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Solid-state fermentation (SSF) with filamentous fungi is a promising technique for the production of a range of biotechnological products and has the potential to play an important role in future biorefineries. The performance of such processes is intimately linked with the mycelial mode of growth of these fungi: Not only is the production of extracellular enzymes related to morphological characteristics, but also the mycelium can affect bed properties and, consequently, the efficiency of heat and mass transfer within the bed. A mathematical model that describes the development of the fungal mycelium in SSF systems at the particle scale would be a useful tool for investigating these phenomena, but, as yet, a sufficiently complete model has not been proposed. This review presents the biological and mass transfer phenomena that should be included in such a model and then evaluates how these phenomena have been modeled previously in the SSF and related literature. We conclude that a discrete lattice-based model that uses differential equations to describe the mass balances of the components within the system would be most appropriate and that mathematical expressions for describing the individual phenomena are available in the literature. It remains for these phenomena to be integrated into a complete model describing the development of fungal mycelia in SSF systems.

Current models that describe the extension of fungal hyphae and development of a mycelium either do not describe the role of vesicles in hyphal extension or do not correctly describe the experimentally observed profile for distribution of vesicles along the hypha. The present work uses the n-tanks-in-series approach to develop a model for hyphal extension that describes the intracellular transport of nutrient to a sub-apical zone where vesicles are formed and then transported to the tip, where tip extension occurs. The model was calibrated using experimental data from the literature for the extension of reproductive aerial hyphae of three different fungi, and was able to describe different profiles involving acceleration and deceleration of the extension rate. A sensitivity analysis showed that the supply of nutrient to the sub-apical vesicle-producing zone is a key factor influencing the rate of extension of the hypha. Although this model was used to describe the extension of a single reproductive aerial hypha, the use of the n-tanks-in-series approach to representing the hypha means that the model has the flexibility to be extended to describe the growth of other types of hyphae and the branching of hyphae to form a complete mycelium.

Solid-state fermentation (SSF) with filamentous fungi is a promising technique for the production of a range of biotechnological products and has the potential to play an important role in future biorefineries. The performance of such processes is intimately linked with the mycelial mode of growth of these fungi: Not only is the production of extracellular enzymes related to morphological characteristics, but also the mycelium can affect bed properties and, consequently, the efficiency of heat and mass transfer within the bed. A mathematical model that describes the development of the fungal mycelium in SSF systems at the particle scale would be a useful tool for investigating these phenomena, but, as yet, a sufficiently complete model has not been proposed. This review presents the biological and mass transfer phenomena that should be included in such a model and then evaluates how these phenomena have been modeled previously in the SSF and related literature. We conclude that a discrete lattice-based model that uses differential equations to describe the mass balances of the components within the system would be most appropriate and that mathematical expressions for describing the individual phenomena are available in the literature. It remains for these phenomena to be integrated into a complete model describing the development of fungal mycelia in SSF systems.

Background Metagenomics is a useful tool in the search for new lipases that might have characteristics that make them suitable for application in biocatalysis. This paper reports the cloning, co-expression, purification and characterization of a new lipase, denominated LipG9, and its specific foldase, LifG9, from a metagenomic library derived from a fat-contaminated soil. Results Within the metagenomic library, the gene lipg9 was cloned jointly with the gene of the foldase, lifg9. LipG9 and LifG9 have 96% and 84% identity, respectively, with the corresponding proteins of Aeromonas veronii B565. LipG9 and LifG9 were co-expressed, both in N-truncated form, in Escherichia coli BL21(DE3), using the vectors pET28a(+) and pT7-7, respectively, and then purified by affinity chromatography using a Ni2+ column (HiTrap Chelating HP). The purified enzyme eluted from the column complexed with its foldase. The molecular masses of the N-truncated proteins were 32 kDa for LipG9, including the N-terminal His-tag with 6 residues, and 23 kDa for LifG9, which did not have a His-tag. The biochemical and kinetic characteristics of the purified lipase-foldase preparation were investigated. This preparation was active and stable over a wide range of pH values (6.5-9.5) and temperatures (10-40°C), with the highest specific activity, of 1500 U mg−1, being obtained at pH 7.5 at 30°C. It also had high specific activities against tributyrin, tricaprylin and triolein, with values of 1852, 1566 and 817 U mg−1, respectively. A phylogenetic analysis placed LipG9 in the lipase subfamily I.1. A comparison of the sequence of LipG9 with those of other bacterial lipases in the Protein Data Bank showed that LipG9 contains not only the classic catalytic triad (Ser103, Asp250, His272), with the catalytic Ser occurring within a conserved pentapeptide, Gly-His-Ser-His-Gly, but also a conserved disulfide bridge and a conserved calcium binding site. The homology-modeled structure presents a canonical α/β hydrolase folding type I. Conclusions This paper is the first to report the successful co-expression of a lipase and its associated foldase from a metagenomic library. The high activity and stability of Lip-LifG9 suggest that it has a good potential for use in biocatalysis. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0171-7) contains supplementary material, which is available to authorized users.

