Ethanol production from glucose and xylose by immobilized Zymomonas mobilis CP4 (pZB5)

Fermentation of glucose-xylose mixtures to ethanol was investigated in batch and continuous experiments using immobilized recombinant Zymomonas mobilis CP4(pZB5). This microorganism was immobilized by entrapment in κ-carrageenan beads having a diameter of 1.5–2.5 mm. Batch experiments showed that the immobilized cells cofermented glucose and xylose to ethanol and that the presence of glucose improved the xylose utilization rate. Batch fermentation of rice straw hydrolysate containing 76 g/L of glucose and 33.8 g/L of xylose gave an ethanol concentration of 44.3 g/L after 24 h, corresponding to a yield of 0.46 g of ethanol/g of sugars. Comparable results were achieved with a synthetic sugar control. Continuous fermentation experiments were performed in a laboratory-scale fluidized-bed bioreactor (FBR). Glucose-xylose feed mixtures were pumped through the FBR at residence times of 2–4 h. Glucose conversion to ethanol was maintained above 98% in all experiments. Xylose conversion to ethanol was highest at 91.5% for a feed containing 50 g/L of glucose and 13 g/L of xylose at a dilution rate of 0.24/h. The xylose conversion to ethanol decreased with increasing feed xylose concentration, dilution rate, and age of the immobilized cells. Volumetric ethanol productivities in the range of 6.5–15.3 g/L·h were obtained. The improved productivities achieved in the FBR compared to other bioreactor systems can help in reducing the production costs of fuel ethanol from lignocellulosic sugars. This article has been authored by a contractor of the US go vernment under contract DE-AC05-96OR22464. Accordingly, the US government retains a nonexclusive, royaltyfree license to publish or reproduce the published form of the contribution, or allow others to do so, for US government purposes.     

Ethanol Production from Wet-Exploded Wheat Straw Hydrolysate by Thermophilic Anaerobic Bacterium Thermoanaerobacter BG1L1 in a Continuous Immobilized Reactor

Thermophilic ethanol fermentation of wet-exploded wheat straw hydrolysate was investigated in a continuous immobilized reactor system. The experiments were carried out in a lab-scale fluidized bed reactor (FBR) at 70°C. Undetoxified wheat straw hydrolysate was used (3–12% dry matter), corresponding to sugar mixtures of glucose and xylose ranging from 12 to 41 g/l. The organism, thermophilic anaerobic bacterium Thermoanaerobacter BG1L1, exhibited significant resistance to high levels of acetic acid (up to 10 g/l) and other metabolic inhibitors present in the hydrolysate. Although the hydrolysate was not detoxified, ethanol yield in a range of 0.39–0.42 g/g was obtained. Overall, sugar efficiency to ethanol was 68–76%. The reactor was operated continuously for approximately 143 days, and no contamination was seen without the use of any agent for preventing bacterial infections. The tested microorganism has considerable potential to be a novel candidate for lignocellulose bioconversion into ethanol. The work reported here also demonstrates that the use of FBR configuration might be a viable approach for thermophilic anaerobic ethanol fermentation.     

A simple model for the continuous production of butanol by immobilized clostridia: I: Clostridium beyerinckii on glucose

A simple model has been developed to describe the continuous production of butanol and isopropanol from glucose by Clostridium beyerinckii cells immobilized in calcium alginate beads. The model contains linear butanol inhibition kinetics and assumes the biomass to be homogeneously distributed throughout the alginate beads. For design purposes this model, containing five parameters, satisfactorily predicts the values of three output variables (the concentrations of glucose, butanol and isopropanol) as a function of three input variables (the glucose feed concentration, the dilution rate and the fraction of beads in the reactor). For process design a high bead fraction and well mixed conditions seem advantageous.     

Continuous production of butanol with immobilized cells ofClostridium acetobutylicum

Spores ofClostridium acetobutylicum were immobilized in calcium alginate. An active gel preparation was obtained after outgrowth of the spores to vegetative cells within the gel matrix. A 100 mL column containing the immobilized cells was used for continuous production. At steady-state conditions the productivity of butanol was 67 g/L reactor volume/day.     

