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Citrus pulp is a waste product of orange juice processing. Pectin can be extracted from this pulp and used in the food industry. However, citrus pulp is produced in such large amounts that the pectin it contains is far in excess of the current world pectin market. An alternative strategy would be to hydrolyze this excess pectin to liberate D-galacturonic acid (D-galA), which can then be converted into several platform chemicals. We report, for the first time, the hydrolysis of pectin by the direct addition of “pectinolytic fermented solids”. These solids were produced by solid-state fermentation of a 30:70 mixture, by dry mass, of ground sugarcane bagasse and orange peels, using a strain of Aspergillus oryzae isolated from rotting passion fruit peels. With the addition of this lyophilized fermented solid to a 10% w v −1 pectin solution, we obtained 247 mmol L −1 of D-galA in the hydrolysate, this being the highest D-galA concentration yet reported. Since the direct addition of fermented solid to the reaction mixture avoids the need for extraction and recovery steps, our process has potential to provide low cost of pectinases for use in citrus-waste biorefineries.

In recent years, metagenomic strategies have been widely used to isolate and identify new enzymes from uncultivable components of microbial communities. Among these enzymes, various lipases have been obtained from metagenomic libraries from different environments and characterized. Although many of these lipases have characteristics that could make them interesting for application in biocatalysis, relatively little work has been done to evaluate their potential to catalyze industrially important reactions. In the present article, we highlight the latest research on lipases obtained through metagenomic tools, focusing on studies of activity and stability and investigations of application in biocatalysis. We also discuss the challenges of metagenomic approaches for the bioprospecting of new lipases.

In this review, we describe our experience in building a pilot-scale packed-bed solid-state fermentation (SSF) bioreactor, with provision for intermittent mixing, and the use of this bioreactor to produce pectinases and lipases by filamentous fungi. We show that, at pilot scale, special attention must be given to several aspects that are not usually problematic when one works with laboratory-scale SSF bioreactors. For example, it can be a challenge to produce large amounts of inoculum if the fungus does not sporulate well. Likewise, at larger scales, the air preparation system needs as much attention as the bioreactor itself. Sampling can also be problematic if one wishes to avoid disrupting the bed structure. In the fermentations carried out in the pilot bioreactor, when the substrate bed contained predominantly wheat bran, the bed shrank away from the walls, providing preferential flow paths for the air and necessitating agitation of the bed. These problems were avoided by using beds with approximately 50% of sugarcane bagasse. We also show how a mathematical model that describes heat and water transfer in the bed can be a useful tool in developing appropriate control schemes. Graphical Abstract.

We review the application of dry fermented solids (DFS) containing naturally immobilized enzymes as catalysts in synthesis and in hydrolysis reactions. The most studied application is the use of DFS containing lipases in the synthesis of biodiesel esters, by transesterification of oils or by esterification of fatty acids with short-chain alcohols in solvent-free reaction media. Other applications of DFS that have been studied include the use of DFS containing pectinases to liberate D-galacturonic acid from pectin and the production of high-value compounds by DFS containing lipases, such as the synthesis of sugar esters and the production of pure enantiomers by resolution of racemic mixtures. To date, studies are limited to proof of concept, and there are still many challenges to be faced in the development of industrial-scale processes using DFS as catalysts. A key challenge is the relatively low activity of DFS compared to commercial enzyme preparations. Attention needs to be given to scale up, not only of the bioreactor for the application of the DFS but also for the production of the fermented solids. There is also a need for economic feasibility studies to determine whether the production of DFS and their use as catalysts can be competitive at industrial scale. Graphical Abstract.

Solid-state cultivation (SSC) involves the cultivation of microorganisms on moist solid particles surrounded by a continuous gas phase. Although SSC has been used for centuries in the production of traditional fermented foods, most biotechnological products are currently produced by submerged culture. However, SSC will become of increasing importance for processing solid residues in biorefineries, especially when filamentous fungi are involved. This chapter presents the basic design and operating principles of the four bioreactor types available for SSC processes: trays, packed beds, rotating (or stirred) drums, and forcefully aerated agitated bioreactors. Control of bed temperature is the main challenge, with the selection of an appropriate bioreactor depending on the specific growth rate of the process organism and also its tolerance of agitation. The bioreactors differ with respect to whether or not the substrate bed is agitated and whether or not the substrate bed is forcefully aerated, offering different combinations of conductive, convective, and evaporative cooling. The best tools for guiding the scale-up of SSC bioreactors are mathematical models that integrate growth kinetics with energy and water balances and which recognize and describe the gradients that occur across the bed during periods of static operation. Due to the complexity of the microscopic-scale phenomena involved in growth of the microorganism on the substrate particles, the kinetic equations used in these models are usually simple empirical equations, such as the logistic equation.

In this review, we describe our experience in building a pilot-scale packed-bed solid-state fermentation (SSF) bioreactor, with provision for intermittent mixing, and the use of this bioreactor to produce pectinases and lipases by filamentous fungi. We show that, at pilot scale, special attention must be given to several aspects that are not usually problematic when one works with laboratory-scale SSF bioreactors. For example, it can be a challenge to produce large amounts of inoculum if the fungus does not sporulate well. Likewise, at larger scales, the air preparation system needs as much attention as the bioreactor itself. Sampling can also be problematic if one wishes to avoid disrupting the bed structure. In the fermentations carried out in the pilot bioreactor, when the substrate bed contained predominantly wheat bran, the bed shrank away from the walls, providing preferential flow paths for the air and necessitating agitation of the bed. These problems were avoided by using beds with approximately 50% of sugarcane bagasse. We also show how a mathematical model that describes heat and water transfer in the bed can be a useful tool in developing appropriate control schemes. Graphical Abstract.