Lipases (glycerol ester hydrolases, E.C. 3.1.1.3) are enzymes of great industrial interest due to their ability to catalyze a broad range of hydrolytic and synthetic reactions. They find applications in the synthesis of compounds used in clinical, nutritional, environmental, pharmaceutical and chemical fields. For example, lipases are used to catalyze key intermediate steps in the synthesis of biologically active compounds such as Naproxen, Ibuprofen and Atenolol [1]. Depending on the application, lipases may need to be purified and characterized biochemically before they can be used. However, the purification of microbial lipases is often made difficult by the presence of high molecular weight aggregates. These aggregates can form due to the presence, in the fermentation medium, of lipids used to induce the production of the enzyme by the microorganism or simply due to hydrophobic interactions amongst the enzyme molecules themselves [2]. In previous work, we characterized a new lipase produced by Bacillus megaterium CCOC P2637. The enzyme eluted in the void volume during gel filtration chromatography, indicating that it was present in the form of a high molecular weight aggregate. This aggregate was dispersed when a gradient of 60% (v/v) isopropanol was used, but formed again when the enzyme was injected in a gel filtration column for further purification, even when the elution buffer contained 20% (v/v) isopropanol. Further, when the enzyme was diluted in buffer (phosphate pH 7.0 20 mM) containing 30% isopropanol, its specific activity was double the activity obtained by diluting in buffer without isopropanol [3].

We present a model for the kinetics of microbial growth that incorporates the influence of the temperature variations that typically occur during solid-state culture in large-scale bioreactors. The model proposes that the specific growth rate constant depends on the level of an essential component within the biomass, with the rate of the synthesis and denaturation reactions of this component depending on temperature according to the Arrhenius equation. Model predictions were compared to literature data for the growth of Rhizopus oligosporus in three different situations: (1) incubation of cultures at different but constant temperatures; (2) initial incubation of cultures at 37 °C, followed by incubation at 50 °C; and (3) tray culture. The model agreed reasonably with all three sets of experimental results with the use a single set of parameter values. This approach to modeling has good potential for application in models of solid-state culture bioreactors.

The kinetics of hydrolysis of triolein and tributyrin and of the synthesis of ethyl-laurate by the lipase of Thermomyces lanuginosa , contained in a commercial preparation (Lipolase®), were studied in AOT/Isooctane reverse micelles. Lipolytic activity against triolein depended strongly on the water content in the system (W0 = [H2O]/[AOT]), in a bell-shaped manner, with a maximum at a W0 of 15. The best conditions for enzyme activity were pH 8.0 and 37 ◦ C. The enzyme did not show Michaelis–Menten kinetics for the hydrolysis of either triolein or tributyrin. The enzyme was unstable at temperatures of 37–60 ◦ C, losing approximately 50% of its activity after 30 min. The catalysis of ethyl-laurate synthesis by T. lanuginosalipase in reverse micelles was studied using factorial designs to optimize the reaction conditions. The most important variables were pH and temperature and their combined effect. The best conditions for ester synthesis were a W0 of 10, a pH of 5.6, a molar ratio of alcohol to acid of 5.0 and a temperature of 30 ◦ C. The specific enzymatic activity under these conditions was 220 U mg −1 and the ester yield 92% after 60 min of reaction. This high yield, obtained in a relatively short time, justifies further exploration of the potential of this system in biocatalysis. © 2004 Elsevier B.V. All rights reserved.

