Cell growth and Trametes versicolor laccase production in transformed Pichia pastoris cultured by solid‐state or submerged fermentations

BACKGROUND: Growth kinetics of Pichia pastoris and heterologous expression of Trametes versicolor laccase were compared. This is the first study of its kind between solid‐state yeast cultures done on polyurethane foam (PUF) and submerged liquid fermentations (SmF). RESULTS: The maximum values of biomass were similar for SSF (solid‐state fermentation) and SmF experiments when the BOD (biochemical oxygen demand) was lower than 100 g L^?1. For higher BOD levels, the maximum values of biomass were 55 g L^?1 (SSF) and 35 g L^?1 (SmF). Micrographs of PUF preparations showed yeast growing within liquid lamellae, thinner than 100 µm, forming large horizontal aggregates. Yeast aggregates were much smaller in SmF than in SSF experiments; however, laccase expression was lower in PUF than in SmF, unless the methanol concentration was increased to 63 g L^?1, which was inhibitory only to the SmF system. CONCLUSION: The results show that oxygen mass transfer is more efficient in SSF, which is related to the higher area/volume ratio compared with SmF. Induction differences may also be due to hindered diffusion of methanol within large yeast aggregates. Copyright © 2009 Society of Chemical Industry     

Improvement of heat removal in solid‐state fermentation tray bioreactors by forced air convection

BACKGROUND: Heat removal is one of the major constraints in large‐scale solid‐state fermentation (SSF) processes. The effect of internal air circulation by forced convection on heat and water transfer has not been studied in SSF tray bioreactors. Formulation of a mathematical model for SSF requires a good estimation of the mass and heat transfer coefficients. RESULTS: A stainless steel tray bioreactor (80.6 L capacity) was used. Aspergillus niger C28B25 was cultivated under SSF conditions on an inert support. Temperature, moisture content, biomass and substrate concentrations were measured. Water and energy integral balances were used to estimate the heat and mass transfer coefficients involved in the process. The Reynolds number (N_Re) in the headspace of the tray bioreactor ranged from 2.5 to 2839, which increased the global heat transfer coefficient from 4.2 to 6.9 (W m^?2 K^?1) and the mass transfer coefficient from 1.0 to 2.1 (g m^?2 s^?1). Mathematical model predictions of the temperature and moisture content of the fermentation bed showed a high goodness‐of‐fit with the experimental results. CONCLUSIONS: This is the first report describing the effect of N_Re of air in the headspace of a SSF tray bioreactor on the heat and mass transfer coefficients and temperature regulation in SSF. Copyright © 2011 Society of Chemical Industry     

Optimization of inulinase production by solid‐state fermentation in a packed‐bed bioreactor

BACKGROUND: This work is focused on inulinase production by solid‐sate fermentation (SSF) using sugarcane bagasse, corn steep liquor (CSL), pre‐treated cane molasses, and soybean bran as substrates in a 3‐kg (dry basis) packed‐bed bioreactor. SSF was carried out by the yeast Kluyveromyces marxianus NRRL Y‐7571 and response surface methodology was used to optimize the temperature, air flow rate and initial mass of cells. RESULTS: The optimum inulinase activity (436.7 ± 36.3 U g^?1 dry substrate) was obtained at 24 h at an inlet air temperature of 30 °C, air flow rate 2.2 m³ h^?1 and 22 g of cells for fermentation. Inulinase productivity at these conditions was 18.2 U gds^?1 h^?1. Kinetic evaluation at the optimized conditions showed that the maximum inulinase production was verified at 24 h of fermentation. The carbon dioxide and the metabolic heat generation are directly associated with the consumption of total reducing sugars present in the medium. CONCLUSION: The high productivity achieved in this work shows the technical viability of inulinase production by SSF in a packed‐bed bioreactor. Copyright © 2009 Society of Chemical Industry     

Statistical optimization of process parameters for the production of vanillic acid by solid‐state fermentation of groundnut shell waste using response surface methodology