We review the application of dry fermented solids (DFS) containing naturally immobilized enzymes as catalysts in synthesis and in hydrolysis reactions. The most studied application is the use of DFS containing lipases in the synthesis of biodiesel esters, by transesterification of oils or by esterification of fatty acids with short-chain alcohols in solvent-free reaction media. Other applications of DFS that have been studied include the use of DFS containing pectinases to liberate D-galacturonic acid from pectin and the production of high-value compounds by DFS containing lipases, such as the synthesis of sugar esters and the production of pure enantiomers by resolution of racemic mixtures. To date, studies are limited to proof of concept, and there are still many challenges to be faced in the development of industrial-scale processes using DFS as catalysts. A key challenge is the relatively low activity of DFS compared to commercial enzyme preparations. Attention needs to be given to scale up, not only of the bioreactor for the application of the DFS but also for the production of the fermented solids. There is also a need for economic feasibility studies to determine whether the production of DFS and their use as catalysts can be competitive at industrial scale. Graphical Abstract.

The high cost of lipases is a barrier to producing biodiesel through the enzymatic route. Costs can be reduced by producing the lipases by solid-state fermentation and simply drying the fermented solids to produce the biocatalyst. When dry fermented solids (DFS) are used to catalyze the ethyl esterification of fatty acids, they preferentially sorb hydrophilic components of the reaction medium, thereby complicating description of the reaction kinetics. Here, we use a classical equation from heterogeneous catalysis, the two-site Langmuir-Hinshelwood-Hougen-Watson (LHHW) equation, to describe the ethyl esterification of oleic acid catalyzed by DFS, using Aspen Plus®. The parameters were determined using literature data for the ethyl esterification of bidistilled olein in a closed-loop system with recirculation of reaction medium from a well-mixed reservoir through a packed-bed bioreactor containing DFS. The model was validated with data obtained for ethyl esterification in the same bioreactor, but with the following changes: (1) use of fatty acids from soybean soapstock acid oil as the feedstock and (2) use of bidistilled olein as the feedstock, but with a reservoir with a separation barrier, so that only the organic phase is recirculated through the bed. We conclude that the two-site LHHW equation is suitable for modeling the ethyl esterification of fatty acids in techno-economic analyses of biodiesel production catalyzed by DFS that contain lipases.

Processes for the enzymatic hydrolysis of polysaccharides in biorefineries are becoming increasingly important. The complex network of reactions involved in polysaccharide hydrolysis can be described by stochastic models that advance in steps of time. Such models have the potential to be important tools for guiding process design and operation, and several have been developed over the last two decades. We evaluate these models. Many of the current stochastic models for the hydrolysis of colloidal polysaccharides use empirical parameters that have no recognized biological meaning. Only one model uses classical parameters of enzyme kinetics, namely specificity constants and saturation constants. Recent stochastic models for the hydrolysis of insoluble cellulose give valuable insights into the molecular-level phenomenon that limit hydrolysis rates. We conclude that, if stochastic models of enzymatic polysaccharide hydrolysis are to become widely used tools for guiding process development, then further improvements are required.

Although mathematical models of heat and water transfer in packed-bed bioreactors for solid-state fermentation have been available for several decades, there have been few efforts to determine the heat and mass transfer coefficients between the solids and air phases experimentally. In the current work, we undertook drying, cooling and heating experiments in a pilot packed-bed bioreactor, using a solid substrate prepared as for a fermentation, but uninoculated. We then used the solids-air heat and mass transfer coefficients as fitting parameters to adjust a heat and mass transfer model to temperature data obtained at different bed heights. The model fit the data well, but only if the air flow rate was allowed to vary as a function of position, indicating that the air flow in the bed was not uniform. In a sensitivity analysis, the air flow rate had a greater effect on the predicted temperature profiles than did the solids-air heat and mass transfer coefficients themselves. However, despite the lesser effect of these coefficients, it is not possible to treat the solids and air phases as being in thermal and hydric equilibrium. Rather, it is essential to use models that recognize the solids and air as separate phases and use expressions based on driving forces to describe evaporation and convective heat removal. The experimentally determined heat and mass transfer coefficients can be substituted into a mathematical model that also describes microbial growth and metabolic heat production and this model can be used to guide the scale-up from our pilot bioreactor to larger scales.

Model-based strategies for scaling up traditional packed-bed bioreactors used in solid-state fermentation have been available for over two decades. However, these strategies are theoretical, not being based on experimental data. A recently proposed strategy did use experimental data; however, it was based on data from a slow-growing fungus and also considered heat transfer in both the axial and radial directions, ignoring the fact that radial heat transfer is negligible in large-scale packed-bed bioreactors. The current work demonstrates a scale-up strategy based on a model describing heat transfer only in the axial direction. The strategy is demonstrated for the cultivation of Aspergillus niger, a fast-growing filamentous fungus. In the model, the kinetics of production of metabolic heat are based on an experimental time-course profile for the O2 uptake rate. The model is validated by comparing its predictions for temperature profiles in the bed with temperatures measured experimentally in a pilot-scale bioreactor. Finally, the validated model is used to explore strategies for optimizing the performance of large-scale traditional packed-bed bioreactors. We conclude that a key strategy is to decrease the temperature of the inlet air during periods of peak heat generation.