The potential for thermal denaturation to cause enzyme losses during solid-state fermentation processes for the production of enzymes was examined, using the protease of Penicillium fellutanum as a model system. The frequency factor and activation energies for the first-order denaturation of this enzyme were determined as 3.447 × 10 59 h −1 and 364,070 J mol −1 , respectively. These values were incorporated into a mathematical model of enzyme deactivation, which was used to investigate the consequences of subjecting this protease to temporal temperature profiles reported in the literature for mid-height in a 34 cm high packed-bed bioreactor of 150 mm diameter. In this literature source, temperature profiles were measured for 5, 15 and 25 liters per minute of air and enzyme activities were measured as a function of time. The enzyme activity profiles predicted by the model were distributed similarly, one relative to the other, as had been found in the experimental study, with substantial amounts of denaturation being predicted when the substrate temperature exceeded 40 °C, which occurred at the lower two airflow rates. A mathematical model of a well-mixed bioreactor was used to explore the difficulties that would be faced at large scale. It suggests that even with airflows as high as one volume per volume per minute, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation. This work highlights the extra care that must be taken in scaling up solid-state fermentation processes for the production of thermolabile products.

The effects of various environmental conditions and chemical compounds on the activity and stability of the lipolytic preparation obtained from a wild strain of Penicillium aurantiogriseum were characterized during a preliminary evaluation of its potential for use in biocatalysis. In aqueous solution, the optimum pH for activity was 8.0 and the enzyme was stable between pH 6.0 and pH 9.0. In assays of 1 min duration carried out at pH 8.0, enzyme activities were quite high from 37 to 70 °C, with a maximum at 60 °C. However, thermal stability was rather low at temperatures higher than 28 °C. Hydrolytic activity was enhanced by Mg 2+ , Zn 2+ , Co 2+ and Mn 2+ , but was inhibited by Cu 2+ , Ba 2+ and Hg 2+ , while Ca 2+ had no effect. Sodium azide activated the enzyme. Triton X-100 caused an inhibition of 52%, while Tween 80 and SDS had negligible effects on enzymatic activity. Despite the low ratio of the activity towards p -nitrophenyl palmitate (pNPP) in organic medium to that in aqueous medium ( R O/A =4.3×10 −2 ), the enzyme showed a good stability in organic solvents with high log P values, the best result being in n -heptane (114% residual activity). These promising results with the crude preparation justify the undertaking of purification studies and the use of the pure enzyme in a more in-depth investigation for its potential in biocatalysis in organic solvents.

Mathematical models are important tools for optimizing the design and operation of solid-state fermentation (SSF) bioreactors. Such models must describe the kinetics of microbial growth, how this is affected by the environmental conditions and how this growth affects the environmental conditions. This is done at two levels of sophistication. In many bioreactor models the kinetics are described by simple empirical equations. However, other models that address the interaction of growth with intraparticle diffusion of enzymes, hydrolysis products and O2 with the use of mechanistic equations have also been proposed, and give insights into how these microscale processes can potentially limit the overall performance of a bioreactor. The current article reviews the advances that have been made in both the empirical- and mechanistic-type kinetic models and discusses the insights that have been achieved through the modeling work and the improvements to models that will be necessary in the future.

Biocatalysis has become a useful alternative to chemical transformations for the production of a range of compounds with applications in the food, feed, chemical and pharmaceutical industries. However, it is not necessarily an easy task to obtain the desired levels of performance in terms of rate, yield and selectivity of the reaction. One strategy for optimizing biocatalyst performance is to use non-conventional media, such as non-aqueous heterogeneous systems. In this article, we highlight some of the current trends in biocatalysis in such systems, focusing on reverse micelles, supercritical fluids and ionic liquids.

The study was carried out in order to determine and establish the functional characters of starch extracted from yam bean (Pachyrhizus erosus (L) Urban) compared with cassava starch. Yam bean is a tropical tuber legume easily grown and holds a great potential as a new source of starch. Yam bean starch shows functional properties which are peculiar to those of most starch root crops. Gelatinization temperature (53-63 degrees C) and the pasting temperature (64.5 degrees C) are less than those of cereal starch, however, the swelling power is high (54.4 g gel/g dried starch). Yam bean starch paste presents a high viscosity profile, high retrogradation tendency and low stability on cooking. The functional properties of yam bean starch, similar to those of cassava starch, allows yam bean to be used as a potential new source of starch.

Mathematical models are important tools for optimizing the design and operation of solid-state fermentation (SSF) bioreactors. Such models must describe the transport phenomena within the substrate bed and mass and energy exchanges between the bed and the other subsystems of the bioreactor, such as the bioreactor wall and headspace gases. The sophistication with which this has been done for SSF has improved markedly over the last decade or so. The current article reviews these advances, showing how the various transport phenomena have been modeled. It also discusses the insights that have been achieved through the modeling work and the improvements to models that will be necessary in order to make them even more powerful tools in the optimization of bioreactor performance.

