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.     

The effect of pretreatments on surfactin production from potato process effluent by Bacillus subtilis

Pretreatments of low-solids potato process effluent were tested for their potential to increase surfactin yield. Pretreatments included heat, removal of starch particulates, and acid hydrolysis. Elimination of contaminating vegetative cells was necessary for surfactin production. After autoclaving, 0.40 g/L of surfactin was produced from the effluent in 72 h, vs 0.24 g/L in the purified potato starch control. However, surfactin yields per carbon consumed were 76% lower from process effluent. Removal of starch particulates had little effect on the culture. Acid hydrolysis decreased growth and surfactant production, except 0.5 wt% acid, which increased the yield by 25% over untreated effluent.     

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.     

Characterization of surfactin from Bacillus subtilis for application as an agent for enhanced oil recovery

Surfactin produced by Bacillus subtilis (ATCC 21332) was used to examine the effect of altering salt concentration, pH, and temperature on surfactin activity (as measured by reductions in surface tension). These parameters are some of the conditions that define oil reservoir characteristics and can affect the application of surfactants. The Biotechnology for Oilfield Operations research program at the Idaho National Engineering and Environmental Laboratory (INEEL) has successfully produced surfactin from potato process effluents for possible use as an economical alternative to chemical surfactants for improved oil recovery. Surfactants enhance the recovery of oil through a reduction of the interfacial tension between the oil and water interfaces, or by mediating changes in the wettability index of the system. We investigated changes in surfactin activity under a range of conditions by measuring surface tension. Surface tension was determined using video image analysis of inverted pendant drops. Experimental variables included NaCl (0–10%), pH (3.0–10.0), and temperature (21–70°C). Each of these parameters, as well as selected combinations, resulted in discrete changes in surfactin activity. It is therefore important to consider the exploration of the studied surfactin as an enhanced oil recovery agent.     

Production of bacterial cellulose from alternate feedstocks

Production of bacterial cellulose by Acetobacter xylinum ATCC 10821 and 23770 in static cultures was tested from unamended food process effluents. Effluents in cluded low-solids (LS) and high-solids (HS) potato effluents, cheese whey permeate (CW), or sugar beet raffinate (CSB). Strain 23770 produced 10% less cellulose from glucose than did strain 10821 and diverted more glucose to gluconate. Unamended HS, CW, and CSB were unsuitable for cellulose production by either strain, and LS was unsuitable for production by strain 10821. However, strain 23770 produced 17% more cellulose from LS than from glucose, indicating that unamended LS could serve as a feedstock for bacterial cellulose.     

Biosurfactant production by Bacillus subtilis using cassava-processing effluent

A cassava flour-processing effluent (manipueira) was evaluated as a substrate for surfactant production by two Bacillus subtilis strains. B. subtilis ATCC 21332 reduced the surface tension of the medium to 25.9 mN/m, producing a crude biosurfactant concentration of 2.2 g/L. The wild-type strain, B. subtilis LB5a, reduced the surface tension of the medium to 26.6 mN/m, giving a crude biosurfactant concentration of 3.0 g/L. A decrease in surfactant concentration observed for B. subtilis ATCC 21332 seemed to be related to an increase in protease activity. The biosurfactant produced on cassava effluent medium by B. subtilis LB5a was similar to surfactin.     

Sustainable Surfactin Production by Bacillus subtilis Using Crude Glycerol from Different Wastes

Most biosurfactants are obtained using costly culture media and purification processes, which limits their wider industrial use. Sustainability of their production processes can be achieved, in part, by using cheap substrates found among agricultural and food wastes or byproducts. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of surfactin production by Bacillus subtilis #309. The culture medium containing soap-derived waste glycerol led to the best surfactin production, reaching about 2.8 g/L. To the best of our knowledge, this is the first report describing surfactin production by B. subtilis using stearin and soap wastes as carbon sources. A complete chemical characterization of surfactin analogs produced from the different waste glycerol samples was performed by liquid chromatography–mass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR). Furthermore, the surfactin produced in the study exhibited good stability in a wide range of pH, salinity and temperatures, suggesting its potential for several applications in biotechnology.     

Comparison of different strains of the yeastYarrowia lipolytica for citric acid production from glucose hydrol

Four commercial strains and two mutants of the yeast species Yarrowia lipolytica were screened using batch fermentation. Strain Y. lipolytica A-101-1.14 (induced with UV irradiation) was found to be the most suitable for citric acid production from glucose hydrol (39.9% glucose and 2.1% other sugars), a byproduct of glucose production from potato starch. The specific rate of total citric and isocitric acid production was 0.138 g/g.h, the yield on consumed glucose 0.93 g/g, and the productivity achieved was as high as 1.25 g/L.h. All of the tested yeast strains were able to utilize only the glucose from the glucose hydrol medium. Thus, some residual higher oligosaccharides remained in the process effluent.     

Effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A

The effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A was studied in a bench-scale bioreactor using a previously optimized growth medium (0.65% yeast extract and 0.1% corn oil, pH7.0) at 37°C and natural pH. Airflow rates of 0.3, 1.0, and 1.5 vvm and a fixed agitation of 200 rpm were initially studied followed by 1.0 vvm and 200, 300, 400, and 500 rpm. The use of 1.0 vvm and 400 rpm increased enzyme concentration 1.8-fold (100–180 U/L) and process productivity 4.8-fold (1.4–6.7 U/[L·h]) in comparison with the use of 200 rpm and 0.3 vvm. The inexpensive corn oil, used as carbon source, besides its antifoam properties, proved to be nonrepressive for enzyme production.     