Continuous Production of Thermostable α-Amylase by Immobilized Bacillus Cells in a Fluidized-Bed Reactor

The exponential cells ofBacillus cereus immobilized in calcium alginate and spun into microcylindrical particles were used in a fluidized-bed reactor for continuous synthesis of thermostable α-amylase. The reactor was operated over a period of 30 d with a dilution rate of 0.33 h^-1, producing 1000–1200 U/mL of enzyme. The productivity of the reactor was in the range of 330–396 kU/h. A 20-fold increase in the productivity with respect to batch fermentation with free cells was attained.     

The Use of Microporous Divinyl Benzene Copolymer for Yeast Cell Immobilization and Ethanol Production in Packed-Bed Reactor

Microporous divinyl benzene copolymer (MDBP) was used for the first time as immobilization material for Saccharomyces cerevisiae ATCC 26602 cells in a bed reactor and ethanol production from glucose was studied as a model system. A very homogenous thick layer of yeast cells were seen from the scanning electron micrographs on the outer walls of biopolymer. The dried weight of the cells was found to be approximately 2 g per gram of cell supporting material. Hydrophobic nature of polymer is an important factor increasing cell adhesion on polymer pieces. The dynamic flow conditions through the biomaterial due to its microporous architecture prevented exopolysaccharide matrix formation around cells and continuous washing out of toxic metabolites and dead and degraded cells from the reactor provided less diffusional limitations and dynamic living environment to the cells. In order to see the ethanol production performance of immobilized yeast cells, a large initial concentration range of glucose between 6.7 and 300 g/l was studied at 1 ml/min in continuous packed-bed reactor. The inhibition effect of glucose with increasing initial concentration was observed at above 150 g/l, a relatively high substrate concentration. The continuous fluid flow around the microenvironment of the attached cells and mass transferring ability of cell immobilized on MDBP can help in decreasing the inhibition effect of ethanol accumulation and high substrate concentration in the vicinity of the cells.     

Immobilization of growing cells by polyethyleneimine-modified alginate

A unique polymer matrix that is suitable for immobilizing growing cells has been developed. Alginate was chemically modified with polyethyleneimine (PEI), and the resultant polymer aggregate was evaluated as a cell carrier. Our method of immobilization depends on reversible gelation of the PEI-modified alginate. Our hypothesis is that immobilized cells grow by dissolving the surrounding gel matrix; the dissolved polymer adduct is displaced peripherally and gelled again by the influx of calcium ion from the surrounding fermentation broth, retaining both cells and carrier polymer in the gel beads. Thus, the immobilized cells gain space for growth by expanding the carrier matrix. The PEI modification offers the following advantages: (1) improved mechanical strength; (2) improved cell retention; (3) increased catalyst life; (4) ease of pelletization; and (5) an apparent bacteriostatic capability. When immobilized yeast cells were applied to a continuous ethanol fermentation, 94% theoretical conversion of glucose to ethanol was observed, with a reactor productivity of 15–30 g/L/h in a nonsterile reactor. A 3-mo catalyst life and minimal cell washout were observed.     

Continuous potable alcohol production by immobilizedSaccharomyces cerevisiae on mineral kissiris

A biocatalyst prepared by the immobilization of Saccharomyces cerevisiae on the surface of the mineral kissiris was used in the present study for continuous potable-alcohol production. An ethanol productivity (calculated on the basis of liquid volume) of 10.5 g/L/h was obtained at a 0.7/h dilution rate, 121 g/L sucrose content, and 29.6% conversion employing molasse as feed material. Glucose, raisin extracts, and molasse were successively used as feed materials without stopping the operation of the reactor for 6 mo. The ethanol productivity and yield remained constant during the operational-stability study of the reactor, carried out for 44 d. Biomass productivity, yield, and free-cell concentration in glucose, raisin extracts, and molasse were examined. Finally, a system with two continuous reactors joined successively was also studied in the present investigation.     

