Biosurfactants from potato process effluents

High-solids (HS) and low-solids (LS) potato process effluents were tested as substrates for surfactin production. Tests used effluents diluted 1∶10, unamended and amended with trace minerals or corn steep liquor. Heat pretreatment was necessary for surfactin production from effluents due to indigenous bacteria, whose spores remained after autoclaving. Surfactin production from LS surpassed HS in all cases. Surfactin yields from LS were 66% lower than from a pure culture in an optimized potatostarch medium. LS could potentially be used without sterilization for surfactin production for low-value applications such as environmental remediation or oil recovery.     

Bacterial Cellulose Production by Acetobacter xylinum Strains from Agricultural Waste Products

Bacterial cellulose is a biopolysaccharide produced from the bacteria, Acetobacter xylinum. Static batch fermentations for bacterial cellulose production were studied in coconut and pineapple juices under 30 °C in 5-l fermenters by using three Acetobacter strains: A. xylinum TISTR 998, A. xylinum TISTR 975, and A. xylinum TISTR 893. Experiments were carried out to compare bacterial cellulose yields along with growth kinetic analysis. Results showed that A. xylinum TISTR 998 produced a bacterial cellulose yield of 553.33 g/l, while A. xylinum TISTR 893 produced 453.33 g/l and A. xylinum TISTR 975 produced 243.33 g/l. In pineapple juice, the yields for A. xylinum TISTR 893, 975, and 998 were 576.66, 546.66, and 520 g/l, respectively. The strain TISTR 998 showed the highest productivity when using coconut juice. Morphological properties of cellulose pellicles, in terms of texture and color, were also measured, and the textures were not significantly different among treatments.     

Utilization of Makgeolli Sludge Filtrate (MSF) as Low-Cost Substrate for Bacterial Cellulose Production by Gluconacetobacter xylinus

Search for efficient low-cost substrate/additives are gaining significant impetus in bacterial cellulose (BC) production. Makgeolli sludge (a traditional Korean wine distillery waste) is enriched with organic acid, alcohol, and sugar. Using makgeolli sludge filtrate (MSF) and Hestrin–Schramm (HS) medium (g/l of distilled water: glucose, 10.0; peptone, 5.0; yeast extract, 5.0; disodium phosphate, 2.7; citric acid, 1.15; pH 5.0), two different media—namely the modified HS media (ingredients of HS media except glucose dissolved in MSF) and mixed modified HS media (equal volume mixture of original and modified HS media)—were formulated. BC production with Gluconacetobacter xylinus was studied using the two above referred medium. Keeping HS medium as reference, effect of initial pH, glucose, ethanol, and organic acid concentration on BC production was also studied. It suggests that increasing initial glucose (up to 25 g/l) though improves BC production but results in poor BC yield above 15 g/l of glucose. However, addition of alcohol (up to 1%v/v) or citric acid (up to 20 mM) escalate productivity up to four and two times, respectively. In both modified HS media and mixed modified HS medium, BC production was four to five times higher than that of original HS medium. Even MSF alone surpassed HS medium in BC production. Scanning electron microscopy showed that BC microfibrils from MSF based media were several micrometers long and about 25–60 nm widths. X-ray diffraction patterns suggested the produced BC were of cellulose I polymorph.     

Development of continuous surfactin production from potato process effluent by Bacillus subtilis in an airlift reactor

The biosurfactant surfactin has the potential to aid in the recovery of subsurface organic contaminants (environmental remediation) or crude oils (oil recovery). However, high medium and purification costs limit its use in these high-volume applications. In previous work, we showed that surfactin can be produced from an inexpensive low-solids (LS) potato process effluent with minimal amendments or pretreatments. Previous research has also shown that 95% or more of the surfactin in Bacillus subtilis cultures can be recovered by foam fractionation. In this work, we present the results of research to integrate surfactin production with foam fractionation. Experiments were performed in an airlift reactor, with continuous collection of the foam through a tube at the top of the column. Preliminary results using both purified potato starch and unamended low-solids potato process effluent as substrates for surfactin production indicate that the process is oxygen limited and that recalcitrant indigenous bacteria in the potato process effluent may hamper continuous surfactin production.     

