A high load of inorganics in raw lignocellulosic biomass is known to inhibit the yield of bio-oil and alter the chemical reactions during fast pyrolysis of biomass. In this study, palm kernel shell (PKS), an agricultural residue from palm oil production, and two other woody biomass samples (mahogany (MAH) sawdust and iroko (IRO) sawdust) were pretreated with distilled water or an acidic solution (either acetic, formic, hydrochloric (HCl) or sulfuric acid (H2SO4)) before fast pyrolysis in order to investigate its effect on the primary products and pyrolysis reaction pathways. The raw and pretreated PKS, MAH and IRO were pyrolysed at 600 °C and 5 s with a micro-pyrolyser connected to a gas chromatograph–mass spectrometer/flame ionisation detector (GC-MS/FID). Of the leaching solutions, HCl was the most effective in removing inorganics from the biomass and enhancing the primary pyrolysis product formed compared to the organic acids (acetic and formic acid). The production of levoglucosan was greatly improved for all pretreated biomasses when compared to the original biomass but especially after HCl pretreatment. Additionally, the relative content of the saccharides was maximised after pretreatment with H2SO4, which was due to the increased production of levoglucosenone. The relative content of the saccharides increased by over 70%. This increase may have occurred due to a possible reaction catalysed by the remaining acid in the biomass. The production of furans, especially furfural, was increased for all pretreatments but most noticeable when H2SO4 was used. However, the relative content of acids and ketones was generally reduced for PKS, MAH and IRO across all leaching solutions. The relative content of the phenol-type compound decreased to a large extent during pyrolysis after acid pretreatment, which may be attributed to dehydration and demethoxylation reactions. This study shows that the production of valuable chemicals could be promoted by pretreatment with different acid solutions.

Co-pyrolysis is one possible method to handle different biomass leftovers. The success of the implementation depends on several factors, of which the quality of the produced bio-oil is of the highest importance, together with the throughput and constraints of the feedstock. In this study, the fast co-pyrolysis of palm kernel shell (PKS) and woody biomass was conducted in a micro-pyrolyser connected to a Gas Chromatograph–Mass Spectrometer/Flame Ionisation Detector (GC–MS/FID) at 600 °C and 5 s. Different blend ratios were studied to reveal interactions on the primary products formed from the co-pyrolysis, specifically PKS and two woody biomasses. A comparison of the experimental and predicted yields showed that the co-pyrolysis of the binary blends in equal proportions, PKS with mahogany (MAH) or iroko (IRO) sawdust, resulted in a decrease in the relative yield of the phenols by 19%, while HAA was promoted by 43% for the PKS:IRO-1:1 pyrolysis blend, and the saccharides were strongly inhibited for the PKS:MAH-1:1 pyrolysis blend. However, no difference was observed in the yields for the different groups of compounds when the two woody biomasses (MAH:IRO-1:1) were co-pyrolysed. In contrast to the binary blend, the pyrolysis of the ternary blends showed that the yield of the saccharides was promoted to a large extent, while the acids were inhibited for the PKS:MAH:IRO-1:1:1 pyrolysis blend. However, the relative yield of the saccharides was inhibited to a large extent for the PKS:MAH:IRO-1:2:2 pyrolysis blend, while no major difference was observed in the yields across the different groups of compounds when PKS and the woody biomass were blended in equal amounts and pyrolysed (PKS:MAH:IRO-2:1:1). This study showed evidence of a synergistic interaction when co-pyrolysing different biomasses. It also shows that it is possible to enhance the production of a valuable group of compounds with the right biomass composition and blend ratio. 

In this study, predominant bacterial and archaeal populations and their roles during anaerobic mono-digestion of food waste (FW) and co-digestion of FW with straw pellets (SP) at thermophilic temperature (53 ± 1 °C) were assessed by Next Generation Sequencing (NGS) analysis at organic loading rates (OLRs) of 3.0 and 7.0 gVS/L/d. Depending on the seed; results revealed that Firmicutes, Bacteroidetes, and Proteobacteria were, respectively the most prevalent bacterial phyla at both OLRs investigated. On the other hand, Euryarchaeota was dominated by methanogens playing crucial role in biogas production and correlated mainly with the activities of Methanobacteria and Methanomicrobia at class level. Acetoclastic Methanosaetae was the predominant genus at OLR = 3.0 gVS/L/d; however, shared the same predominance with hydrogenotrophic methanogens Methanospirillium at the highest OLR. Although no clear effect in response to straw addition at OLR of 3.0 gVS/L/d could be seen in terms of methanogenic archaea at genus level, hydrogenotrophic methanogens revealed some shift from Methanobacterium to Methanospirillium at higher OLR. Nevertheless, no prominent microbial shift in the presence of wheat straw at increased OLR was likely due to adapted inoculation at start-up which was also demonstrated by relatively stable biogas yields during co-digestion.