Biological Hydrogen Production Using Chloroform-treated Methanogenic Granules

In fermentative hydrogen production, the low-hydrogen-producing bacteria retention rate limits the suspended growth reactor productivity because of the long hydraulic retention time (HRT) required to maintain adequate bacteria population. Traditional bacteria immobilization methods such as calcium alginate entrapment have many application limitations in hydrogen fermentation, including limited duration time, bacteria leakage, cost, and so on. The use of chloroform-treated anaerobic granular sludge as immobilized hydrogen-producing bacteria in an immobilized hydrogen culture may be able to overcome the limitations of traditional immobilization methods. This paper reports the findings on the performance of fed-batch cultures and continuous cultures inoculated with chloroform-treated granules. The chloroform-treated granules were able to be reused over four fed-batch cultures, with pH adjustment. The upflow reactor packed with chloroform-treated granules was studied, and the HRT of the upflow reactor was found to be as low as 4 h without any decrease in hydrogen production yield. Initial pH and glucose concentration of the culture medium significantly influenced the performance of the reactor. The optimum initial pH of the culture medium was neutral, and the optimum glucose concentration of the culture medium was below 20 g chemical oxygen demand/L at HRT 4 h. This study also investigated the possibility of integrating immobilized hydrogen fermentation using chloroform-treated granules with immobilized methane production using untreated granular sludge. The results showed that the integrated batch cultures produced 1.01 mol hydrogen and 2 mol methane per mol glucose. Treating the methanogenic granules with chloroform and then using the treated granules as immobilized hydrogen-producing sludge demonstrated advantages over other immobilization methods because the treated granules provide hydrogen-producing bacteria with a protective niche, a long duration of an active culture, and excellent settling velocity. This integrated two-stage design for immobilized hydrogen fermentation and methane production offers a promising approach for modifying current anaerobic wastewater treatment processes to harvest hydrogen from the existing systems.     

Production of acetic acid by immobilized whole cells ofClostridium thermoaceticum

Immobilized cells ofClostridium thermoaceticum for acetic acid production has been investigated. Using κ-carrageenan gel as the immobilization-matrix, high cell concentration within the gel could be achieved and thus lead to high volumetric acetic acid productivity. Batch experiments using 3% gel showed that cell concentration up to 65 g (dry cell weight)/L gel could be achieved. These dry weight cell concentrations in the gel through immobilization are typically 10–15 times greater than what can be obtained in free-cell fermentations. The specific growth rate and acetic acid formation rate were similar to those observed for the free cells. Continuous culture experiments using a feed medium containing 20 g/L of glucose were performed where the reactor contained 50% by volume of the carrageenan gel and the pH was controlled at 6.9. Different steady states were acheived at dilution rates ranging from 0.061 to 0.399 h^?1. Cells grew mainly near the surface of the gel and reached maximum concentration within the matrix of approximately 35 g/L. Dilution rates much greater than the maximum specific growth rate were obtained, which resulted in volumetric productivity up to 4.9 g/L-h. This value was significantly greater than that for the conventional continuous culture with free cells. Using a 40 g/L feed glucose concentration, steady states could be achieved between dilution rates of 0.12–0.4 h^?1. The maximum productivity further increased to 6.9 g/L-h at a dilution rate of 0.37 h^?1 and at an acetic acid concentration of 19 g/L. The cell concentration was 60 g (dry weight)/L gel at steady state.     

Production of Lactic Acid from Raw Sweet Potato Powders by Rhizopus Oryzae Immobilized in Sodium Alginate Capsules

Rhizopus oryzae immobilized in calcium alginate was applied in lactic acid fermentation with unhydrolyzed raw sweet potato powders as the sole carbon source. The effects of sodium alginate concentration, calcium chloride concentration, and the immobilized bead diameter on lactic acid production were investigated. Increase in sodium alginate concentration during the gelation process would harden the immobilized capsule, which led to a decrease in lactic acid production. The increase in calcium chloride would increase the thickness of the immobilized capsule, which would increase the mass transfer resistance. Nevertheless, while the calcium chloride was lower than 15%, it would not have obvious effects on lactic acid production. A larger bead could have more space for cell growth, which led to the maximum lactic acid production observed at the 5-mm bead diameter. Moreover, results of repeated-batch operation suggested that immobilized cells could have higher stability in lactic acid production than free cells. The total cumulative lactic acid in immobilized-cell operation could increase by 55% as compared with free-cell operation after 216 h (seven repeated-batches), and no loss of amylolytic activity was observed. The results indicated that immobilized R. oryzae by Ca-alginate could be suitable for lactic acid production from unhydrolyzed raw potato powders.     

Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor

Acetone butanol ethanol was produced in a continuous immobilized cell (biofilm) plug-flow reactor inoculated with Clostridium beijerinckii BA101. To achieve high reactor productivity, C. beijerinckii BA101 cells were immobilized by adsorption onto clay brick. The continuous plug-flow reactor offers high productivities owing to reduced butanol inhibition and increased cell concentration. Although high productivity was achieved, it was at the expense of low sugar utilization (30.3%). To increase sugar utilization, the reactor effluent was recycled. However, this approach is complicated by butanol toxicity. The effluent was recycled after removal of butanol by pervaporation to reduce butanol toxicity in the reactor. Recycling of butanolfree effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2g/(L·h) at a dilution rate of 1.5 h⁻¹. A dilution rate of 2.0h⁻¹ resulted in a reactor productivity of 16.2g/(L·h) and sugar utilization of 101.4%. It is anticipated that this reactor-recovery system would be economical for butanol production when using C. beijerinckii BA101.     

Experimental studies of immobilized-yeast,packed-bed reactors with reduced CO2 entrapment

A horizontal packed-bed reactor with baffles (HPBR) and a continuously stirred tank reactor with intermittent paddle agitation have been shown to considerably reduce the CO₂ entrapment when glucose is fermented with immobilized baker’s yeast in calcium alginate beads. Using high cell contents in the gel resulted in internal mass transfer hindrance. The highest productivity was obtained with the HPBR giving 29 g EtOH/Lh at an ethanol yield of 90%. The substrate used was 100 g/L glucose. Fermentation of lactose and deproteinized whey by coimmobilized baker’s yeast and |3-galactosidase resulted in much lower productivity-about 5 g EtOH/L.h because of the slow fermentation of galactose.     

Hydrolysis of starch with immobilized glucoamylase

Glucoamylase (E.C.3.2.1.3) covalently immobilized onto chitin particles (d_st = 0.37 mm) was examined in two types of continuous bench-scale reactors (180 mL) fed with hydrolyzed manioc starch (15%, w/v): a two-phase reactor (liquid expanded-bed) and a threephase reactor (air expanded-bed). Several conditions of continuous operation were investigated, varying the biocatalyst load (16.7, 37.2, and 54 g/L) into the reactor and the hydraulic residence time. The best results were achieved with the two-phase reactor, which operated continuously for 20 d and showed a decrease of only 6% in conversion (starch to glucose). Conversion levels of 96% were obtained with a hydraulic residence time of about 4 h. A simple mathematical model was able to describe the experimental results of the two types of reactors considering biocatalyst deactivation.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Production of cyclodextrins in a fluidized-bed reactor using cyclodextrin-glycosyl-transferase

Cyclodextrin-glycosyl-transferase (EC2.4.1.19), produced by Wacker (Munich, Germany), was purified by biospecific affinity chromatography with β-cyclodextrin (β-CD) as ligand, and immobilized into controlled pore silica particles (0.42 mm). This immobilized enzyme (IE) had 4.7 mg of protein/g of support and a specific activity of 8.6 μmol of β-CD/(min·g_IF) at 50°C, pH 8.0. It was used in a fluidized-bed reactor (FBR) at the same conditions for producing cyclodextrins (CDs) with 10% (w/v) maltodextrin solution as substrate. Bed expansion was modeled by the Richardson and Zaki equation, giving a good fit in two distin ctranges of bed porosities. The minimum fluidization velocity was 0.045 cm/s, the bed expansion coefficient was 3.98, and the particle terminal velocity was 2.4 cm/s. The FBR achieved high productivity, reaching in only 4 min of residence time the same amount of CDs normally achieved in a batch reactor with free enzyme after 24h of reaction, namely, 10.4 mM β-CD and 2.3 mM γ-CD.     

Effect of Concentration and Substrate Flow Rate on Isomaltulose Production from Sucrose by <Emphasis Type="Italic">Erwinia</Emphasis> sp. Cells Immobilized in Calcium-Alginate Using Packed Bed Reactor

Isomaltulose was obtained from sucrose solution by immobilized cells of Erwinia sp. D12 using a batch and a continuous process. Parameters for sucrose conversion into isomaltulose were evaluated using both experimental design and response surface methodology. Erwinia sp. D12 cells were immobilized in different alginates, and the influence of substrate flow rate and concentration parameters to produce isomaltulose from sucrose were observed. Response surface methodology demonstrated that packed bed columns containing cells immobilized in low-viscosity sodium alginate (250 cP) presented a mean isomaltulose conversion rate of 47%. In a continuous process, both sucrose substrate concentration and substrate flow rate parameters had a significant effect (p < 0.05) and influenced the conversion of sucrose into isomaltulose. Higher conversion rates of sucrose into isomaltulose, from 53–75% were obtained using 75 g of immobilized cells at a substrate flow rate of 0.6 mL/min.     