Effects of gamma irradiation on cell-wall constituents of some agricultural residues

The effects of 150 kilogray (kGy) of γ irradiation on cell-wall constituents of cottonwood (CW), lentils straw (LS), apple pruning products (AP) and olive cake (OC) were investigated. Samples were irradiated by γ irradiation at a dose level of 150 kGy under identical conditions of temperature and humidity and analyzed for crude fibre (CF), neutral-detergent fibre (NDF), acid detergent fibre (ADF) and acid-detergent lignin (ADL). The results indicate that γ irradiation decreased CF contents by about 29% for CW, LS and AP and by 17% for OC. NDF values were also decreased by about 4% for CW and OC, and by about 12% for LS and AP. γ Irradiation treatment also decreased ADF values only for CW by 8%. ADL contents decreased by 8% for CW and 5% for OC with no effects for LS and AP. The percentage of cellulose (CL): CF ratio increased by 30, 34, 38 and 20% for CW, LS, AP and OC, respectively. Also, the percentage of hemicellulose (HCL): CF increased by 57% for CW and 16% for OC and decreased by 7% for LS and AP. The percentage of HCL: ADL increased by 22% for CW but decreased by 33% for LS and AP with no changes for OC. There were no changes in CL: ADL ratio for all residues.     

Production of insoluble exopolysaccharide of Agrobacterium sp. (ATCC 31749 and IFO 13140)

Agrobacterium isolated from soil samples produced two extracellular polysaccharides: succinoglycan, an acidic soluble polymer, and curdlan gum, a neutral, insoluble polymer. Maize glucose, cassava glucose, and maize maltose were used in fermentation medium to produce insoluble polysaccharide. Two Agrobacterium sp. strains which were used (ATCC 31749 and IFO 13140) in the production of insoluble exopolysaccharide presented equal or superior yields compared to the literature. The strain ATCC 31749 yielded better production when using maize maltose, whose yield was 85%, whereas strain IFO 13140 produced more when fed maize glucose, producing a yield of 50% (on reducing sugars).     

Enhanced bacterial cellulose production from <Emphasis Type="Italic">Gluconobacter xylinus</Emphasis> using super optimal broth

The bacterial cellulose (BC) produced by Gluconobacter xylinus due to its versatile properties, is used in healthcare and industrial applications. However, its use is restricted owing to the limited yield from the existing culture protocols. In the current study, BC production is studied in the presence of Super Optimal Broth with catabolite repression (SOC) medium which is used to revive Escherichia coli cells after electroporation or chemoporation. In SOC medium, Gluconobacter xylinus produces cellulose pellicles within 5 days of incubation with an enhanced conversion of the carbon source to cellulose compared to traditional Hestrin–Schramm (HS) medium. SOC medium also maintains the pH close to 7.0 in static cultures unlike in HS medium where the pH is acidic. The physico-chemical and morphological characteristics of the BC produced in SOC are determined using powder X-ray diffraction (pXRD), thermo gravimetric analysis (TGA), Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH), and scanning electron microscopy (SEM) analyses. Our results indicate that SOC enhance the yield of bacterial cellulose and allows conversion of 50% of the carbon source to bacterial cellulose, compared to only 7% conversion in the case of traditional HS medium after 7 days of interaction. We also observe an increase in hydration capacity of BC produced using SOC as compared to HS media.     

Production of bacterial cellulose by Gluconacetobacter sp. RKY5 isolated from persimmon vinegar

The optimum fermentation medium for the production of bacterial cellulose (BC) by a newly isolated Gluconacetobacter sp. RKY5 was investigated. The optimized medium composition for cellulose production was determined to be 15 g/L glycerol, 8 g/L yeast extract, 3 g/L K₂HPO₄, and 3 g/L acetic acid. Under these optimized culture medium, Gluconacetobacter sp. RKY5 produced 5.63 g/L of BC after 144 h of shaken culture, although 4.59 g/L of BC was produced after 144 h of static culture. The amount of BC produced by Gluconacetobacter sp. RKY5 was more than 2 times in the optimized medium found in this study than in a standard Hestrin and Shramm medium, which was generally used for the cultivation of BC-producing organisms.     