BACKGROUND: Vanillic acid is a flavoring agent and also serves as precursor for vanillin production. Culture medium and fermentation condition for the single step production of vanillic acid from Phanerochaete chrysosporium using lignocellulosic waste as a substrate under solid state fermentation (SSF) were optimized using response surface methodology. RESULTS: The process parameters were chosen by borrowing methodology, and L‐asparagine, pH and moisture content of the solid medium during SSF were identified as the most significant variables. The optimum value of selected variable and their mutual interactions were determined by response surface methodology. The result demonstrated that a yield of 73.58 mg vanillic acid g^?1 substate was predicted under optimum conditions (L‐asparagine 5.98 mmol L^?1 (2.37 mg g^?1 groundnut shell), pH of solid medium 4.51 and moisture content 74.83%). The predicted response was experimentally validated and resulted in a maximum vanillic acid yield of 73.69 mg g^?1 after 8 days of SSF. CONCLUSION: The optimization of fermentation variables resulted in a maximum 10‐fold increase in vanillic acid yield compared with that observed under sub‐optimal conditions (from 7.2 mg g^?1 to 73.69 mg g^?1). Copyright © 2011 Society of Chemical Industry     

Partitioning behaviour of cephalexin and 7‐aminodeacetoxicephalosporanic acid in PEG/ammonium sulfate aqueous two‐phase systems

In order to develop an aqueous two‐phase system (ATPS) for cephalexin synthesis with extractive bioconversion, the partitioning behaviour of cephalexin and 7‐aminodeacetoxicephalosporanic acid (7‐ADCA) in poly(ethylene glycol) (PEG)/salt ATPS were examined. Parameters such as PEG size, salt type and tie line length were investigated to find a primary extraction system. In PEG400/ammonium sulfate and PEG400/magnesium sulfate systems, the partition coefficient of cephalexin (K_C) was larger than 1 while that of 7‐ADCA (K_A) deviated about 1.5. Addition of neutral salts, surfactants and water‐miscible solvents were also investigated in the primary ATPS in order to improve the separation efficiency. K_C greatly increased when neutral salts and surfactants were added to the PEG400/ammonium sulfate primary systems whereas K_A was only slightly higher than that of the additive‐free ATPS. In an improved ATPS for extractive bioconversion, consisting of PEG400 (20% w/w), ammonium sulfate (17.5% w/w), methanol (5% w/w) and NaCl (3% w/w), a K_C value of up to 15.2 was achieved; K_A was 1.8; K_P (partition coefficient of phenylglycine methyl ester) was 1.2 and the recovery yield of cephalexin was 94.2%. The results obtained from the extractive bioconversion of cephalexin in the improved ATPS showed that it is feasible to perform such an enzymatic process in an ATPS and the system offers the potential as a model for enzymatic synthesis of some water soluble products. © 2001 Society of Chemical Industry     

Production of α‐amylase under solid‐state fermentation utilizing coffee waste

Coffee industry substrates such as coffee pulp, coffee cherry husk, silver skin, spent coffee and mixtures of these coffee wastes (MC) were evaluated for their efficacy as sole carbon source for the synthesis of α‐amylase in solid‐state fermentation (SSF) using a fungal strain of Neurospora crassa CFR 308. For SSF with coffee pulp and with MC, α‐amylase activity of 3908 U g^?1 ds (units per gram of dry substrate) and 3870 U g^?1 ds, respectively, was observed. Parameters such as moisture (60%), pH (4.6), temperature (28 °C), particle size (1.0 mm), inoculum size (10⁷ spores g^?1 ds), and fermentation time (5 days) were optimized for enzyme synthesis, wherein 4981 and 4324 U g^?1 U g^?1 ds of α‐amylase activity was obtained in SSF with coffee pulp and MC, respectively. The enzyme production was further improved when the substrates were subjected to pre‐treatment by steaming. Accordingly, maximum α‐amylase activity of 7084 U g^?1 ds and 6342 U g^?1 ds was obtained with steam‐pretreated coffee pulp and MC, respectively, demonstrating them to be excellent sole carbon sources for synthesis of α‐amylase production. Copyright © 2009 Society of Chemical Industry     

