cycle fermentation. As for the effect of the amount CZAPEK ‐DOX medium, the water constituent contributed more to activity increase than did the salt component. The ARRHENIUS activation energies were 23 and 7.9 kcal/mole below and above the optimum of 33°C, respectively. In all the studies, along with protease production, variation of protein content and specific activity were also observed. Attempts were made to explain the effects and also gauge their implications for large‐scale production.     

Effect of different cultivation conditions and inducers on the production of laccase by the litter-dwelling fungal isolate Fusarium incarnatum LD-3 under solid substrate fermentation

The litter-dwelling fungus Fusarium incarnatum LD-3 has been identified as a novel producer of laccase. The present work was oriented towards the optimization of various cultivation conditions for maximizing laccase production under solid substrate fermentation. The process parameters were optimized by the “one factor at a time” approach. Maximum laccsase production was obtained at pH 5.0 and at a temperature of 28 °C with 60 % moisture content using rice bran as a substrate. The laccase production was enhanced in the presence of aromatic inducer, i.e. ortho-dianisidine at a concentration of 0.5 mM. Laccase production was further increased by 52.56 % when the medium was supplemented with 2 % (v/v) alcohol. Among the various amino acids tested as a growth factor and nitrogen source, D-Serine and DL-2 Amino n-butyric acid, DL-Alanine and L-Glycine were found to be the most suitable for laccase production. The highest laccase production (1,352.64 U/g) was achieved under optimized conditions, and was 2.1-fold higher than the unoptimized conditions. Thus, the novel litter-dwelling fungal isolate Fusarium incarnatum LD-3 seems to be an efficient producer of laccase and can be further exploited for biotechnological applications. This is the first report on the optimization of cultivation conditions and inducers for laccase production from Fusarium incarnatum LD-3.     

Methods of assessing microbial activity and inhibition under anaerobic conditions: a literature review

This work reviews the existing methodologies for assessing microbial activity and inhibition under anaerobic conditions. The anaerobic digestion process consists of several metabolic steps–the Anaerobic Digestion Model No. 1 (ADM1) has attempted to describe these steps in the form of a mathematical model with the intention of providing a reference base for all further efforts in the modelling of anaerobic processes. The existence of a reference point for modelling has highlighted the fact that there is a lack of coherence between the many different methodologies for experimentally assessing anaerobic activity and inhibition.A working group of the International Water Association was recently founded to harmonise the existing methodologies with the ultimate intention of developing a unified reference procedure– a primary objective of the group will be the establishment of a standard terminology in the field of anaerobic digestion, activity and inhibition assessment. Secondly, it will compare the existing methodologies and develop standard protocols for assessing the kinetic parameters (e.g. maximum uptake rate, half-saturation constant) of anaerobic processes that may be entered directly into ADM1 and its successors.This paper revises and enlarges a contribution presented by the authors at the workshop Harmonisation of anaerobic biodegradation, activity and inhibition assays (Ligthart & Nieman 2002, Proc. workshop held in Orta (Italy) June 7–8, 2002) and aims to promote a clear understanding of the currently established methodology.Numerous methods have been developed over the past 30 years, since Van den Berg et al. (1974, Biotechnol Bioeng 16(11)– 1459–1469) measured methanogenic activity, by using a manometric device equipped with a photoelectric sensor to quantify the gas production. Methanogenesis is often the rate limiting step of the entire process and since the quantification of gas flowrate is relatively easy to perform, most of the methods reported in literature monitor the production of biogas. These methods can be termed volumetric or manometric methods, as the volume of biogas produced or the pressure increase due to gas production inside a close vessel are assessed, respectively. However, this same concept can be employed to assess activity or inhibition of individual metabolic steps preceding the methanogenic one, providing that they are rate limiting for the whole process. The reliability of activity assessment through gas measurement has been proven to be strongly dependent on the equilibrium between liquid and gas phase in a closed vessel. This can be influenced by many factors, e.g. the amount and characteristics of the test substrate; the concentration of the biomass; the gas-to-liquid ratio– all these aspects will need to be addressed in the standard procedure. Other direct or indirect methods, targeting physico-chemical or microbiological parameter exist and have been investigated by many authors. Besides the interest for research purposes, the definition of reference methods to assess activity and inhibition can be of great interest for engineers, both phy. Specific reference procedures might be needed for particular applications, e.g. the (kinetic) study of rate limiting microbial steps and might require ad-hoc methodologies to be devised. A microbiological technique such as FISH, coupled with microsensors have been reported to have a great potential in the near future.Passed away on April 7th 2003.     