In previous work, a new lipase and its cognate foldase were identified and isolated from a metagenomic library constructed from soil samples contaminated with fat. This new lipase, called LipG9, is a true lipase that shows specific activities that are comparable to those of well-known industrially-used lipases with high activity against long-chain triglycerides. In the present work, LipG9 was co-expressed and co-immobilized with its foldase, on an inert hydrophobic support (Accurel MP1000). We studied the performance of this immobilized LipG9 (Im-LipG9) in organic media, in order to evaluate its potential for use in biocatalysis. Im-LipG9 showed good stability, maintaining a residual activity of more than 70% at 50 °C after incubation in n-heptane (log P 4.0) for 8 h. It was also stable in polar organic solvents such as ethanol (log P -0.23) and acetone (log P -0.31), maintaining more than 80% of its original activity after 8 h incubation at 30 °C. The synthesis of ethyl esters was tested with fatty acids of different chain lengths in n-heptane at 30 °C. The best conversions (90% in 3 h) were obtained for medium and long chain saturated fatty acids (C8, C14 and C16), with the maximum specific activity, 29 U per gram of immobilized preparation, being obtained with palmitic acid (C16). Im-LipG9 was sn-1,3-specific. In the transesterification of the alcohol (R,S)-1-phenylethanol with vinyl acetate and the hydrolysis of the analogous ester, (R,S)-1-phenylethyl acetate, Im-LipG9 showed excellent enantioselectivity for the R-isomer of both substrates (E> 200), giving an enantiomeric excess (ee) of higher than 95% for the products at 49% conversion. The results obtained in this work provide the basis for the development of applications of LipG9 in biocatalysis.

We studied the production of rhamnolipids by Pseudomonas aeruginosa UFPEDA 614 in submerged culture, using glycerol as the carbon source. A rhamnolipid yield of 15.9 g/L was obtained with 40 g/L glycerol and 5 g/L sodium nitrate as nitrogen source after 7 days of cultivation. Structural analysis carried out at different cultivation periods showed that the four major mono-rhamnolipid homologues are present in higher proportion in the first 48 h. Over time, the corresponding four major di-rhamnolipid homologues predominated, representing about 75 % of the total rhamnolipids after 96 h. Physicochemical analysis of the rhamnolipid mixtures obtained at different cultivation periods showed that the sample obtained from the first day of cultivation had the lower critical micelle concentration (15.6 mg/L), which is probably related to the higher proportion of mono-rhamnolipids. The results presented here show that the composition of the mixture of rhamnolipid homologues produced by P. aeruginosa UFPEDA 614 varies over time and that this variation influences the physicochemical properties of the mixture. These findings can be used in order to produce rhamnolipid mixtures that have suitable properties for different applications.

Citrus processing waste is a leftover from the citrus processing industry and is available in large amounts. Typically, this waste is dried to produce animal feed, but sometimes it is just dumped. Its main component is the peel, which consists mostly of pectin, with D-galacturonic acid as the main monomer. Aspergillus niger is a filamentous fungus that efficiently produces pectinases for the hydrolysis of pectin and uses the resulting D-galacturonic acid and most of the other components of citrus peel for growth. We used engineered A. niger strains that were not able to catabolise D-galacturonic acid, but instead converted it to L-galactonic acid. These strains also produced pectinases for the hydrolysis of pectin and were used for the conversion of pectin in orange peel to L-galactonic acid in a consolidated process. The D-galacturonic acid in the orange peel was converted to L-galactonic acid with a yield close to 90%. Submerged and solid-state fermentation processes were compared.

Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.

Conventional techniques of enzymes relies on isolation and cultivation of microorganism. Since it is estimated that only 1-10% of microorganisms are culturable different approaches have been used for obtaining new enzymes of biotechnological interest. Metagenomic technique obviate this need by directly extracting of DNA fragments from environment and subsequent sequencing, analysis and overexpression into a host microorganism culturable in the laboratory 1 . Since lipases obtained by metagenomic have not been evaluated for their applicability in organic synthesis, we decided to evaluate the lipases LipC6G9 and LipC12, wich were obtained from a metagenomic library constructed from a sample of animal fat contaminated soil 2 , in the kinetic resolution of secondary alcohols and respective esters 1a-1d.