Mathematical model of heat transfer during solid‐state fermentation in well‐mixed rotating drum bioreactors

We present a mathematical model that describes heat and mass transfer during solid‐state fermentation (SSF) of Aspergillus oryzae in a well‐mixed rotating drum bioreactor (RDB). In addition to the substrate bed and the headspace, the model recognises the bioreactor wall as a subsystem, allowing it to identify the role of this subsystem in heat removal from the bed. Model predictions agree well with previously published experimental data obtained in a rotating drum bioreactor of 0.19 m diameter and 0.85 m length, with maximum temperatures up to 15 °C greater than the incubation temperature being reached. The model offers insights into the rate limiting steps in heat removal, and how SSF performance might be improved in the experimental system. Copyright © 2003 Society of Chemical Industry     

Straw bed priming enhances the methane yield and speeds up the start‐up of single‐stage,high‐solids anaerobic reactors treating plant biomass

A simple and potentially inexpensive implementation of a high‐solids reactor is a single‐stage, stratified bed reactor, in which the bed is made up of the plant biomass fed into the system. In the present study, the stratified bed was started up for a period of four weeks by either direct feeding of sugar beet leaves at four different feeding rates, or by introducing a straw bed primer which was batch digested without feeding. During weeks five to six both systems were fed with sugar beet leaves at such a rate that the total amount of beet leaves added at the end of week six was the same in each of the four corresponding pairs of straw and ‘no‐straw’ reactors. Straw bed priming enhanced the methane yield of the sugar beet leaves, with 0.33–0.37 m³ kg^?1 VS_added (volatile solids) accumulated at average solid retention times as short as 11–25 days, while the ‘no‐straw’ reactors had lower yields at longer average solid retention times. The levels and speciation of the organic acids suggested that both the rate and extent of the anaerobic digestion of the sugar beet leaves added in the straw reactors were improved. At the highest loading rate, the straw reactor failed, while the ‘no‐straw’ reactor did not. It is hypothesised that the microbial biomass was better established in the straw reactors than in the ‘no‐straw’ reactors. Copyright © 2006 Society of Chemical Industry     

Software for Simulation of Second‐Generation Ethanol Production by a Simultaneous Saccharification and Fermentation Process

Environmental impacts associated with the consumption of fossil fuels and the need to generate power through renewable resources demands the usage of alternative materials. The objective is the production of clean energy from materials like lignocellulosic biomass to produce second‐generation (2G) ethanol. A software in the Matlab program is elaborated to simulate the simultaneous saccharification and fermentation (SSF) process of lignocellulosic biomass for the 2G ethanol production in batch reactors. Studying the effects of the process variables, it was found that the higher interference is caused by cellulose concentration. Higher concentrations of the product in batch processes are obtained with the maximum cellulose concentrations, cells, and enzyme.     

Conversions of olive mill wastewater‐based media by Saccharomyces cerevisiae through sterile and non‐sterile bioprocesses

BACKGROUND: Olive mill wastewaters (OMWs) are an important residue and several methods have been proposed for their treatment. RESULTS: Remarkable decolorization (~63%) and phenol removal (~34% w/w) from OMW was achieved. In glucose‐based flask sterile cultures, enrichment with OMWs increased ethanol and biomass production compared with cultures without OMWs added. Flask sterile and un‐sterilized cultures demonstrated similar kinetic results. Batch‐bioreactor trials performed showed higher ethanol and lower biomass quantities compared with the respective shake‐flask experiments, while cultures used under un‐sterilized conditions revealed equivalent results to the sterile ones. In non‐sterile bioreactor cultures, OMWs addition enhanced biomass production in comparison with culture with no OMWs added, whereas ethanol biosynthesis was not affected. The maximum ethanol quantity achieved was 52 g L^?1 (conversion yield per sugar consumed of 0.46 g g^?1) in a batch bioreactor non‐sterilized trial with OMW–glucose enriched medium used as substrate, that presented initial reducing sugars concentration at ~115 g L^?1. Fatty acid analysis of cellular lipids demonstrated that in OMW‐based media, cellular lipids containing increased concentrations of oleic and linoleic acid were produced in comparison with cultures with no OMWs added. CONCLUSIONS: S. cerevisiae simultaneously produced bio‐ethanol and biomass and detoxified OMWs, under non‐sterile conditions. © 2012 Society of Chemical Industry     