Influence of protective agents for preservation of Gluconacetobacter xylinus on its cellulose production

Gluconacetobacter xylinus produces extracellularly cellulose under aerobic conditions. The formed bacterial cellulose has gained much attention as temporary substitute for human skin, artificial blood vessels applicable in microsurgery or new material for cartilage replacement. Therefore, it is important to preserve the chosen bacteria strain to guarantee reproducibility of the work as well as to shorten the preparation time. In the present paper freezing in suspension using glycerol, DMSO and skim milk as protective agents and drying in gelatine drops were evaluated. A useful preserving method should provide high survival rates of G. xylinus and should have no influence on the cellulose formation. As it is known that addition of substances can have significant effects on formation and structure of bacterial cellulose produced by G. xylinus, the effect of the protective agents and of the storage time on these parameters was studied here. The use of glycerol and skim milk as protective agents for freezing was considered as not recommendable as both altered the structure of cellulose produced by G. xylinus and showed influence on the bacterial metabolism. Freezing in suspension with DMSO proved best in our investigations. DMSO guaranteed high survival rates and had no determinable influence on the structure of the formed bacterial cellulose. Drying of the bacteria cells in gelatine drops had no effect on the morphological structure and kinetic parameters but showed a very low survival rate.     

Transgenic expression of <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis> strain ATCC 53582 cellulose synthase genes in the cyanobacterium <Emphasis Type="Italic">Synechococcus leopoliensis</Emphasis> strain UTCC 100

We report the transfer of cellulose synthesis genes (acsABΔC) from the heterotropic alpha proteobacterium, Gluconacetobacter xylinus strain ATCC 53582 to a photosynthetic microbe (Synechococcus leopoliensis strain UTCC 100). These genes were functionally expressed in this cyanobacterium, resulting in the production of non-crystalline cellulose. Although the cellulose lacks the structural integrity of the product synthesized by G. xylinus, the non-crystalline nature of the cyanobacterial cellulose makes it an ideal potential feedstock for biofuel production.     

Rotation of Cellulose Ribbons During Degradation with Fungal Cellulase

Degradation of bacterial cellulose with a commercial cellulase, Celluclast 1.5L (Novo Nordisk), from the fungus Trichoderma reesei, causes a rotational movement of the cellulose microfibrils. Purified cellulases (CBH I, CBH II, and EG II) do not induce rotation of bacterial cellulose, however, ratios of CBH I and EG II do cause rotation of bacterial cellulose. Equimolar amounts of CBH I or CBH II and EG II do not result in motion during degradation. Based on these observations, we provide further evidence supporting, at least on theoretical grounds, the hypothesis that cellulose chains have intrinsic chirality. As the cellulase enzymes interact with and degrade the cellulose fibrils, the crystalline structure of the cellulose is altered, allowing the linear cellulose polymers to relax into a lower energy state, thus relieving the strain induced by crystallization of the nascent -glucan chains during the biogenesis of the microfibril. This conversion of crystalline bacterial ribbons into more relaxed conformations produces the rotation observed during the treatment of bacterial cellulose with cellulase.     

Production of laccase by immobilized cells of Agaricus sp.

Laccase was produced in the supernatant of culture of a local isolate of Agaricus sp. obtained from decaying Ficus religiosa wood. The enzyme was produced at a constitutive level when growing the fungus in a nitrogenlimited medium supplemented with either glycerol, glucose, fructose, mannitol, arabinose, maltose, sacch arose, cellulose, or cellobiose. Atwo-to sixfold increase in enzyme specific activity was observed when growing the strain in the presence of straw, xylan, xylose, lignosulfonate, veratryl alcohol, and ferulic and veratric acid. Experimentsare consistent with the existence of an induction control on laccase and the absence of a form of carbon catabolite repression mediated by noninducing carbon sources. Immobilization of the Agaricus sp. on several supports, including polyurethane foam, textilestrips, and straw, resulted in an increase of enzyme production as compared to cultivation in liquid medium.     