Over the last decade there has been a significant improvement in understanding how to design, operate and scale-up solid-state fermentation bioreactors. The key to these advances has been the application of mathematical modeling techniques to describe the biological and transport phenomena within the system. This review focuses on the advances in understanding that have come from this modeling work, and the insights it has given us into bioreactor design, operation and scale-up. It also highlights two promising bioreactor designs that have emerged over the last decade or so. For processes in which the substrate bed must remain static throughout the fermentation, the most promising design is the Zymotis design of ORSTOM at Montpellier, France, which involves closely spaced internal heat transfer plates within a packed-bed bioreactor. For those processes in which mixing can be tolerated, the stirred bioreactor developed at INRA, in Dijon, France, has been successfully demonstrated at scales of 1–25 t of substrate. Theoretical work suggests that mathematical models will be useful tools in the scale-up process, however, there are no reports that they have been used in the development of any current large-scale process. Rather, the models have been validated against data obtained from laboratory-scale bioreactors. There is an urgent need to test the accuracy and robustness of the models by applying them within real process development.

Microbial inulinases are an important class of industrial enzymes that have gained much attention recently. Inulinases can be produced by a host of microorganisms, including fungi, yeast, and bacteria. Among them, however, Aspergillus sp. (filamentous fungus) and Kluyveromyces sp. (diploid yeast) are apparently the preferred choices for commercial applications. Among various substrates (carbon source) employed for their production, inulin-containing plant materials offer advantages in comparison to pure substrates. Although submerged fermentation has been universally used as the technique of fermentation, attempts are being made to develop solid-state fermentation technology also. Inulinases catalyze the hydrolysis of inulin to D-fructose (fructose syrup), which has gained an important place in human diets today. In addition, inulinases are finding other newer applications. This article reviews more recent developments, especially those made in the past decade, on microbial inulinases--its production using various microorganisms and substrates. It also describes the characteristics of various forms of inulinases produced as well as their applications.

A lipase from a wild strain of Penicillium citrinum was purified by ammonium sulphate precipitation, gel filtration chromatography on a Superose 6 column and hydrophobic interaction chromatography (HIC) on a Phenyl Superose column. The yield and purification factor were 15.2% and 379 fold, respectively. The gel filtration step was efficiently scaled-up in a Superose 6 preparative grade column and after this step, the lipase was recovered in the form of a high molecular weight aggregate. The partial disaggregation of the complex was achieved by HIC and elution with 1.0% (w/v) CHAPS. The lipase produced by Penicillium citrinum forms a dimmer of 63 000 Da, as determined by SDS-PAGE, and it accumulates in the fermentation broth as high molecular weight aggregates (>2 00 000 Da). The analysis of the dimmer showed two subunits with similar molecular weights (31 000–33 000 Da) and isoelectric points (4.8–5.0).

A lipase from a wild strain ofPenicillium citrinum was encapsulated in AOT/isooctane-reversed micelles, and the kinetic parameters were studied relative to triolein hydrolysis. Lipolytic activity was strongly dependent on the water amount in the system (Wo) and presented a bell-shaped curve for this parameter, with a maximum in the range of Wo 10–15. Optimum conditions for enzyme activity were pH 8.0 and 45‡C. The influence of substrate concentration was also studied. The enzyme showed a Michaelis-Menten behavior and the apparent kinetics constants were calculated as beingV max.app. - 120 U/mg and Kmapp = 49.2 mM.

This work describes the extraction and back-extraction of a lipase from crude extract of Penicillium citrinum using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration on the protein forward and backward transfer at 20°C was studied. The maximum protein forward extraction (32·0%) was achieved at pH 4·0 with a 50 mmol dm−3 acetate buffer containing 100 mmol dm−3 KCl and 100 mmol dm−3 AOT in isooctane. Proteins were back-extracted (82·7%) to a new aqueous phase containing 100 mmol dm−3 pH 8·0 phosphate buffer and 1000 mmol dm−3 KCl. No enzyme activity could be detected either in the micellar phase or in the aqueous phase after protein back-extraction. However, the lipolytic activity was recovered after hydrophobic interaction chromatography on a Phenyl Superose column. The yield obtained for the overall process was 68% for activity, 26·4% for protein recovery and the purification factor was 810-fold. A single protein band at 33000 Da was obtained for SDS–PAGE analysis for the recovered and purified enzyme. © 1997 SCI.