This study aimed to establish primary reactions and identify gaseous products during fast pyrolysis of low-density polyethylene (LDPE), polypropylene (PP) and polystyrene (PS). Fast pyrolysis was performed by using Py-GC/MS/FID at 574 ± 22 °C for 5 s. Gaseous fractions formed during pyrolysis of LDPE, PP and PS were 14 ± 1 wt%, 31 ± 3 wt% and 103 ± 12 wt%, respectively. The main gaseous compounds from LDPE were butane, 1-pentane and 1-hexene. PP pyrolysis gave propene, pentane and 2,4-dimethyl-1-heptene as the main gaseous compounds. Styrene monomer was the dominant gas from PS. The results showed that polyolefin (PP and PE) produced aliphatic hydrocarbons, while PS formed aromatic hydrocarbons. Furthermore, the proposed mechanism suggests that both inter- and intra-molecular hydrogen transfer occur during PP and PE pyrolysis. PS pyrolysis involves a C-C cleavage at the aliphatic side chain. This work is important to understand the mechanism of gas formation of primary reactions from pyrolysis of common plastics.

Fast pyrolysis is an industrially attractive method to produce fuels and chemicals from biomass; however, to gain better control over the process, the reactions and interactions between the components and decomposition products need elucidation. This study investigated primary reactions during fast pyrolysis of biomass. Pyrolysis of the three main biomass components (cellulose, hemicellulose and lignin) and their blends was carried out with a micro-pyrolyser connected to a Gas Chromatograph-Mass Spectrometer/Flame Ionisation Detector (GC–MS/FID). The blends of the individual components were prepared in similar proportions to that of native biomass (birchwood) and were pyrolysed at 600 °C for 2 s. The results showed that the two-component blends decrease the production of saccharides to a large extent. This was especially noticeable for levoglucosan when cellulose was mixed with either hemicellulose or lignin. Similarly, in the presence of cellulose, the formation of phenolic compounds from lignin was inhibited by 62 %. However, no differences were found in yields of the main products for the xylan-lignin blend compared to those from the individual components. The yields of volatile products from the cellulose-xylan blend were promoted for a majority of the product categories and were most pronounced for the aldehydes. Furthermore, while the formation of the phenols and saccharides was slightly inhibited for the three-component blend, the aldehydes, ketones and furans showed an increased production compared to the weighed sum of products expected, based on the pyrolysis of the individual components. The native biomass showed a similar trend as the three-component blend in all product categories except for the saccharides, which were inhibited to a large extent. This study provides a better understanding of the interactions occurring between different components during fast pyrolysis of biomass.

Raw syngas contains tar contaminants including toluene and naphthalene, which inhibit its conversion to methane. Cell encasement in a hydrophilic reverse membrane bioreactor (RMBR) could protect the cells from hydrophobic contaminants. This study aimed to investigate the inhibition of toluene and naphthalene and the effect of using RMBR. In this work, toluene and naphthalene were added at concentrations of 0.5?1.0 and 0.1?0.2 g/L in batch operation. In continuous operation, concentration of 0?6.44 g/L for toluene and 0?1.28 g/L for naphthalene were studied. The results showed that no inhibition was observed in batch operation for toluene and naphthalene at concentrations up to 1 and 0.2 g/L, respectively. In continuous operation of free cell bioreactors (FCBRs), inhibition of toluene and naphthalene started at 2.05 and 0.63 g/L, respectively. When they were present simultaneously, inhibition of toluene and naphthalene occurred at concentrations of 3.14 and 0.63 g/L, respectively. In continuous RMBRs, no inhibition for toluene and less inhibition for naphthalene were observed, resulting in higher methane production from RMBR than that of FCBR. These results indicated that RMBR system gave a better protection effect against inhibitors compared with FCBR.