Production of Keratinase by Free and Immobilized Cells of <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Strain PPKS-2: Partial Characterization and Its Application in Feather Degradation and Dehairing of the Goat Skin

An extremely alkaliphilic bacterial strain, Bacillus sp. PPKS-2, was isolated from rice mill effluents and screened for the production of extracellular keratinase. The maximum production of keratinase occurred after 48 h in shaking culture at pH 11.0 and 37 °C in a medium containing 0.5% soybean flour. The strain grew and produced alkaline keratinase using chicken feather and horn meal as the sole source of carbon and nitrogen. An addition of 0.1% soybean flour or feather hydrolysate and sodium sulfite to feather medium increased the production and complete solubilization of feather took place within 5 days under solid-state fermentation conditions. The partially purified enzyme displayed maximum activity at pH 11.0 and 60 °C in a broad range of NaCl, 0–16%, and was not inhibited by sodium dodecyl sulfate (10%), ethylenediaminetetraacetic acid (10 mM), H₂O₂ (15%), and other commercial detergents. Immobilization of the whole cells proved to be useful for continuous production of keratinase and feather degradation. The enzyme was effectively used to remove hair from goat hide. The strain PPKS-2 can be effectively used for solid waste management of poultry feather in submerged as well as solid-state fermentation.     

Medium Initial pH and Carbon Source Stimulate Differential Alkaline Cellulase Time Course Production in Stachybotrys microspora

The production profile of cellulases of the mutant strain A19 from the filamentous fungus Stachybotrys microspora was studied in the presence of various carbon sources (glucose, lactose, cellulose, carboxymethylcellulose (CMC), and wheat bran) and a range of medium initial pH (5, 7, and 8). Two extracellular cellulases from the Stachybotrys strain (endoglucanases and β-glucosidases) were monitored by enzymatic assay, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and zymogram analysis. Glucose and lactose repressed CMCase time course production while they permitted a strong β-glucosidase one. On Avicel cellulose, CMC, and wheat bran, both activities were highly produced. Wheat bran (WB) is the best carbon source with an optimum of production at days 5 and 6. The production kinetics of both activities were shown to depend on the medium initial pH, with a preference for neutral or alkaline pH in the majority of conditions. The exception concerned the β-glucosidase which was much more produced at acidic pH, on glucose and cellulose. Most interestingly, a constitutive and conditional expression of an alkaline endoglucanase was revealed on the glucose-based medium at an initial pH of 8 units. The zymogram analysis confirmed such conclusions and highlighted that carbon sources and the pH of the culture medium directed a differential induction of various endoglucanases and β-glucosidases.     

Hydrogen production from paper sludge hydrolysate

The main objective of this study was to develop a system for the production of “renewable” hydrogen. Paper sludge is a solid industrial waste yielding mainly cellulose, which can be used, after hydrolysis, as a feedstock in anaerobic fermentation by (hyper)thermophilic organisms, such as Thermotoga elfii and Caldicellulosiruptor saccharolyticus. Tests on different medium compositions showed that both bacteria were able to produce hydrogen from paper sludge hydrolysate, but the amount of produced hydrogen and the requirement for other components differed. Hydrogen production by T. elfii strongly depended on the presence of yeast extract and salts. By contrast, C. saccharolyticus was less dependent on medium components but seemed to be inhibited by a component present in the sludge hydrolysate. Utilization of xylose was preferred over glucose by C. saccharolyticus.     