An alternative source of starch obtained from jacatupe (Pachyrhizus erosus L. Urban) a tropical tuber has been characterized enzymatically using amylolytic enzymes. The best hydrolysis conditions were obtained using α-amylase of 13.56U/ml at pH 7.0 and 80°C; amyloglucosidase of 177.5U/ml at a pH range from 3.5 to 4.0 and 70°C, respectively. Michaelis constants were determined in terms of substrate concentration; Km and Vmax for α-amylase and amyloglucosidase were found to be 16.8g/1, 1.49g/1, 11.2μmol/h and 3.10μmol/h, respectively. Scanning electron microscopy studies suggest that the enzymes tunnelled into the granular interior of the starch granules and then hydrolysed from within, along concentric holes - a well established phenomenon in the starch granules from classical crops. Enzymatische Hydrolyse der Starke von Jacatupe (Pachyrhizus erosus L. Urban) durch thermostabile amylolytische Enzyme. Eine alternative Quelle von Jacatupe (Pachyrhizus erosus L. Urban), einer tropischen Wurzel, wurde enzymatisch unter Anwendung von amylolytischen Enzymen charakterisiert. Die besten Amylolyse-Bedingungen wurden erhalten durch Verwendung von α-Amylase mit 13,56U/ml bei pH 7,0 und 80°C, bzw. 177,5U/ml Amyloglucosidase im pH-Bereich von 3,5 bis 4,0 und 70°C. Die Michaelis-Konstanten wurden in Abhangigkeit von der Substratkonzentration bestimmt. Km und Vmax fur α-Amylase und Amyloglucosidase betrugen 16,8g/1, 1,49g/1, 11,2μmol/h und 3,10μmol/h. Raster-Elektronenmikroskopie-Untersuchungen lassen vermuten, das die Enzyme durch Tunnelbildung in das Innere der Starkekorner gelangt sind und von innen her hydrolysiert haben in Richtung konzentrischer Locher, ein gut etabliertes Phanomen in den Starkekornern klassischer Starkelieferanten.

In recent years biosurfactants have attracted attention because of their low toxicity, biodegradability and ecological acceptability. However, their use is currently extremely limited due to their high cost in relation to that of chemical surfactants. Solid-state cultivation represents an alternative technology for biosurfactant production that can bring two important advantages: firstly, it allows the use of inexpensive substrates and, secondly, it avoids the problem of foaming that complicates submerged cultivation processes for biosurfactant production. In this chapter we show that, despite its potential, to date relatively little attention has been given to solid-state cultivation for biosurfactant production. We also note that this cultivation technique brings its own challenges, such as the selection of a bioreactor type that will allow adequate heat removal, of substrates with appropriate physico-chemical properties and of methods for monitoring of the cultivation process and recovering the biosurfactants from the fermented solid. With suitable efforts in research, solid-state cultivation can be used for large-scale production of biosurfactants.

LipC12, a true lipase from family I.1 of bacterial lipases which was previously isolated through a metagenomics approach, contains 293 amino acids. Among lipases of known three-dimensional structure, it has a sequence identity of 47% to the lipase fromPseudomonas aeruginosaPAO1. Recombinant N-terminally His6-tagged LipC12 protein was expressed inEscherichia coli, purified in a homogenous form and crystallized in several conditions, with the best crystals being obtained using 2.0 Msodium formate and 0.1 Mbis-tris propane pH 7.0. X-ray diffraction data were collected to 2.70 Å resolution. The crystals belonged to the tetragonal space groupP4122, with unit-cell parametersa=b= 58.62,c = 192.60 Å.

Background Metagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1) and lipases (triacylglycerol lipases, EC 3.1.1.3). In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant. Results In preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v). Conclusions The combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range of pH values, good thermal stability and stability in water-miscible organic solvents and at high salt concentrations. These characteristics suggest that this lipase has potential to perform well in biocatalytic processes, such as for hydrolysis and synthesis reactions involving long-chain triacylglycerols and fatty acid esters.

Solid-state fermentation is a cultivation technique in which microorganisms are cultivated on moist solid particles, in beds within which there is a continuous gas phase between the particles. This cultivation technique will become of increasing importance in the future, as population growth puts an ever greater pressure on world resources, stimulating the development of biorefineries: the use of solid-state fermentation in many of the biological processing steps in the biorefinery will help to minimize water use. However, in order to fulfill this potential, it will be necessary to have reliable large-scale solid-state fermentation bioreactors and strategies for optimizing their operation. This article evaluates the current state of our knowledge in this area. It presents the various bioreactors that have been used to date, classifying them on the basis of aeration and agitation strategies: trays, packed beds, rotating/stirred drums, and forcefully aerated agitated bioreactors. It gives an overview of the state of the art in the application of engineering principles to the design and operation of these bioreactor types, identifying where further work is necessary.