This study focused on the effect of temperature and residence time on the primary thermal decomposition reactions during a fast pyrolysis of softwood alkali lignin. The use of Py-GC/MS/FID (Micropyrolyser-Gas Chromatography/Mass Spectrometry/Flame Ionization Detector) allowed for rapid heating of the sample and detailed identification and quantification of the pyrolysis products at a temperature range of 400–600 °C, with residence times from 0.5–5 s. The identified primary pyrolysis products were mainly volatile guaiacyl-type compounds. There was a general increase in yield for the majority of the volatile compounds with increased temperature and time. The cleavage of the lignin polymer to linear carbonyl (acetaldehyde) and guaiacyl-type aromatic compounds increased with temperature, while that of catechol and cresol type was mainly favoured at 500 and 600 °C. Based on these results, a mechanistic pathway for the pyrolytic process was proposed, drawing a linkage from structural units of lignin to the formed primary products. In summary, our findings suggest that the primary decomposition reactions that occur under the fast pyrolysis conditions can be controlled by varying the process temperature and residence time, and deliver mechanistic insight into the product distribution from structurally complex lignin material.

The aim of this study was to investigate the effects of temperature and reaction time on the primary pyrolysis of cellulose and xylan. Fast pyrolysis of cellulose and xylan was carried out with a micropyrolyser connected to a gas chromatograph-mass spectrometer/flame ionisation detector (GC–MS/FID) to separate and identify volatile components, both qualitatively and quantitatively. This set-up meant a minimum amount of secondary reactions, low impact of the heating period and at the same time provided rapid and accurate analyses. The two biomass components investigated were: cellulose and hemicellulose (represented by xylan). They were pyrolysed during 0.5, 1, 2 and 5 s (s) and within a temperature range of 400–600 °C. The results showed that levoglucosan (1, 6-anhydro β-D-glucopyranose) is the main chemical compound released during cellulose pyrolysis. It increased with increasing temperature and time. The main volatile compounds produced from pyrolysis of xylan are: 1-hydroxy-2-butanone, 4-hydroxy-5, 6-dihydro-(2 H)-pyran-2-one, 1-hydroxy-2-propanone (acetol), acetaldehyde and hydroxyacetaldehyde (HAA). HAA was the most abundant chemical compound released during xylan pyrolysis, increasing with higher temperatures and time. Acetol and acetaldehyde also showed similar behaviour. The chemical compounds released from cellulose and xylan fast pyrolysis are primary products and assumed to be produced directly from both cellulose and xylan molecules and not from secondary degradation. In this study, possible reaction routes during biomass primary pyrolysis are also suggested based on the product distribution from the thermal decomposition of cellulose and xylan.

Biogas reactors can be utilized more efficiently when straw and food waste are digested together instead of separately. In the present study, straw in the form of pellets and briquettes has been used in experiments and calculations. Co-digestion of different substrates can give a more optimal substrate composition and a more efficient utilization of available digester volume. The pelleting and briquetting process has been shown to be an adequate pretreatment method of the straw. Digesting food waste and straw together showed synergistic effects with improved degradation of the food waste as well as a higher total volumetric methane production as compared to when food waste was used as the sole substrate. Energy produced through increased biogas production was higher than the energy needed for the pelleting and briquetting process. The positive effect in regard to gas production was mainly seen for the straw pellets, results supported by both chemical and microbiological analysis. These effects were observed in both mesophilic and thermophilic conditions. In conclusion, this study illustrates that straw is a suitable co-digestion substrate to food waste and can be used to improve gas yields as well as for more efficient utilization of the digester volume. These results show the biogas potential of straw, today not yet used as a substrate to a large extent.