Degradation of 2,4,6-trichlorophenol (2,4,6-TCP) by co-immobilization of anaerobic and aerobic microbial communities in an upflow reactor under air-limited conditions

The co-immobilization and the culture of anaerobic and aerobic communities was tested for the mineralization of 2,4,6-trichlorophenol (2,4,6-TCP). At first, the anaerobic microorganisms (aggregated into granules) were cultivated in an upflow anaerobic sludge blanket (UASB) reactor, in a continuous mode, with glucose, propionate, acetate (COD loading rate = 0.5-2.0 g COD/l per day, ratio 1:1:1) and 2,4,6-TCP (2,4,6-TCP loading rate = 25-278 micromol/l per day) as substrates. 2,4,6-TCP was degraded into 2,4-DCP and 4-CP, but it was not mineralized because of the low degradation rates of 4-CP. Furthermore, the highest loading rates of 2,4,6-TCP (>126 micromol/l per day) caused the inhibition of the strains degrading the propionate. The granules were therefore tested in association with the aerobic community. They were immobilized in kappa-carrageenan/gelatin [2% (w/w) of each polymer] gel beads and cultivated in a reactor, on their own (to test the influence of the gel), and then with the aerobic community, under anaerobic and air-limited conditions, respectively. The results showed that (1) the gel did not influence the activity of the granules, (2) the anaerobic and aerobic communities could be easily co-immobilized in gel beads and cultivated in a reactor, (3) the mineralization of 2,4,6-TCP (2,4,6-TCP loading rate = 10-506 micromol/l per day), its intermediates of degradation and the other substrates [glucose + acetate + propionate (ratio 1:1:1) = COD loading rate = 500 mg COD/l per day] could be obtained under air-limited conditions if the culture parameters were strictly controlled [airflow = 36-48 vvd (volume of air/volume of liquid in the reactor per day), pH value at around 7.5]. Finally, the gel did not retain its structure during the whole culture (263 days) in the air-limited reactor, but the anaerobic and aerobic communities retained their activities and worked together for the mineralization.     

Macrokinetic basis for the model of microbial growth in a limited volume under constant conditions with a single leading substrate

Within the framework of the macrokinetic approach and continuum and chemical/biochemical gross reaction conceptions, an equation describing the complete dynamics of microbial growth and decline as function of a variable concentration of the leading substrate was deduced. This equation allows us to distinguish quantitatively and qualitatively the stages of microbial growth and the intervals of microbial tolerance to the initial concentration of the leading substrate. Adequacy of the model was confirmed by comparison with experimental dynamics of aerobic microorganisms in the samples of groundwater collected from a region polluted with uranium.     

High yield of poly-gamma-glutamic acid from Bacillus subtilis by solid-state fermentation using swine manure as the basis of a solid substrate

Solid-state fermentations (SSF), using swine manure as the basis of a solid substrate, were carried out for high yield of poly-gamma-glutamic acid (gamma-PGA) by Bacillus subtilis CCTCC202048. Fermentation medium and process parameters were optimized through three orthogonal array designs. The optimal medium consisted of 62.3% (w/w, dry weight basis) swine manure, 25.0% soybean cake, 5.0% wheat bran, 5.0% glutamic acid, 2.5% citric acid and 0.2% MnSO4.H2O. The optimal process parameters were 15.0 g medium with initial moisture content 60% and initial pH 9.0 in 250 ml flask, inoculation at mid-log phase with a 4% inoculum level and cultivation for 48 h at 37 degrees C. The average-PGA yield (6.0%) in triplicate under optimal conditions was obtained on the laboratory scale while it was 4.5% at compost experiment. These would lay a foundation for lessening the pollution of swine manure, increasing fertilizer efficiency and exploring a late-model organic fertilizer that retains water and nutrients.     

Influence of the water activity of a solid substrate on the growth rate and sporogenesis of filamentous fungi

The water activity (a(w)) of substrates has been related to the mycelial growth and the sporogenesis of two molds. In the absence of other limiting factors, optimal a(w) values were determined for growth and sporogenesis as 0.99 and 0.98, respectively, for Trichoderma viride TS and 0.97 and 0.96 for Penicillium roqueforti. In all cases, the accuracy of the optimal value would justify the regulation of this parameter. A model was proposed which establishes a relationship between the mycelial growth and the water activity value of the substrate.     

The analysis of handsheets from wheat straw following solid substrate fermentation by Streptomyces cyaneus and soda cooking treatment

The recent interest in the utilisation of agricultural fibres has promoted research into their potential as raw materials for the pulp and paper industry. In the current study, we report on the effect of biological pretreatment of wheat straw by Streptomyces cyaneus on the performance of the handsheets produced from the treated pulps. The pre-treatment of wheat straw with S. cyaneus had a positive effect on both the burst and tear indexes of the pulps but had a negative impact on tensile index. No significant variation in permeability and in folding endurance was observed. Manipulation of handsheets from wheat straw through biological treatment may therefore result in improved quality traits.