Rhamnolipid biosurfactants are attracting attention due to their low toxicity, high biodegradability, and good ecological acceptability. However, production in submerged culture is made difficult by severe foaming problems. Solid-state cultivation (SSC) is a promising alternative production method. In the current work, we report the optimization of rhamnolipid production by Pseudomonas aeruginosa UFPEDA 614 on a solid substrate containing sugarcane bagasse and corn bran. The best rhamnolipid production, 45 g/l of impregnating solution used, was obtained with a 50:50 (m/m) mixture of sugarcane bagasse and corn bran supplemented with an impregnating solution containing 6% (v/v) of each of glycerol and soybean oil. This level is comparable with those of previous studies undertaken in solid-state cultivation; the composition of the biosurfactant is similar, but our medium is cheaper. Our work therefore provides a suitable basis for future studies of the development of an SSC-based process for rhamnolipid production.

In recent years biosurfactants have attracted attention because of their low toxicity, biodegradability and ecological acceptability. However, their use is currently extremely limited due to their high cost in relation to that of chemical surfactants. Solid-state cultivation represents an alternative technology for biosurfactant production that can bring two important advantages: firstly, it allows the use of inexpensive substrates and, secondly, it avoids the problem of foaming that complicates submerged cultivation processes for biosurfactant production. In this chapter we show that, despite its potential, to date relatively little attention has been given to solid-state cultivation for biosurfactant production. We also note that this cultivation technique brings its own challenges, such as the selection of a bioreactor type that will allow adequate heat removal, of substrates with appropriate physico-chemical properties and of methods for monitoring of the cultivation process and recovering the biosurfactants from the fermented solid. With suitable efforts in research, solid-state cultivation can be used for large-scale production of biosurfactants.

In recent years biosurfactants have attracted attention because of their low toxicity, biodegradability and ecological acceptability. However, their use is currently extremely limited due to their high cost in relation to that of chemical surfactants. Solid-state cultivation represents an alternative technology for biosurfactant production that can bring two important advantages: firstly, it allows the use of inexpensive substrates and, secondly, it avoids the problem of foaming that complicates submerged cultivation processes for biosurfactant production. In this chapter we show that, despite its potential, to date relatively little attention has been given to solid-state cultivation for biosurfactant production. We also note that this cultivation technique brings its own challenges, such as the selection of a bioreactor type that will allow adequate heat removal, of substrates with appropriate physico-chemical properties and of methods for monitoring of the cultivation process and recovering the biosurfactants from the fermented solid. With suitable efforts in research, solid-state cultivation can be used for large-scale production of biosurfactants.

E-mail Addresses: thais_salum@hotmail.com, villeneuve@cirad.fr, bruno.barea@cirad.fr, ciyama@ufpr.br, lilian.cocco@ufpr.br, davidmitchell@ufpr.br, nkrieger@ufpr.br; International audience; We produced a lipase from Burkholderia cepacia in solid-state fermentation and used it to catalyze the synthesis of biodiesel in a fixed-bed reactor. In the solid-state fermentation step, a 50:50 (by mass) mixture of sugarcane bagasse and sunflower seed meal gave 234 units of pNPP-hydrolyzing activity per gram of dry solids at 96 h. This fermented solid was lyophilized and delipidated, packed into a column and used to catalyze the synthesis of biodiesel through the ethanolysis of soybean oil in a medium free of co-solvents, with the reaction mixture being continuously circulated through the column. The best conversion was 95% after 46 h, which was obtained at 50 C, with an alcohol:oil molar ratio of 3:1, alcohol addition in two steps and the addition of 1% of (m/m) water to the reaction medium. This system has potential to decrease the costs of enzyme-catalyzed transesterification reactions