Processes involving the enzymatic hydrolysis of polysaccharides in biorefineries are receiving increased attention. Mathematical models of these processes can guide process design and operation. For complex reaction networks, stochastic models are preferable to deterministic models. To be useful for guiding process design and operation, stochastic models cannot advance in steps of degree of reaction, they must advance in steps of time. Several current stochastic models for the hydrolysis of polysaccharides do advance in steps of time. However, some use parameters that have no recognized biological meaning; in others, the time axis is fitted to experimentally determined profiles. In the current work, we propose stochastic models that advance in time in a manner that is consistent with classical enzyme kinetic theory: the parameters of these models are specificity constants and saturation constants. We present three case studies. (1) We use the hydrolysis of thiophenyl N-acetyl-β-(1,6)-pentaglucosamine by DispersinB to demonstrate that the predictions of the stochastic model are equal to those of a deterministic model based on differential equations with classical kinetic expressions. (2) We model the hydrolysis of a mixture of oligomers of D-galacturonic acid by an endopolygalacturonase, a system for which it is not convenient to use a deterministic model. (3) We extend the method to a system with two enzymes and demonstrate how the model can be used to optimize the ratio of the enzymes. Our approach can lay the basis for the development of a new family of stochastic models describing the hydrolysis of colloidal suspensions of polysaccharides.

Lipases are widely used as biocatalysts in the resolution of racemates, where high chemo-, regio- and enantioselectivity are required. In this work, we produced lipases by culturing Rhizopus microsporus in solid-state fermentation. After the fermentation, the solids were dried, producing “Rhizopus microsporus fermented solids” (RMFS). The lipases in RMFS had high activity in the synthesis of ethyl oleate in n-heptane, retaining 100 % activity after 23 successive esterification cycles, each for 4 h at 40 °C. In the kinetic resolution of three aromatic secondary alcohols by transesterification with isopropenyl acetate, in n-heptane, RMFS showed preference for the (S)-enantiomers, in other words, an anti-Kazlauskas enantiopreference. The best results were a conversion of 23 % and an enantiomeric ratio (ESR, i.e. S relative to R) of 26 for resolution of (RS)-1-phenyl-1-ethanol, after 96 h at 40 °C. Since it is rare for lipases to have an (S)-enantiopreference, RMFS have the potential as low-cost biocatalysts for the resolution of racemates of secondary alcohols.

Ibead-CELipC12 is a metagenomic lipase, LipC12, immobilized directly from a crude extract onto Immobead 150. We evaluated its solvent and thermal stability and its potential for producing flavor esters and structured lipids. Ibead-CELipC12 had residual activities above 88% after 24 h at 40 °C in toluene, iso-octane, n-hexane and n-heptane and 72% after 24 h at 60 °C in n-hexane. In esterification of palmitic acid, Ibead-CELipC12 gave higher activities with ethanol, propanol and butanol than with isopropanol. In esterification of caprylic acid with ethanol, propanol and butanol, Ibead-CELipC12 performed best with butanol, giving 62% conversion in 6 h. Ibead-CELipC12 was sn-1,3-specific during triolein hydrolysis and was therefore used to convert olive oil into an MLM structured lipid containing 23% of caprylic acid in the sn-1 and sn-3 positions. These results demonstrate that Ibead-CELipC12 has good potential to be used in the enzymatic synthesis of flavor esters and structured lipids.

Among all hyphal types formed on substrate particles during solid-state fermentation, penetrative hyphae are the least studied. Important questions regarding penetrative hyphae remain to be answered: for example, what determines the final length of a penetrative hypha and why do some fungi produce two types of penetrative hyphae, long and short penetrative hyphae? In this work, we develop a discrete model to describe the biomass distribution of penetrative hyphae as a function of depth inside solid particles and apply this model to Rhizopus oligosporus and Aspergillus oryzae, growing in different media. We use our model to suggest different modes of growth and roles for long and short penetrative hyphae: the long ones, also called scouting hyphae, explore the composition of the particle and search for new colonization sites across the particle, while the short hyphae, also called vegetative hyphae, act as anchors for the colony and as enzyme producers. The same factors, namely the firmness of the solid particle and the pressure drop inside the hypha, may determine the final hyphal length of both hyphae. Scouting hyphae may be longer due to a lower pressure drop along the hypha. These hypotheses regarding growth of penetrative hyphae now require experimental investigation.

We present three case studies, based on literature data, in which the “fingerprinting” method of determining the relative specificities of an enzyme is applied to branched reaction schemes. The first case study involves the hydrolysis of maltoheptaose by a β-amylase and shows that the fingerprinting method can be applied to schemes involving processivity. The analysis reveals that the native β-amylase has a 1.26-fold preference for attacking maltoheptaose by the processive route over the non-processive route, but that for a mutant enzyme this preference is 0.18-fold. The second case study involves the hydrolysis of β-1,6-N-acetylglucosamine oligomers by DispersinB. Our set of relative specificity constants is more consistent with the results of initial rate experiments than is the set that the authors obtained by fitting a pseudo-first order model to their data. The third case study involves the hydrolysis of galacturonic acid oligomers by an endopolygalacturonase. This enzyme can catalyze a total of 11 different reactions with a mixture of tri-, tetra-, penta-, hexa- and heptagalacturonates. We determined the relative specificity constants for these 11 reactions. The fingerprinting method has advantages over the methods that have been previously used to determine specificity constants for branched reaction schemes, being able to use a single experimental reaction profile for determination of all relative specificity constants.

Pectinases can be used in citrus waste biorefineries to hydrolyze the pectin in citrus pulp to produce d -galacturonic acid, a potential platform chemical. Solid-state fermentation has the potential to produce low-cost pectinases for such biorefineries, but it is difficult to control the process at large scales. In the current work, Aspergillus oryzae was cultivated in a pilot-scale packed-bed bioreactor, on 15 kg of a substrate containing 51.6% citrus pulp and 48.4% sugarcane bagasse (w/w, dry basis). The sugarcane bagasse gave a high bed porosity and ensured a stable bed structure, avoiding problems of bed shrinkage and the formation of compact agglomerates within the bed. As a result, bed temperatures were controlled to within 1 °C of the inlet air temperature and pectinase yields of 33–41 U g −1 were obtained across the bed. When the fermented solids were dried and added directly to a pectin solution, they gave a profile for the release of d -galacturonic acid similar to that obtained with a commercial pectinase. These results show the potential for using solid-state fermentation to produce pectinases in a citrus waste biorefinery, with subsequent direct addition of the fermented solids to produce d -galacturonic acid from the pectin contained in the citrus pulp.