Successive solution fractionation mechanisms for high‐density polyethylene

BACKGROUND: Preparative fractionation techniques are currently used in order to obtain large amounts of polyethylene fractions. Preparative successive solution fractionation (SSF) and temperature rising elution fractionation (TREF) are compared as regards obtaining, at a multi‐gram scale, low‐dispersity fractions of high‐density polyethylene (HDPE). The operative separation mechanisms during a SSF of a broad HDPE, which are not yet totally elucidated, are also studied in this work. RESULTS: SSF and TREF approaches lead to the separation of HDPE macromolecules according to their molar masses. If very homogeneous fractions (dispersities from 1.1 to 1.3) are isolated in TREF at the lowest elution temperature, the collected mass is too low. At higher elution temperatures, the fractions have too broad a molar mass distribution (dispersities from 2.7 to 3.7). With the SSF procedure, dispersities are not as low as for the first TREF fractions. But, the relative weight fraction is better distributed between the different extraction temperatures. The molar mass distribution exhibits a dispersity of around 1.9. CONCLUSION: The SSF method is the most suitable way to obtain large gram amounts of low‐dispersity (ca 2) HDPE fractions over a wide molar mass range. Complementary gram‐scale rheological characterization is thus possible enabling a better comprehension of the SSF mechanism. Liquid–liquid demixing is the main mechanism in SSF, but its relative importance depends on polymer characteristics and solvent quality. Copyright © 2008 Society of Chemical Industry     

Sugarcane bagasse and leaves: foreseeable biomass of biofuel and bio‐products

Sugarcane is among the principal agricultural crops cultivated in tropical countries. The annual world production of sugarcane is ～1.6 billion tons, and it generates ～279 million metric tons (MMT) of biomass residues (bagasse and leaves). Sugarcane residues, particularly sugarcane bagasse (SB) and leaves (SL) have been explored for both biotechnological and non‐biotechnological applications. For the last three decades, SB and SL have been explored for use in lignocellulosic bioconversion, which offers opportunities for the economic utilization of residual substrates in the production of bioethanol and value‐added commercial products such as xylitol, specialty enzymes, organic acids, single‐cell protein, etc. However, there are still major technological and economic challenges to be addressed in the development of bio‐based commercial processes utilizing SB and SL as raw substrates. This article aims to explore SB and SL as cheaper sources of carbohydrates in the developing world for their industrial implications, their use in commercial products including commercial evaluation, and their potential to advance sustainable bio‐based fuel systems. Copyright © 2011 Society of Chemical Industry     

Lactic acid fermentation of food waste using integrated glucoamylase production

Commercial enzyme is usually needed for the bioconversion of organic waste or biomass. The overall cost could be reduced very significantly if enzyme production could be integrated with its application, avoiding unnecessary steps in enzyme production (such as concentration, recovery and transportation). This investigation attempted to integrate crude glucoamylase production with lactic acid fermentation of food waste. A maximum glucoamylase activity of 1850 U g^?1 was obtained with Aspergillus nigerduring solid‐state fermentation (SSF) of food waste, 14.8 times more than that obtained during submerged fermentation (SmF). The optimum pH for producing glucoamylase was 4.6, and glucoamylase retained 83.5% of peak activity at pH 3.0. Without any recovery treatment, the glucoamylase produced by SSF could be used directly for lactic acid fermentation of food waste. Lactic acid concentration reached 45.5 g L^?1 with the addition of the crude enzyme, 72% higher than the control. No side‐effects were caused by the viable A. niger in the crude enzyme. This work successfully integrated glucoamylase production with lactic acid fermentation. The enzyme produced by SSF of food waste had sufficient activity to be used directly without any treatment. The integrated process proposed in this study was very economical and may be helpful to other bioconversions. Copyright © 2008 Society of Chemical Industry     