The novel concept of reverse membrane bioreactors (rMBR) introduced in this review is a new membrane-assisted cell retention technique benefiting from the advantageous properties of both conventional MBRs and cell encapsulation techniques to tackle issues in bioconversion and fermentation of complex feeds. The rMBR applies high local cell density and membrane separation of cell/feed to the conventional immersed membrane bioreactor (iMBR) set up. Moreover, this new membrane configuration functions on basis of concentration-driven diffusion rather than pressure-driven convection previously used in conventional MBRs. These new features bring along the exceptional ability of rMBRs in aiding complex bioconversion and fermentation feeds containing high concentrations of inhibitory compounds, a variety of sugar sources and high suspended solid content. In the current review, the similarities and differences between the rMBR and conventional MBRs and cell encapsulation regarding advantages, disadvantages, principles and applications for biofuel production are presented and compared. Moreover, the potential of rMBRs in bioconversion of specific complex substrates of interest such as lignocellulosic hydrolysate is thoroughly studied.[on SciFinder (R)]

Integrated permeate channel (IPC) flat sheet membranes were examined for use as a reverse membrane bioreactor (rMBR) for lignocellulosic ethanol production. The fermenting organism, Saccharomyces cerevisiae (T0936), a genetically-modified strain with the ability to ferment xylose, was used inside the rMBR. The rMBR was evaluated for simultaneous glucose and xylose utilization as well as in situ detoxification of furfural and hydroxylmethyl furfural (HMF). The synthetic medium was investigated, after which the pretreated wheat straw was used as a xylose-rich lignocellulosic substrate. The IPC membrane panels were successfully used as the rMBR during the batch fermentations, which lasted for up to eight days without fouling. With the rMBR, complete glucose and xylose utilization, resulting in 86% of the theoretical ethanol yield, was observed with the synthetic medium. Its application with the pretreated wheat straw resulted in complete glucose consumption and 87% xylose utilization; a final ethanol concentration of 30.3 g/L was obtained, which corresponds to 83% of the theoretical yield. Moreover, complete in situ detoxification of furfural and HMF was obtained within 36 h and 60 h, respectively, with the rMBR. The use of the rMBR is a promising technology for large-scale lignocellulosic ethanol production, since it facilitates the co-utilization of glucose and xylose; moreover, the technology also allows the reuse of the yeast for several batches.

Lignocellulosic biomass is a potential feedstock for production of sugars, which can be fermented into ethanol. The work presented in this thesis proposes some solutions to overcome problems with suboptimal process performance due to elevated cultivation temperatures and inhibitors present during ethanol production from lignocellulosic materials. In particular, continuous processes operated at high dilution rates with high sugar utilisation are attractive for ethanol fermentation, as this can result in higher ethanol productivity. Both encapsulation and membrane bioreactors were studied and developed to achieve rapid fermentation at high yeast cell density. My studies showed that encapsulated yeast is more thermotolerant than suspended yeast. The encapsulated yeast could successfully ferment all glucose during five consecutive batches, 12 h each at 42 °C. In contrast, freely suspended yeast was inactivated already in the second or third batch. One problem with encapsulation is, however, the mechanical robustness of the capsule membrane. If the capsules are exposed to e.g. high shear forces, the capsule membrane may break. Therefore, a method was developed to produce more robust capsules by treating alginate-chitosan-alginate (ACA) capsules with 3-aminopropyltriethoxysilane (APTES) to get polysiloxane-ACA capsules. Of the ACA-capsules treated with 1.5% APTES, only 0–2% of the capsules broke, while 25% of the untreated capsules ruptured within 6 h in a shear test. In this thesis membrane bioreactors (MBR), using either a cross-flow or a submerged membrane, could successfully be applied to retain the yeast inside the reactor. The cross-flow membrane was operated at a dilution rate of 0.5 h-1 whereas the submerged membrane was tested at several dilution rates, from 0.2 up to 0.8 h-1. Cultivations at high cell densities demonstrated an efficient in situ detoxification of very high furfural levels of up to 17 g L-1 in the feed medium when using a MBR. The maximum yeast density achieved in the MBR was more than 200 g L-1. Additionally, ethanol fermentation of nondetoxified spruce hydrolysate was possible at a high feeding rate of 0.8 h-1 by applying a submerged membrane bioreactor, resulting in ethanol productivities of up to 8 g L-1 h-1. In conclusion, this study suggests methods for rapid continuous ethanol production even at stressful elevated cultivation temperatures or inhibitory conditions by using encapsulation or membrane bioreactors and high cell density cultivations.