Bioprospection for new enantioselective enzymes for application in organic synthesis is a prominent area of investigation in biocatalysis. In this context, here we present the evaluation of an immobilized lipase isolated from a metagenomic library (LipG9) for the enzymatic kinetic resolution (EKR) of aliphatic sec -alcohols, which are still challenging substrates, since low enantioselectivity values are usually observed for these resolutions. LipG9 was successfully employed in EKR of aliphatic alcohols, which were resolved with satisfactory conversions (19–59%) and enantiomeric excesses for alcohols (26–88%) and esters (30–96%) by transesterification reactions, demonstrating that its performance is equal to or better than commercially available enzymes for the same reaction.

Solid-state fermentation can be used to produce pectinases using agro-industrial byproducts. However, heat and mass transfer limitations make it difficult to control the temperature within the bioreactor, especially at large scale, so reliable scale-up strategies are essential. In the current work, we scaled up the production of pectinases in packed-bed bioreactors, from 12 g to 30 kg of dry substrate, the biggest scale yet reported for pectinase production. When compaction occurred, bed temperatures up to 47 °C were recorded and the pectinase activity in different regions of the bed at 26 h varied from 11 to 28 × 10 3 U kg −1 . When compaction was avoided, the maximum bed temperature was 32 °C and the pectinase activity at 26 h varied from 17 to 20 × 10 3 U kg −1 . The best result was obtained with a 40-cm high bed containing 27 kg of wheat bran and 3 kg of sugarcane bagasse, with switching of the temperature of the saturated inlet air between 24 °C and 32 °C. Under these conditions, the maximum productivity was 1840 U kg −1 h −1 at 10 h. We propose that the process can be scaled up to production scale by maintaining the same bed height and operational strategy, while increasing the width of the bed to several meters. If the superficial velocity of the air is maintained constant at 0.1 m s −1 , then the performance at scales involving several tonnes of solid substrate should be similar to that obtained in pilot-scale bioreactor in the current study.

Fatty acid ethyl esters (FAEEs) were produced from waste cooking oil (WCO) using the immobilized Novozym 435 (Novozymes) lipase in a solvent-free system. WCO contained high molar mass degradation compounds that did not affect the lipase activity but remained as a minor component of the resulting FAEEs. The enzyme-catalyzed FAEE production was investigated using a central composite design. The best experimental conditions were obtained with an enzyme loading of 5 wt % and an ethanol/oil molar ratio of 6:1. Under these conditions and at the optimal enzyme temperature of 40 °C, FAEE mixtures with a total ester content of 77.2 and 89.5 wt % were produced after 10 and 24 h, respectively. Treatment of the spent biocatalyst with tert-butanol could restore the transesterification activity of the biocatalyst to its original value.

Burkholderia contaminans LTEB11 is a Gram-negative betaproteobacterium isolated as a contaminant of a culture in mineral medium supplemented with vegetable oil. Here, we report the genome sequence of B. contaminans LTEB11, identifying and analyzing the genes involved in its lipolytic machinery and in the production of other biotechnological products. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00076-4) contains supplementary material, which is available to authorized users.

Genes encoding lipase LipBC (lipA) and foldase LifBC (lipB) were identified in the genome of Burkholderia contaminans LTEB11. Analysis of the predicted amino acid sequence of lipA showed its high identity with lipases from Pseudomonas luteola (91%), Burkholderia cepacia (96%) and Burkholderia lata (97%), and classified LipBC lipase in the lipase subfamily I.2. The genes lipA and lipB were amplified and cloned into expression vectors pET28a(+) and pT7-7, respectively. His-tagged LipBC and native LifBC were co-expressed in Escherichia coli and purified. LipBC and LifBC have molecular weights of 35.9 kDa and 37 kDa, respectively, and remain complexed after purification. The Lip-LifBC complex was active and stable over a wide range of pH values (6.5–10) and temperatures (25–45 °C), with the highest specific activity (1426 U mg−1) being against tributyrin. The Lip-LifBC complex immobilized on Sepabeads was able to catalyze the synthesis of ethyl-oleate in n‑hexane with an activity of 4 U g−1, maintaining high conversion (>80%) over 5 reaction cycles of 6 h at 45 °C. The results obtained in this work provide a basis for the development of applications of recombinant LipBC in biocatalysis.

A fermented solid containing lipases was produced by growing Burkholderia cepacia LTEB11 on a 1:1 mixture, by mass, of sugarcane bagasse and sunflower seed meal. This fermented solid was dried and then used in a packed-bed bioreactor to catalyze the esterification of fatty acids from soybean soapstock acid oil with ethanol in a solvent-free system. The bioreactor was operated in batch mode, with the medium being recirculated from the reservoir through the bed in a closed-loop system. During the reaction, the bulk reaction medium separated into organic and aqueous phases. Additionally, up to 30% of the reaction medium was held as a sorbed phase on the fermented solids. The composition of this sorbed phase, which represents the microenvironment of the lipases, was significantly different from the compositions of the bulk organic and aqueous phases. It was predominantly polar, with water or ethanol as the major component, but also contained significant amounts of ester. Importantly, the molar ratios of ethanol to fatty acid in the sorbed phase were significantly higher than those in the bulk reaction medium; this has implications for the reaction, since ethanol is known to cause inhibition and denaturation of lipases.