AnSBBR Applied to a Personal Care Industry Wastewater Treatment: Effects of Fill Time,Volume Treated Per Cycle,and Organic Load

A study was performed regarding the effect of the relation between fill time, volume treated per cycle, and influent concentration at different applied organic loadings on the stability and efficiency of an anaerobic sequencing batch reactor containing immobilized biomass on polyurethane foam with recirculation of the liquid phase (AnSBBR) applied to the treatment of wastewater from a personal care industry. Total cycle length of the reactor was 8 h (480 min). Fill times were 10 min in the batch operation, 4 h in the fed-batch operation, and a 10-min batch followed by a 4-h fed batch in the mixed operation. Settling time was not necessary since the biomass was immobilized and decant time was 10 min. Volume of liquid medium in the reactor was 2.5 L, whereas volume treated per cycle ranged from 0.88 to 2.5 L in accordance with fill time. Influent concentration varied from 300 to 1,425 mg COD/L, resulting in an applied volumetric organic load of 0.9 and 1.5 g COD/L.d. Recirculation flow rate was 20 L/h, and the reactor was maintained at 30 °C. Values of organic matter removal efficiency of filtered effluent samples were below 71% in the batch operations and above 74% in the operations of fed batch followed by batch. Feeding wastewater during part of the operational cycle was beneficial to the system, as it resulted in indirect control over the conversion of substrate into intermediates that would negatively interfere with the biochemical reactions regarding the degradation of organic matter. As a result, the average substrate consumption increased, leading to higher organic removal efficiencies in the fed-batch operations.     

Operability and feasibility of ethanol production by immobilizedZymomonas mobilis in a fluidized-bed bioreactor

Studies have been carried out using immobilized Z.mobilis in fluidized-bed bioreactors and have emphasized operation during high productivity and conversion. The bacteria are immobilized within small uniform beads (~1 to 1.5-mm diam) of K-carrageenan at cell loadings of 15-50 g (dry wt)/L. Conversion and productivity were measured under a variety of conditions, including feedstocks, flow rates, temperature, pH, and column sizes (up to 2.5 m tall). Volumetric productivities of 50-120 g EtOH/h-L reactor volume have been achieved. Productivities of 60 g/h-L are demonstrated from a 15% feed with residual glucose concentrations of less than 0.1% and 7.4% EtOH in the tallest fermentor. Among feeds of 10, 15, and 20% dextrose, the 15% gave the highest productivity and avoided substrate inhibition. A temperature of 30°C and pH 5 were the optimum conditions. The ethanol yield was shown to be nearly constant at 0.49 g EtOH/g glucose, or 97% of the theoretical under a variety of conditions and transients. The biocatalyst beads have been shown to remain active for two months. Nonsterile feed has been used for weeks without detrimental contamination. The advantages of this advanced bioreactor system over conventional batch technology are discussed.     

Characteristics of an Immobilized Yeast Cell System Using Very High Gravity for the Fermentation of Ethanol

The characteristics of ethanol production by immobilized yeast cells were investigated for both repeated batch fermentation and continuous fermentation. With an initial sugar concentration of 280?g/L during the repeated batch fermentation, more than 98% of total sugar was consumed in 65?h with an average ethanol concentration and ethanol yield of 130.12?g/L and 0.477?g ethanol/g consumed sugar, respectively. The immobilized yeast cell system was reliable for at least 10 batches and for a period of 28?days without accompanying the regeneration of Saccharomyces cerevisiae inside the carriers. The multistage continuous fermentation was carried out in a five-stage column bioreactor with a total working volume of 3.75?L. The bioreactor was operated for 26?days at a dilution rate of 0.015?h^?1. The ethanol concentration of the effluent reached 130.77?g/L ethanol while an average 8.18?g/L residual sugar remained. Due to the high osmotic pressure and toxic ethanol, considerable yeast cells died without regeneration, especially in the last two stages, which led to the breakdown of the whole system of multistage continuous fermentation.