The release of inhibitory concentrations of acetic acid from lignocellulosic raw materials during hydrolysis is one of the main concerns for 2nd generation ethanol production. The undissociated form of acetic acid can enter the cell by diffusion through the plasma membrane and trigger several toxic effects, such as uncoupling and lowered intracellular pH. The effect of acetic acid on the ethanol production was investigated in continuous cultivations by adding medium containing 2.5 to 20.0 g·L−1 acetic acid at pH 5.0, at a dilution rate of 0.5 h−1. The cultivations were performed at both high (~25 g·L−1) and very high (100–200 g·L−1) yeast concentration by retaining the yeast cells inside the reactor by a cross-flow membrane in a membrane bioreactor. The yeast was able to steadily produce ethanol from 25 g·L−1 sucrose, at volumetric rates of 5–6 g·L−1·h−1 at acetic acid concentrations up to 15.0 g·L−1. However, the yeast continued to produce ethanol also at a concentration of 20 g·L−1 acetic acid but at a declining rate. The study thereby demonstrates the great potential of the membrane bioreactor for improving the robustness of the ethanol production based on lignocellulosic raw materials.

The release of inhibitory concentrations of acetic acid from lignocellulosic raw materials during hydrolysis is one of the main concerns for 2nd generation ethanol production. The undissociated form of acetic acid can enter the cell by diffusion through the plasma membrane and trigger several toxic effects, such as uncoupling and lowered intracellular pH. The effect of acetic acid on the ethanol production was investigated in continuous cultivations by adding medium containing 2.5 to 20.0 g•L−1 acetic acid at pH 5.0, at a dilution rate of 0.5 h−1. The cultivations were performed at both high (~25 g•L−1) and very high (100–200 g•L−1) yeast concentration by retaining the yeast cells inside the reactor by a cross-flow membrane in a membrane bioreactor. The yeast was able to steadily produce ethanol from 25 g•L−1 sucrose, at volumetric rates of 5–6 g•L−1•h−1 at acetic acid concentrations up to 15.0 g•L−1. However, the yeast continued to produce ethanol also at a concentration of 20 g•L−1 acetic acid but at a declining rate. The study thereby demonstrates the great potential of the membrane bioreactor for improving the robustness of the ethanol production based on lignocellulosic raw materials.

A submerged membrane bioreactor (sMBR) was developed to ferment toxic lignocellulosic hydrolyzate to ethanol. The sMBR achieved high cell density of Saccharomyces cerevisiae during continuous cultivation of the hydrolyzate by completely retaining all yeast cells inside the sMBR. The performance of the sMBR was evaluated based on the ethanol yield and productivity at the dilution rates 0.2, 0.4, 0.6, and 0.8 h-1 with the increase of dilution rate. Results show that the yeast in the sMBR was able to ferment the wood hydrolyzate even at high dilution rates, attaining a maximum volumetric ethanol productivity of 7.94 ± 0.10 g L-1 h-1 at a dilution rate of 0.8 h-1. Ethanol yields were stable at 0.44 ± 0.02 g g-1 during all the tested dilution rates, and the ethanol productivity increased from 2.16 ± 0.15 to 7.94 ± 0.10 g L-1 h-1. The developed sMBR systems running at high yeast density demonstrates a potential for a rapid and productive ethanol production from wood hydrolyzate.

Abstract: A membrane bioreactor was developed to counteract the inhibition effect of furfural in ethanol production. Furfural, a major inhibitor in lignocellulosic hydrolyzates, is a highly toxic substance which is formed from pentose sugars released during the acidic degradation of lignocellulosic materials. Continuous cultivations with complete cell retention were performed at a high dilution rate of 0.5 h−1. Furfural was added directly into the bioreactor by pulse injection or by addition into the feed medium to obtain furfural concentrations ranging from 0.1 to 21.8 g L−1. At all pulse injections of furfural, the yeast was able to convert the furfural very rapidly by in situ detoxification. When injecting 21.8 g L−1 furfural to the cultivation, the yeast converted it by a specific conversion rate of 0.35 g g−1 h−1. At high cell density, Saccharomyces cerevisiae could tolerate very high furfural levels without major changes in the ethanol production. During the continuous cultures when up to 17.0 g L−1 furfural was added to the inlet medium, the yeast successfully produced ethanol, whereas an increase of furfural to 18.6 and 20.6 g L−1 resulted in a rapidly decreasing ethanol production and accumulation of sugars in the permeate. This study show that continuous ethanol fermentations by total cell retention in a membrane bioreactor has a high furfural tolerance and can conduct rapid in situ detoxification of medium containing high furfural concentrations.