We immobilized a new metagenomic lipase, LipC12, and studied its application in the synthesis of ethyl-oleate, a model ester for biodiesel studies. Initially, we purified His-tagged LipC12 on a nickel column and immobilized it by adsorption on Accurel MP-1000 and by covalent bonding on Immobead 150. Preparations obtained with 10 mg of protein per gram of support were compared with respect to the synthesis of ethyl-oleate in n -hexane, using an ethanol to oleic acid molar ratio of 3:1. LipC12 gave a better conversion of oleic acid when immobilized on Immobead 150 (95% in 4 h) than when immobilized on Accurel MP-1000 (80% in 6 h). This led us to investigate the immobilization of His-tagged LipC12 directly from the crude extract on Immobead 150. At a protein loading of 200 mg g −1 , the immobilized preparation (“Ibead-CELipC12”) gave a conversion of oleic acid of 99% in 60 min, for reactions performed in n -hexane, again using an ethanol to oleic acid molar ratio of 3:1. We obtained conversions of over 90% in ten successive 60-min reactions using the same batch of immobilized enzyme. When Ibead-CELipC12 was used to catalyze the esterification of oleic acid with ethanol in a solvent-free system at an ethanol to oleic acid molar ratio of 1:1, all activity was lost within 12 h, with a conversion of oleic acid of around 30%. However, when the reaction was repeated with the addition of ethanol in six equal aliquots during the course of the reaction, a conversion of oleic acid of 85% in 48 h was achieved. These results demonstrate that LipC12 has good potential to be used in the enzymatic synthesis of biodiesel. The results are particularly encouraging because it was possible to immobilize His-tagged LipC12 directly from the crude extract, thereby avoiding the need to purify the enzyme prior to immobilization.

We obtained liquid–liquid equilibrium (LLE) data for the systems involved in biodiesel production from a low-value feedstock, namely fatty acids from soybean soapstock acid oil (FA-SSAO), at 303.2 and 323.2 K. The systems studied were water + ethanol + FA-SSAO and water + ethanol + fatty acid ethyl esters (EE-SSAO). The mass fractions of the compounds in equilibrium in the two phases were determined analytically, whereas the binodal curves were obtained by the cloud-point method. The increase in temperature from 303.2 and 323.2 K slightly increased the miscibility of the EE-SSAO system. We found that ethanol is more soluble in water than in either FA-SSAO or EE-SSAO, and that an increase in ethanol content in either the FA-SSAO-rich phase or the EE-SSAO-rich phase enhances water solubility in these phases. The experimental data were satisfactorily modeled by the NRTL and UNIQUAC approaches.

A fermented solid containing lipases was produced by solid-state fermentation of Rhizopus microsporus on sugarcane bagasse enriched with urea, soybean oil, and a mineral solution. The dry fermented solid produced using R. microsporus (RMFS) was used to catalyze the synthesis of alkyl-esters by esterification in a solvent-free system containing ethanol and oleic acid (as a model system) or a mixture of fatty acids obtained from the physical hydrolysis of soybean soapstock acid oil (FA-SSAO) in subcritical water. The conversions were 93.5 and 84.1%, for oleic acid and FA-SSAO, respectively, at 48 h and 40 °C, at a molar ratio (MR) of ethanol to fatty acid of 5:1. A further increase in the MR to 10:1 improved the production of ethylic-esters, giving conversions at 48 h of 98 and 86% for oleic acid and FA-SSAO, respectively. The results obtained in this work foster further studies on scaling-up of an environmentally friendly process to produce biofuels.

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candida rugosa lipase, metagenomic lipase, β-galactosidase and β-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

Biocatalysts with lipase activity were produced by Penicillium corylophilum under basal and olive oil-induced conditions. In the presence of olive oil, both the extracellular and the mycelium-bound lipase activities were increased significantly. The four biocatalysts maintained a residual lipase activity after incubation in a wide pH range showing high stability in a defined acidic to neutral pH range. Some differences between biocatalyst residual activities were also observed after incubation at various temperatures or in the presence of organic solvents such as methanol, ethanol, acetone, butanol, hexanol, n-hexane, and heptane. Enzymes which are stable in the presence of ethanol or butanol could be useful in ecofriendly processes for biodiesel production. Fil: Romero, Cintia Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Baigori, Mario Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Baron, Alessandra. Universidade Federal do Paraná; Brasil Fil: Krieger, N.. Universidade Federal do Paraná; Brasil Fil: Pera, Licia Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina

Burkholderia cepacia LTBE11 lipase was used in the kinetic resolution of four secondary allylic alcohols by transesterification reactions with vinyl acetate: rac-1 [1-phenylprop-2-en-1-ol], rac-2 [1-(4-chlorophenyl)prop-2-en-1-ol], rac-3 [1-(3-methoxyphenyl)prop-2-en-1-ol] and rac-4 [5-methylhex-1-en-3-ol]. Greater enantioselectivities, expressed as the enantiomeric ratio (ERS or ESR, as appropriate), were obtained with the addition of the lipase immobilized on a polypropylene support (Accurel) than with the addition of a powdered free enzyme preparation. The fast-reacting enantiomer was the R-enantiomer for the alcohols with aromatic substituents (rac-1, rac-2 and rac-3) and the S-enantiomer for the alcohol with an aliphatic substituent (rac-4). The ERS values obtained for rac-1, rac-2 and rac-3 were greater than the ESR value obtained for rac-4. With the increasing temperature, ERS decreased for rac-2 and rac-3, while ESR increased for rac-4. Thermodynamic analysis of the difference in activation energies based on the temperature data showed that ΔR−SΔH# and ΔR−SΔS# were negative in all cases. For rac-2 and rac-3, the selectivity for the R-enantiomer was due to a predominance of enthalpic effects (i.e. ∣ΔR−SΔH#∣>∣TΔR−SΔS#∣). For rac-4, the selectivity for the S-enantiomer was due to a predominance of entropic effects (i.e. ∣TΔR−SΔS#∣>∣ΔR−SΔH#∣). The resolution of rac-2 was investigated in different solvents. ERS enhanced with the increasing values of log P and molecular volume of the solvent but decreased with the increasing dielectric constant. The highest ERS value, 110, was obtained in n-heptane. This elevated enantioselectivity suggests that the lipase of B. cepacia LTBE11 has good potential for the application in the resolution of secondary allylic alcohols.