Solid-state cultivation involves the growth of microorganisms in beds of moist solid particles that have a minimum of free water between the particles. The chapter describes environmentally-related solid-state cultivation processes. For example, some processes use substrates that are residues of agriculture, forestry, or food-processing, thereby reducing the environmental impact of the residue. Other processes do not use residues, but produce products that have environmental applications. Still other processes use environmental-friendly biotransformations that have the potential to replace current industrial processes. Finally, some solid-state cultivation processes can be used to remove pollutants from soil or waste streams. Typically, environmental applications of solid-state cultivation involve large-scale processing of organic solids. The current chapter addresses the design and operation of bioreactors for these processes. It shows how the various bioreactor types can be classified according to the aeration strategy, namely whether the bed of solids is forcefully aerated or not, and according to the agitation strategy, namely the frequency of mixing of the bed of solids. It discusses the current state-of-the-art in optimizing the design and operation of the various bioreactor types, showing how mathematical models that combine microbial growth kinetics and heat and mass transfer phenomena are the most powerful tools that we have available for this task. The chapter concludes by highlighting the necessity to convert current mathematical models into user-friendly computer programs that can guide design and operation decisions for large-scale solid-state cultivation bioreactors.

In recent years, biosurfactants have attracted attention because of their low toxicity, high biodegradability, and good ecological acceptability. However, their production in submerged liquid culture is hampered by the severe foaming that occurs. Solid-state cultivation can avoid this problem. In the current work, we optimized the production of a rhamnolipid biosurfactant by Pseudomonas aeruginosa UFPEDA 614, grown on a solid medium impregnated with a solution containing glycerol. During the study, we increased the production of the biosurfactant over tenfold, with levels reaching 172 g of rhamnolipid per kilogram of dry initial substrate after 12 days. On the basis of the volume of impregnating solution added to the solid support, this yield is of the order of 46 g/L, which is comparable with the best results that have been obtained to date in submerged liquid cultivation. Our results suggest that there is a great potential for using solid-state cultivation for the production of rhamnolipids.

We present a general formulation of a model that can be used to analyze reaction profiles in systems in which a single enzyme catalyzes several sequential reactions with the same molecular backbone. The analysis of these so-called “repeated-attack systems” allows estimation of the specificities that the enzyme has for the various intermediate substrates that appear in the reaction mixture, relative to the specificity that it has for the initial substrate. Our analytical method has the important advantage that it is not affected by competitive or uncompetitive inhibition, nor by denaturation of the enzyme during the reaction. We carry out case studies in three different systems, the lipase-catalyzed alcoholysis of triacylglycerols, the phytase-catalyzed removal of phosphate groups from phytic acid and the β-amylase-catalyzed removal of maltose units from maltoheptaose. Our model fits well to all reaction profiles in which the phenomenon of processivity does not occur. It can therefore be used as a general tool for characterizing the relative specificities of “repeated-attack enzymes”.

This chapter addresses the design and operation of rotating-drum bioreactors and those stirred-drum bioreactors in which the air is blown into the headspace and not forcefully through the substrate bed itself. This type of bioreactor might be chosen for continuous processes, which will be discussed in Chap. 11. It can also be used for batch processes, which will be the focus of this chapter. Note that there are several bioreactors that are very similar in appearance to rotating-and stirreddrum bioreactors in which air is introduced directly into the bed. This forced aeration would tend to place them in the Group IVa bioreactors considered in Chap. 9 (continuously-agitated, forcefully-aerated bioreactors), however, whether the bioreactor performs more closely to this type of bioreactor or to a rotating-drum or stirred-drum bioreactor depends on the effectiveness of this forced aeration.

The layer of substrate in trays is limited to a bed height of around 5 cm by considerations of heat and O2 transfer within the bed. Therefore scale-up of the process cannot be achieved by increasing the bed height. The only manner to scale up a tray process to large scale is to increase the surface area of the trays, which is equivalent to saying that the large-scale process must use a large number of trays of the same size as those in which the laboratory studies were done. The use of large numbers of trays implies the necessity either for manual handling or highly sophisticated robotic systems, both of which can be inordinately expensive. However, in regions in which manual labor costs are low, such tray-type processes may find applications.

This chapter has shown how it is possible to plan experiments in order to determine the kinetic profile and, in the case that the biomass is estimated on the basis of measurements of a biomass component, how these measurements can be processed in order to obtain an estimated biomass profile. The next chapter shows how the kinetic equation determined from this profile can be written in differential form within a kinetic model of growth.

This chapter has shown how the basic empirical kinetic equation is written, and how the parameters of the equation can be written as functions of the key environmental variables. The next chapter extends the discussion to how we can model the effects that growth has on the environment of the organism.