Fermentation using encapsulated yeast leads to more robust ethanol production from lignocellulose hydrolyzates. Encapsulated yeast is much more tolerant to inhibitors present in hydrolyzates, and fermentation is faster due to increased total cell density. For industrial applications, capsules must be made robust enough to endure long periods and numerous cultivations without breaking. Liquid core alginate–chitosan–alginate (ACA) capsules containing Saccharomyces cerevisiae were produced by the liquid-droplet-forming method and treated with hydrolyzed 3-aminopropyltrietoxysilane (hAPTES) forming very glossy capsules. Capsules produced with 3.0% hAPTES showed the best mechanical robustness but no ethanol could be produced in dilute-acid spruce hydrolyzate using these capsules. Untreated ACA capsules gave the highest ethanol production but demonstrated poor mechanical robustness. 25% of the ACA capsules ruptured within 6 h in the shear test. Capsules treated with 1.5% hAPTES were significantly stronger, since only 0–2% of these capsules broke. Moreover, the ethanol production in the fifth consecutive cultivation in lignocellulose hydrolyzate was nearly as high as for untreated ACA capsules. The mechanical robustness of ACA capsules can be easily improved by treating the capsules with hAPTES. ACA capsules treated with 1.5% hAPTES showed excellent mechanical robustness and a similar ethanol production profile to untreated ACA capsules. © 2012 Society of Chemical Industry

Companies developing and producing membranes for different separation purposes, as well as the market for these, have markedly increased in numbers over the last decade. Membrane and separation technology might well contribute to making fuel ethanol and biogas production from lignocellulosic materials more economically viable and productive. Combining biological processes with membrane separation techniques in a membrane bioreactor (MBR) increases cell concentrations extensively in the bioreactor. Such a combination furthermore reduces product inhibition during the biological process, increases product concentration and productivity, and simplifies the separation of product and/or cells. Various MBRs have been studied over the years, where the membrane is either submerged inside the liquid to be filtered, or placed in an external loop outside the bioreactor. All configurations have advantages and drawbacks, as reviewed in this paper. The current review presents an account of the membrane separation technologies, and the research performed on MBRs, focusing on ethanol and biogas production. The advantages and potentials of the technology are elucidated.

Robust polysiloxane-ACA capsules for prolonged ethanol production from wood hydrolyzate by Saccharomyces cerevisiae Päivi Ylitervo,a,b Carl Johan Franzén b and Mohammad J. Taherzadeh a a University of Borås, School of Engineering, Sweden b Chalmers University of Technology, Industrial Biotechnology, Sweden The recalcitrance of lignocellulose makes it difficult to hydrolyze and toxic inhibitors are formed during its decomposition. The formed inhibitors can severely affect the fermentability of the hydrolyzate. Encapsulating the fermenting yeast can be a potential option to make the cells more inhibitor and stress tolerant when compared with suspended yeast. In the encapsulation process the yeast is enclosed in a thin semi-permeable membrane surrounding the cells in the liquid core. To apply encapsulation for industrial applications the capsules need to be mechanically stable for long periods. Therefore, a new encapsulation method was developed were alginate-chitosan-alginate (ACA) capsules were treated with hydrolyzed 3-aminopropyltrietoxysilane (hAPTES) to reinforce capsules with polysiloxane (PS). PS-ACA-capsules treated with 1.5% and 3.0% hAPTES were very robust and only 0-1% capsules broke during the mechanical shear test performed after five batch cultivations. Of the untreated capsules, 25% burst within 6 h. The yeast in 3.0% hAPTES treated PS-ACA-capsules did not produce any ethanol during cultivations. However, capsules treated with 1.5% hAPTES were significantly stronger and showed similar ethanol production profile to untreated ACA-capsules cultivated in hydrolyzate. The produced PS-ACA-capsules were easily prepared and demonstrated high stability, reusability, and good ethanol production which are crucial features to make capsules the applicable at large scale for ethanol production.