We demonstrate, for the first time, the lipase-catalyzed ethanolysis of castor oil in a solvent-free system. The lipase used was that of Burkholderia cepacia LTEB11, immobilized on Accurel EP 100. With a loading of 4.5% (mass of enzyme preparation in relation to the mass of triacylglycerols in the castor oil) and an ethanol to oil molar ratio of 3:1, 90% conversion of fatty acids to their corresponding ethyl esters was achieved in 6 h. These results are comparable to results reported in the literature for the lipase-catalyzed ethanolysis of castor oil in supercritical CO2 and in systems containing n-hexane as the solvent. Our system has the advantage of being simpler to operate.

Layered double hydroxides (LDHs) are cheap materials suitable for immobilization of enzymes. In this study, we prepared Zn/Al-Cl LDHs with different Zn:Al molar ratios for immobilization of the lipase from Pseudomonas cepacia. The best values for activity retention (188%), immobilization efficiency (96%) and hydrolytic activity in organic medium (279 U g−1) were obtained with a molar ratio of Zn:Al of 4:1, a protein loading of 162 mg g−1 and Tris-HCl buffer (10 mmol L−1, pH 7.5) as the solvent for preparing the lipase solution. The immobilized lipase keeps its activity when stored at 4 °C during 30 days. The immobilized lipase gave a conversion of 50% in 1 h for the kinetic resolution of the alcohol rac-1-phenylethanol, with both ees and eep higher than 99% and E higher than 200. In the reutilization study, 30 successive 1-h kinetic resolutions were done with the same batch of immobilized enzyme. For all 30 resolutions, 50% conversion was maintained, with ees and eep higher than 99% and E higher than 200. These are promising results that lay the basis for further studies of immobilization of lipases onto LDHs for applications in organic media.

Enzymes are the main biocatalysts of biological systems and nowadays they play an important role in asymmetric organic synthesis. Microorganisms are the main source for enzymes, however, just a very small portion of them are culturable at lab conditions and, as an alternative, metagenomics approaches allow new enzymes to be accessed from so-called “non-culturable” microorganisms. Several classes of metagenomic enzymes have been described in literature. Nevertheless, studies about their potential for asymmetric biotransformation are underexploited. Therefore, we present our recent efforts to establish the substrate-scope of LipG9, a metagenomic lipase, in enzymatic kinetic resolution (EKR) of chiral substances. LipG9 was previously isolated, immobilized and successfully applied in EKR of aliphatic alcohols. In this study, a series of resolvable chiral substances were assayed with LipG9, and secondary benzyl alcohols/esters were preferentially resolved in a much superior enantioselectivity (E > 200) than those described for aliphatic alcohols (E from 4 to 63). In an opposite way, Im-LipG9 did not present activity for tertiary alcohols, amines and lactones. When compared to commercial lipases, Im-LipG9 enantioselectivity was superior to Candida rugosa lipase and equivalent to Candida antarctica lipase B. Thus, the chemo and enantioselectivity of LipG9 in EKR reactions were identified and its potential for asymmetric synthetic approaches was demonstrated.

We investigated a new hydroesterification strategy for the production of biodiesel from low-value oil feedstocks: complete hydrolysis of the feedstock to fatty acids in subcritical water, followed by the use of a packed-bed reactor, containing a fermented solid with lipase activity, to convert the fatty acids to their ethyl esters. The fermented solids were produced by cultivating Burkholderia cepacia LTEB11 for 72 h on a 1:1 mixture, by mass, of sugarcane bagasse and sunflower seed meal. The esterification of fatty acids obtained from soybean soapstock acid oil was studied in the packed-bed bioreactor, in a solvent-free system, with the best results being a 92% conversion in 31 h, obtained at 50 °C. When the packed-bed reactor was reused in successive 48-h esterification reactions, conversions of over 84% of the fatty acids to esters were maintained for five cycles at 50 °C and for six cycles at 45 °C. Unlike previous hydroesterification processes that have used lipase-catalyzed hydrolysis followed by chemically-catalyzed esterification, our process does not expose the lipases to contaminants present in low quality feedstocks such as soapstocks. This advantage opens the possibility of operating the packed-bed esterification reactor in continuous mode.

Background Pectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. One possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. However, the ethanol-producing organism of choice, Saccharomyces cerevisiae, is not able to catabolize d-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, namely, 18 % (w/w) from citrus pulp and 16 % (w/w) sugar beet pulp. Results In the current work, we describe the construction of a strain of S. cerevisiae in which the five genes of the fungal reductive pathway for d-galacturonic acid catabolism were integrated into the yeast chromosomes: gaaA, gaaC and gaaD from Aspergillus niger and lgd1 from Trichoderma reesei, and the recently described d-galacturonic acid transporter protein, gat1, from Neurospora crassa. This strain metabolized d-galacturonic acid in a medium containing d-fructose as co-substrate. Conclusion This work is the first demonstration of the expression of a functional heterologous pathway for d-galacturonic acid catabolism in Saccharomyces cerevisiae. It is a preliminary step for engineering a yeast strain for the fermentation of pectin-rich substrates to ethanol. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0544-1) contains supplementary material, which is available to authorized users.