Phytase Production by a Marine Yeast <Emphasis Type="Italic">Kodamea ohmeri</Emphasis> BG3

The marine yeast strain Kodamea ohmeri BG3 isolated from the gut of a marine fish (Hexagrammes otakii) was found to secrete a large amount of phytase into the medium. The crude phytase produced by this marine yeast showed the highest activity at pH 5.0 and 65 °C. The optimal medium for phytase production contained oat 10.0 g/l, ammonium sulfate 15.0 g/l, glucose 30 g/l, and NaCl 20.0 g/l, while the optimal cultivation conditions for phytase production were pH 5.0, a temperature of 28 °C, and a shaking speed of 170 rpm. Under the optimal conditions, over 557.9 mU/ml of phytase activity was produced within 72 h of fermentation at the shake flask level. This is a very high level of phytase activity produced by yeasts. We think that the medium and process for phytase production by the marine yeast strain were very simple, and such marine yeast from the gut of natural marine fish may have a potential application in the maricultural industry and marine environmental protection. The results demonstrate that phytate was actively degraded by the crude phytase within a short period.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca

Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose in lignocellulosic material. Unfortunately, cellulases have a low catalytic efficiency on their substrates when compared to similar enzymes such as amylases, which has led to a strong interest in improving their activities. Thermobifida fusca secretes six cellulose degrading enzymes: two exo- and three endocellulases and an endo/exocellulase Cel9A (formerly called E4). Cel9A shows unique properties because of its endo- and exocellulase characteristics, strong activity on crystalline cellulose, and good synergistic properties. Therefore, it is an excellent target for mutagenesis techniques to improve crystalline cellulose degradation. In this article, we describe research conducted to improve Cel9A catalytic efficiency using a rational design and computer modeling. A computer model of Cel9A was created using the program CHARMM plus its PDB structure and a cellohexose molecule attached to the catalytic site as a starting model. Initially molecular graphics and energy minimization were used to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD). The interaction between this cellulose chain and conserved CBD residues was determined in the model, and mutations likely to improve the binding properties of the CBD were selected. Site-directed mutations were carried out using the pET vector pET26b, Escherichia coli DH5-α, and the QuickChange mutagenesis method. E. coli BL21-DE3 was used for protein production and expression. The purified proteins were assayed for enzymatic activity on filter paper, swollen cellulose, bacterial microcrystalline cellulose, and carboxymethylcellulose (CMC). Mutation of the conserved residue F476 to Y476 gave a 40% improved activity in assays with soluble and amorphous cellulose such as CMC and swollen cellulose.     

Effect of temperature on growth of psychrophilic and psychrotrophic members of Rhodotorula aurantiaca

The thermodependence of growth kinetic parameters was investigated for the Antarctic psychrophilic strain Rhodotorula aurantiaca and a psychrotrophic strain of the same species isolated in Belgium (Ardennes area). Cell production, maximum growth rate (μ_max), and half-saturation constant for glucose uptake (Ks) of both yeasts were temperature dependent. For the two yeasts, a maximum cell production was observed at about 0°C, and cell production decreased when temperature increased. The μ_max values for both strains increased with temperature up to a maximum of 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. For both yeasts, Ks for glucose was relatively constant at low temperatures. It increased at temperatures above 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. Although its glucose affinity was lower, the psychrotrophic strain grew more rapidly than the psychrophilicone. The difference in growth rate and substrate affinity was related to the origin of the strain and the adaptation strategy of R. aurantiaca to environmental conditions.     

Bacterial cellulose films: influence of bacterial strain and drying route on film properties

Structural properties of bacterial cellulose (BC) depend on the microstructure of the material, which in turn is influenced by the bacterial strain. This paper reports the production of BC thin films from two bacterial strains, gluconacetobacter xylinus (GX) and gluconacetobacter europaeus (GE), and three methods of drying the films; at room temperature, freeze drying and supercritical drying. The porosity, transparency, water absorption capacity (WAC) and mechanical properties of the obtained films are further investigated. We conclude that materials with different properties can be fabricated by selecting the bacterial strain or the drying method. Supercritical drying of films of GE achieved mechanically robust and extremely light films, 0.05 g/mL, with up to 96 % of porosity, and with a WAC up 110 times their dried weight. We determined that materials resulting from GE strain are not much affected by the drying method. On the other hand, GX produced BC films more sensitive to the drying method used. Films are denser, 0.6–0.2 g/mL, with tunable porosity from 60 to 90 % and their maximum WAC is 66 times their dried weight.