This paper reviews the latest developments in microbial products by encapsulated microorganisms in a liquid core surrounded by natural or synthetic membranes. Cells can be encapsulated in one or several steps using liquid droplet formation, pregel dissolving, coacervation, and interfacial polymerization. The use of encapsulated yeast and bacteria for fermentative production of ethanol, lactic acid, biogas, l-phenylacetylcarbinol, 1,3-propanediol, and riboflavin has been investigated. Encapsulated cells have furthermore been used for the biocatalytic conversion of chemicals. Fermentation, using encapsulated cells, offers various advantages compared to traditional cultivations, e.g., higher cell density, faster fermentation, improved tolerance of the cells to toxic media and high temperatures, and selective exclusion of toxic hydrophobic substances. However, mass transfer through the capsule membrane as well as the robustness of the capsules still challenge the utilization of encapsulated cells. The history and the current state of applying microbial encapsulation for production processes, along with the benefits and drawbacks concerning productivity and general physiology of the encapsulated cells, are discussed.

The capability of two zygomycetes strains, Mucor indicus and an isolate from tempeh (Rhizopus sp.), to grow on orange peel hydrolysate and their tolerance to its antimicrobial activity, was investigated. Both fungi, in particular M. indicus, tolerated up to 2% d-limonene in semi-synthetic media during cultivation in shake flasks, under aerobic as well as anaerobic conditions. The tolerance of M. indicus was also tested in a bioreactor, giving rise to varying results in the presence of 2% limonene. Furthermore, both strains were capable of consuming galacturonic acid, the main monomer of pectin, under aerobic conditions when no other carbon source was present. The orange peel hydrolysate was based on 12% (dry w/v) orange peels, containing d-limonene at a concentration of 0.6% (v/v), which no other microorganism has been reported to be able to ferment. However, the hydrolysate was utilised by M. indicus under aerobic conditions, resulting in production of 410 and 400 mg ethanol/g hexoses and 57 and 75 mg fungal biomass/g sugars from cultivations in shake flasks and a bioreactor, respectively. Rhizopus sp., however, was slow to germinate aerobically, and neither of the zygomycetes was able to consistently germinate in orange peel hydrolysate, under anaerobic conditions. The zygomycetes strains used in the present study demonstrated a relatively high resistance to the antimicrobial compounds present in orange peel hydrolysate, and they were capable of producing ethanol and biomass in the presence of limonene, particularly when cultivated with air supply.

Nowadays, fuel ethanol is both used as a substitute and an additive to the conventional fossil fuels and the interest in converting lignocellulose to fuel ethanol has expanded in the last few decades. Lignocellulose is attractive as raw material due to its high abundance and low price. However, chemical hydrolysis or pre-treatment of lignocelluloses creates several components that are toxic to fermenting organisms and makes cultivation complicated. By using encapsulated yeast, one can overcome this problem. In encapsulation, the yeast cells are confined inside a capsule composed of an outer semi-permeable membrane and an inner liquid core (Fig. 1). Encapsulation is an attractive method since it can improve the cell stability and inhibitor tolerance, increase the biomass concentration, and decrease the cost of cell recovery, recycling, downstream processing, and fermentation time. Mechanical resistance is a key parameter together with permeability for the success of an encapsulation system. In order to improve the robustness of the capsules we are testing different cross linkers to introduce covalent bonds to the chitosan-alginate matrix. By treating chitosan covered alginate capsules with glutaraldehyde the capsules became harder and less elastic. One big disadvantage in using crosslinking agent is, however, that they are toxic for the yeast. If the encapsulated yeast is treated at too harsh conditions they will die. Although, to improve the capsules mechanical strength the membrane have to be crosslinked to a satisfying degree. We have examined different capsule-treatments and found some encouraging results when applying repetitive treatments with crosslinking agent.

Encapsulated yeast has several advantages for ethanol production from lignocellulosic materials such as enhanced inhibitor tolerance and cell stability, higher biomass concentration inside the reactor, easier cell recovery and shortened fermentation time (Talebnia 2005). During encapsulation, cells are captured inside a spherical capsule composed of an outer semipermeable membrane and an inner liquid core. Compared to entrapment in a porous gel bead, the diffusion resistance is therefore much lower trough the capsule membrane (Talebnia 2005). Encapsulation has in several studies shown to stabilize cells and improve the tolerance for inhibitors (Talebnia 2005, Pourbafrani 2008). The main goal of the present work was to investigate if encapsulation can also improve the termotolerance characteristics of S. cerevisiae in order to produce ethanol at high temperatures. In the experiments glucose conversion and ethanol production was recorded during 24 h in encapsulated and suspended yeast at high temperatures.