Enzymatic processes to produce interesterified fats and oils with desirable properties to incorporate in margarines and shortenings are currently quite expensive. In the current work, fermented solids, produced by growing Rhizopus oryzae and Rhizopus microsporus on a mixture of sugarcane bagasse and sunflower seed meal, were able to catalyze the interesterification of a mixture of palm stearin, palm kernel oil and a concentrate of triacylglycerols enriched with ω-3 polyunsaturated fatty acids (“EPAX 4510TG”). The best conditions for the interesterification reaction with the fermented solid produced by R. oryzae, found by response surface methodology, were a temperature of 65 °C, a palm stearin content of 38% and an EPAX content of 15%. Under these conditions, after 24 h, the product had a solid fat content at 35 °C (SFC35 °C) of 2.3%, making it suitable for the production of margarines and shortenings. If the reaction time can be decreased, this process has good potential to lower the costs of the enzymatic interesterification process, since the fermented solids are reasonably cheap to produce and their use avoids the recovery and immobilization steps that are required for lipases produced by submerged fermentation.

In solid-state fermentation systems, the growth of aerial hyphae into the interparticle spaces increases the pressure drop through forcefully aerated beds. Aerial hyphae can also bind particles into agglomerates, restricting the transfer of O2 to the particle surfaces. Despite these important effects, to date relatively little effort has been made to understand the growth patterns of these hyphae. In the current work we present a discrete lattice-based model that can be used to simulate the growth of the aerial hyphae of filamentous fungi. In the model, the elongation of hyphae involves the successive addition of 10 μm cubes, with random numbers being used to choose the direction of growth. The model was able to describe profiles available in the literature for the density of the aerial hyphae, as a function of height above the surface, for a situation in which the filamentous fungus Rhizopus oligosporus was grown on potato dextrose agar. The model can be modified to describe various different situations involving the growth of filamentous fungi in solid-state fermentation systems, such as the growth of penetrative hyphae and the growth of hyphae within the wet mycelial layer that often forms at the surfaces of particles. It therefore represents a useful tool for investigating phenomena that occur at the micro-scale in solid-state fermentation systems.

We describe a method that we have called SPIL, the ‘simultaneous production and immobilization of lipases’. In this method the lipase of Burkholderia cepacia LTEB11 was immobilized by addition of Accurel EP 100 to the growth medium during culture. A factorially designed experiment was undertaken to optimize the activity adsorbed on the support, with variables being the time of addition of Accurel (0–24 h), the concentration of Accurel (10–30 g L−1) and the concentration of olive oil (0.25–1.0%, v/v). The highest activity loading, of 2600 units of p-nitrophenyl palmitate-hydrolyzing activity per gram of support, was obtained by the addition of 10 g Accurel L−1 at 12 h to a medium containing 1% (v/v) olive oil. This preparation gave threefold higher rates for the esterification of ethyl oleate in n-heptane than did a preparation obtained through the conventional practice of adding Accurel at the end of the fermentation, with a conversion of 95% being attained in 1 h.

We present a new mathematical method for determining the enantiomeric ratio (E) during lipase-catalyzed kinetic resolutions. The method involves the fitting of a model to profiles of adimensionalized concentrations of the two enantiomers of the chiral substrate, plotted against degree of conversion. The model equations are presented for a reversible reaction involving bi-bi ping-pong kinetics in which the chiral substrate enters second and the chiral product leaves second. However, it is also shown that the method is easy to modify for analysis of resolutions involving other chiral substrate-product pairs and of resolutions in which the behavior of the system can be approximated by irreversible uni-uni kinetics. We show that our method retains several advantageous features of existing methods that help to ensure accuracy.

We use a mathematical model, based on the N-tanks-in-series approach, to evaluate the potential advantages that can be obtained in solid-state fermentation processes by operating packed-bed bioreactors as multi-layer beds. We explore classical operation, in which air is blown unidirectionally through a substrate bed that remains immobile, with two strategies that involve movement of the layers. The first strategy involves batch operation, in which the positions of the layers are changed at 1 h intervals, in a cycling motion. The second strategy involves continuous plug-flow of the layers, with the regular addition of new layers at the air outlet and removal of spent layers at the air inlet. Under the conditions of the simulation, the rate of metabolic heat generation during the steady state of the continuous plug-flow process is only 60% of the peak value predicted for classical operation. As a result, the maximum bed temperature in the continuous plug-flow process is 4.5 °C lower than that predicted for classical operation. We conclude that the operation of multi-layer packed beds in the continuous plug-flow mode can improve bioreactor performance significantly.

Nine isolates of Botryosphaeria spp. were screened for lipases when cultivated on eight different plant seed oils and glycerol, and all produced lipases. Botryosphaeria ribis EC-01 produced highest lipase titres on soybean oil and glycerol, while eight isolates of Botryosphaeria rhodina produced significantly lower enzyme titres. B. ribis EC-01 produced lipase when grown on different fatty acids, surfactants, carbohydrates and triacylglycerols, with highest enzyme titres produced on Triton X-100-emulsified stearic (316.7 U/mL), palmitic (283.5 U/mL) and oleic (247.4 U/mg) acids, and soybean oil (105.6 U/mL), as well as castor oil (191.2 U/mg); an enhancement of 9-fold over soybean oil-grown cultures. Glycerol was also a good substrate for lipase production. The crude lipase extract was optimally active at pH 8.0 and 55 °C, stable between 30 and 55 °C and pH 1–10, and tolerant to 50% (v/v) glycerol, methanol and ethanol. The crude lipase showed affinity for substrates of short, average and long-chain fatty acids (different esters of p -nitrophenol and triacylglycerols). Zymograms developed with 4-methylumbelliferyl-butyrate showed two bands of lipolytic activity at 45 and 15 kDa. This is the first report on the production of lipases by B. ribis grown on these different carbon sources.