Influence of solvent quality on successive solution fractionation (SSF) efficiency of high‐density polyethylene

BACKGROUND: Preparative successive solution fractionation (SSF) is a powerful technique for obtaining narrow‐dispersity fractions on a multi‐gram scale of high‐density polyethylene (HDPE). In a previous paper, the operative separation mechanisms during SSF of a broad HDPE in cyclohexanone were studied. Two mechanisms, and not only one as expected from the literature, contribute to the separation of HDPE molecules according to their molar mass (MM). The very low MM chains are separated by a solid–liquid (S–L) mechanism, while the longer chains are isolated by a liquid–liquid (L–L) phase separation. In the present paper, the influence of a poorer solvent, diphenyl ether, is reported. RESULTS: It is shown that the relative importance of the S–L mechanism with respect to the L–L one is altered by the use of this solvent. The L–L temperature range is increased in diphenyl ether while the S–L transition temperature remains unchanged. Consequently, the SSF efficiency is improved. Large amounts (on a gram scale) of narrow‐dispersity fractions are isolated, mainly by the L–L mechanism. Polydispersities are about 1.5 (compared to 2.0 for cyclohexanone) and a broader MM range of closer molar mass distribution fractions is available. CONCLUSION: This work demonstrates that the use of diphenyl ether, a poorer solvent than cyclohexanone (always used as SSF solvent for polyethylene in the literature), leads to an improvement of SSF efficiency for an essentially linear HDPE. The differences of behaviour during the separation with cyclohexanone or diphenyl ether are explained by the establishment of a phase diagram. Copyright © 2009 Society of Chemical Industry     

Production of protein and metabolites by yeast grown in solid state fermentation: present status and perspectives

Culture conditions for the generation of products using yeast have been optimized for fermentative processes in industry involving predominantly submerged medium (SmF). However, solid‐state fermentation (SSF) is now a realistic alternative system for the production of recombinant proteins and metabolites of interest in the market, with great potential in biofuels production, food, chemical and pharmaceutical industries. One of the main advantages of SSF over SmF is the reduction of downstream expenses. Also, the use of artificial and very cheap solid supports for yeast SSF such as polyurethane foam or amberlite helps with study of the physiology of such systems. This mini‐review makes an overview of previous research and emphasizes the major physiological advantages of yeast SSF that can be used for new processes and product development and stresses the need for integrated approaches between adaptive evolution and high‐throughput genetic analysis. © 2015 Society of Chemical Industry     

A bioprocessing mode for simultaneous fungal biomass protein production and wastewater treatment using an external air‐lift bioreactor

A pilot plant investigation for bioprocessing has been undertaken to develop a simple, non‐aseptic, low‐cost single process for production of fungal biomass protein (FBP) and wastewater treatment using starch processing wastewater. It has been confirmed that the newly developed external air‐lift bioreactor was very suitable for bioconversion of starch materials and FBP production by the microfungi Aspergillus oryzae and Rhizopus arrhizus. Bioproduct yields of 8.5 g dm^?3 of FBP that contained 46–50% protein were obtained within a comparatively short retention time. A fungal biomass productivity in a range of 0.85–0.92 g dm^?3 h^?1 and removals of total suspended solids and 95% COD were achieved in batch, semi‐continuous and continuous processes. The operation modes of the semi‐continuous and continuous processes demonstrated a high biological dynamics in fungal biomass productivity and COD reduction. The semi‐continuous process appeared to be the most practical mode. © 2001 Society of Chemical Industry     

Microbial conversion of androst‐1,4‐dien‐3,17‐dione by Mucor racemosus to hydroxysteroid‐1,4‐dien‐3‐one derivatives