Pseudomonas aeruginosa UFPEDA 614 produced a rhamnolipid biosurfactant when grown on sugarcane bagasse impregnated with a solution containing glycerol. Biosurfactant levels reached 40 g of rhamnolipid per kilogram of dry initial substrate after 12 days. On the basis of the volume of liquid used, the biosurfactant levels were similar to those obtained in submerged liquid culture of a medium identical to the impregnating solution. The properties of the biosurfactant were very similar to those obtained with rhamnolipids produced in submerged culture, with a critical micelle concentration of 46.8 mg/L and an emulsification index at 24 h of over 90% against gasoline. The surface properties were maintained after autoclaving of the fermented solids, meaning that it is possible to minimize safety risks by killing the producing organism with a heat treatment of the solids prior to product extraction. The biosurfactant was used in the washing of soils contaminated with gasoline. An aqueous biosurfactant solution was 3.2-fold more efficient than water in leaching organic material from the soil, demonstrating the viability of application of rhamnolipids in the bioremediation of soils contaminated with gasoline.

Crystals of Mg/Al layered double hydroxide were synthesized by alkaline precipitation and treated in an aqueous solution of glutamic acid. The glutamate ions were not intercalated into the interlayer space, but were detected in the material by Fourier transform infrared spectroscopy, suggesting that only the external surfaces of crystals were modified with glutamate ions. The resulting hybrid material was tested as a support for immobilization of the enzyme laccase (Myceliophthora thermophila). The immobilized enzyme preparation was characterized by electronic paramagnetic resonance spectroscopy and by assays of catalytic activity. The activity of the immobilized laccase was 97% of the activity in the free enzyme. Layered double hydroxide is a suitable support for use in remediation of soil studies.

We propose a model for the sequential hydrolysis of ester bonds of triacylglycerols by lipases and use it as the basis for an analytical method for determining the relative specificity of the lipase for the various substrates with which it can react, when the substrates occur simultaneously in a single reaction system. We then apply the method to our own data and literature data involving the hydrolysis of triacylglycerols by lipases. Our model is able to fit well to most of the reaction profiles, enabling the estimation of relative specificities. We discuss the limitations and potential applications of our method.

The synthesis of ethyl-oleate by the lipase from the newly isolated strain Burkholderia cepacia LTEB11 in three different systems has been studied – immobilization on a hydrophobic support (Accurel EP 100®), encapsulation in reverse micelles, and direct addition of powdered free enzyme to the reaction medium. The immobilized enzyme performed best, giving a 70% ester yield in 10 h, this yield being five-fold greater than that obtained for reversed micelles, and two and a half times greater than that obtained for direct addition. An increase in the amount of immobilized enzyme preparation added gave a 100% ester yield in 3 h. The immobilized preparation was quite stable, giving a 100% yield of ethyl-oleate during 11 repeated reactions, and 50% yield after 24 reactions. These results suggest that the lipase of our strain of B. cepacia LTEB11 immobilized on Accurel has good potential for application in biocatalysis in organic media.

Pseudomonas aeruginosa DAUPE 614 produced rhamnolipids (3.9gL(-1)) when cultivated on a medium containing glycerol and ammonium nitrate. These rhamnolipids reduced the surface tension of water to 27.3mNm(-1), with a critical micelle concentration of 13.9mgL(-1). The maximum emulsification index against toluene was 86.4%. The structure of the carbohydrate moiety of the glycolipid was determined by gas chromatography-mass spectroscopy (GC-MS) analysis allied to electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) 1D, 2D (13)C, (1)H spectroscopy. The hydroxyl fatty acids were analyzed by GC-MS as hydroxy-acetylated fatty acid methyl ester derivatives. The positions of the fatty acids in the lipid moiety were variable, with 6 mono-rhamnolipid homologues (Rha-C(10)-C(10); Rha-C(10)-C(8); Rha-C(8)-C(10); Rha-C(10)-C(12:1); Rha-C(12)-C(10); Rha-C(10)-C(12)) and 6 di-rhamnolipid homologues (Rha(2)-C(10)-C(10); Rha(2)-C(10)-C(8); Rha(2)-C(8)-C(10); Rha(2)-C(10)-C(12:1); Rha(2)-C(12)-C(10); Rha(2)-C(10)-C(12)). The ratio of Rha(2)-C(10)-C(10) to Rha-C(10)-C(10) was higher than has been reported in previous studies. Our methodology allowed us to distinguish between the isomeric pairs Rha-C(10)-C(8)/Rha-C(8)-C(10), Rha-C(10)-C(12)/Rha-C(12)-C(10), Rha(2)-C(10)-C(8)/Rha(2)-C(8)-C(10) and Rha(2)-C(12)-C(10)/Rha(2)-C(10)-C(12). For each isomeric pair, the congener with the shorter chain adjacent to the sugar was always more abundant than the congener with longer chain.

We report for the first time both the production of the lipase of Burkholderia cepacia in solid-state fermentation and the biocatalysis of esterification and transesterification reactions through the direct addition of the lyophilised fermented solids to organic reaction media. B. cepacia produced a lipolytic activity equivalent to 108 U of p NPP-hydrolysing activity per gram of dry solids after 72 h growth on corn bran with 5% (v/w) commercial corn oil as the inducer. The fermented solid material was lyophilised and added directly to the reaction medium in esterification and transesterification reactions. A factorial design was used to study the effects on esterification of temperature, alcohol-to-acid molar ratio and amount of lipolytic activity added. All three variables affected the ester yield significantly, with the amount of enzyme being most important. A 94% ester yield was obtained at 18 h at 37 °C, with an alcohol-to-acid molar ratio of 5:1 and 60 U of added lipolytic activity. For the transesterification reaction, a factorial design was undertaken with the variables being the alcohol-to-acid molar ratio and the added lipolytic activity. Ester yields of over 95% were obtained after 120 h. Our results suggest that biocatalysis using direct addition of fermented solids to organic reaction medium should be further explored.