BACKGROUND: A large number of bacterial, fungal and microalgal species are able to bio‐transform steroid compounds. Among them, fungi from the Mucor genus have been shown to mediate hydroxylation, oxidation, and desaturation by the double bond formation and epoxidation of various steroid substances. Mucor racemocus has not been studied for its ability to modify androst‐1,4‐dien‐3,17‐dione, a pharmaceutically important steroid precursor. RESULTS: The filamentous fungus M. racemosus was applied for bioconversion of androst‐1,4‐dien‐3,17‐dione (ADD, I ) in a 5‐day fermentation. Microbial metabolites were purified chromatographically and identified on the basis of their spectral data as 17β‐hydroxyandrost‐1,4‐dien‐3‐one ( II ), 14α‐hydroxyandrost‐1,4‐dien‐3,17‐dione ( III ), 15α‐hydroxyandrost‐1,4‐dien‐3,17‐dione ( IV ), 15α,17β‐dihydroxyandrost‐1,4‐dien‐3‐one ( V ), 14α,17β‐dihydroxyandrost‐1,4‐dien‐3‐one ( VI ), and 6β,17β‐dihydroxyandrost‐1,4‐dien‐3‐one ( VII ). CONCLUSION: Observed modifications included hydroxylation at C‐6β, C‐14α, C‐15α positions and 17‐carbonyl reduction. The best fermentation conditions for production of hydroxysteroid‐1,4‐dien‐3‐one derivatives were found to be 25 °C at 150 rpm for 5 days with a substrate concentration of 0.5 g L^?1. Copyright © 2009 Society of Chemical Industry     

Deoxygenation of Bio‐oil from Calcium‐Rich Paper‐Mill Waste

De‐inking sludge, an ash‐rich recycling paper solid waste, is generated in huge amounts. The catalytic deoxygenation potential of calcium‐based de‐inking sludge in co‐pyrolysis mode with wood and its neat thermal conversion to sustainable biofuels are investigated. Wood, de‐inking sludge, and their blends are processed in a thermocatalytic reforming (TCR) system. In the presence of de‐inking sludge, the oxygen content in the organic phase decreases and the bio‐oil calorific value improves as compared to the neat wood‐derived bio‐oil. The TCR processing of neat de‐inking sludge produces a bio‐oil with low oxygen content and higher calorific value.     

Bioprocess scale‐up: quest for the parameters to be used as criterion to move from microreactors to lab‐scale

Advances in high‐throughput process development and optimization involve the rational use of miniaturized stirred bioreactors, instrumented shaken flasks and microtiter plates. As expected, each one provides different levels of control and monitoring, requiring a compromise between data quantity and quality. Despite recent advances, traditional shaken flasks with nominal volumes below 250 mL and microtiter plates are still widely used to assemble wide arrays of biotransformation/bioconversion data, because of their simplicity and low cost. These tools are key assets for faster process development and optimization, provided data are representative. Nonetheless, the design, development and implementation of bioprocesses can present variations depending on intrinsic characteristics of the overall process. For each particular process, an adequate and comprehensive approach has to be established, which includes pinpointing key issues required to ensure proper scale‐up. Recently, focus has been given to engineering characterization of systems in terms of mass transfer and hydrodynamics (through gaining insight into parameters such as k_La and P/V at shaken and microreactor scale), due to the widespread use of small‐scale reactors in the early developmental stages of bioconversion/biotransfomation processes. Within this review, engineering parameters used as criteria for scaling‐up fermentation/bioconversion processes are discussed. Particular focus is on the feasibility of the application of such parameters to small‐scale devices and concomitant use for scale‐up. Illustrative case studies are presented. Copyright © 2010 Society of Chemical Industry     

Liquid crystals from star‐like clusto‐supramolecular macromolecules

Supramolecular liquid crystals containing inorganic nanoclusters represent a promising avenue in the field of liquid crystals. The main motivation for developing these hybrid materials originates from the value‐added combination between functional properties of inorganic nano‐objects and the self‐assembly behavior of organic liquid crystal molecules. This review highlights the recent progress regarding nanocluster‐containing supramolecular liquid crystals. Important factors affecting the liquid crystalline behaviors are systematically described and summarized. The driving forces behind the molecular self‐assembly are discussed in depth. Finally, potential applications of the liquid‐crystalline nanohybrids are discussed. © 2014 Society of Chemical Industry