Characterization of Surfactin Produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Isolate BS5

Physical and chromatographic characterization of the surfactin biosurfactant produced by Bacillus subtilis isolate BS5 has been conducted to study its potentiality for industrial application. The crude extract of test surfactin appeared as off-white to buff flake-like amorphous residue with bad odor similar to sour pomegranate. Test surfactin showed solubility in aqueous solution at pH>5 with optimum solubility at pH 8-8.5. It was also soluble in organic solvents like ethanol, acetone, methanol, butanol, chloroform, and dichloromethane. Surfactin crystals appeared rectangular with blunt corners and were arranged perpendicular to each other making a plus sign. Extracted surfactin showed high surface activity, as it could lower the surface tension of water from about 70 to 36 mN/m at approximately 15.6 mg/l. Moreover, test surfactin exhibited excellent stabilities at high temperatures (100 degrees C for up to 1 h at and autoclaving at 121 degrees C for 10 min), salinities (up to 6% NaCl), and over a wide range of pH (5-13). Test surfactin in the cell-free supernatant or crude culture broth forms showed high emulsification indices against kerosene (62.5% and 59%, respectively), diesel (62.5% and 66%, respectively), and motor oil (62% and 66%, respectively). These characters can effectively make test surfactin, in its crude forms, a potential candidate for the use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry. Chromatographic characterization of test surfactin, using high-performance liquid chromatography technique, revealed that the extracted surfactin contained numerous isoforms, of which six were found in the standard surfactin preparation (Fluka). Additional peaks appeared in the test surfactin and not in the standard one. These peaks may correspond to new surfactin isoforms that may be present in the test surfactin produced by B. subtilis isolate BS5.     

Effects of Different Amino Acids in Culture Media on Surfactin Variants Produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> TD7

Surfactin produced by Bacillus subtilis has different variants, which are affected by the composition of substrate available. To demonstrate the effects of amino acids on surfactin variants, B. subtilis TD7 was cultivated under the same conditions but with different amino acids supplied in media, respectively, and the type as well as the proportion of surfactin variants produced was analyzed with electrospray ionization mass spectrometry and gas chromatography–mass spectrometry. The result shows that the addition of different amino acids significantly influences the proportion of surfactin variants with different fatty acids. When Arg, Gln, or Val was added to the culture medium of B. subtilis TD7, the proportion of produced surfactin variants with even β-hydroxy fatty acids significantly increased, while the addition of Cys, His, Ile, Leu, Met, Ser, or Thr enhanced the proportion of surfactin variants with odd β-hydroxy fatty acids markedly. This result may be of some reference value in enhancing the production of specific surfactin variants as well as in the research on the relationship between culture media and the corresponding products of a certain bacterium.     

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.     

Effect of the oxygen transfer coefficient on xylitol production from sugarcane bagasse hydrolysate by continuous stirred-tank reactor fermentation

The effect of the oxygen transfer coefficient on the production of xylitol by biocon version of xylose present in sugarcane bagasse hemicellulosic hydrolysate using the yeast Candiada guilliermondii was investigated. Continuous cultivation was carried out in a 1.25-L fermentor at 30°C, pH 5.5, 300 rpm, and a dilution rate of 0.03/h, using oxygen transfer coefficients of 10,20, and 30/h. The results showed that the microbial xylitol production (11 g/L) increased by 108% with the decrease in the oxygen volumetric transfer coefficient from 30 to 20/h. The maximum values of xylitol productivity (0.7g/[L…h]) and yield (0.58 g/g) were obtained at k _L a 20/h.     

Xanthan gum production from cassava bagasse hydrolysate with Xanthomonas campestris using alternative sources of nitrogen

Cassava bagasse was hydrolyzed using HCl and the hydrolysate was used for the production of xanthan gum using a bacterial culture of Xanthomonas campestris. Cassava bagasse hydrolysate with an initial concentration of approx 20 g of glucose/L proved to be the best substrate concentration for xanthan gum production. Among the organic and inorganic nitrogen sources tested to supplement the medium—urea, yeast extract, peptone, potassium nitrate, and ammonium sulfate—potassium nitrate was most suitable. Ammonium sulfate was the least effective for xanthan gum production, and it affected sugar utilization by the bacterial culture. In media with an initial sugar concentration of 48.6 and 40.4 g/L, at the end of fermentation about 30 g/L of sugars was unused. Maximum xanthan gum (about 14 g/L) was produced when fermentation was carried out with a medium containing 19.8 g/L of initial reducing sugars supplemented with potassium nitrate and fermented for 72 h, and it remained almost the same until the end of fermentation (i.e., 96 h).     

High-Cell-Density cultivation of pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates

We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHA_MCL) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65h⁻¹. Limitation and inhibition owing to NH₄ ⁺ ions were observed, respectively, at 400 and 3500 mg of NH₄ ⁺/L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHA_MCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHA_MCL. Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHA_MCL content. Using the last strategy, 21% PHA_MCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH₄ ⁺ limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHA_MCL and a productivity of 0.8 g/(L·h) were obtained in 42 h of fed-batch operation. The PHA_MCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.     

Production of 2,3- butanediol from pretreated corn cob byKlebsiella oxytoca in the presence of fungal cellulase

A simple and effective method of treatment of lignocellulosic material was used for the preparation of corn cob for the production of 2,3-butanediol byKlebsiella oxytoca ATCC 8724 in a simultaneous saccharification and fermentation process. During the treatment, lignin, and alkaline extractives were solubilized and separated from cellulose and hemicellulose fractions by dilute ammonia (10%) steeping. Hemicellulose was then hydrolyzed by dilute hydrochloric acid (1%, wJv) hydrolysis at 100°C at atmospheric pressure and separated from cellulose fraction. The remaining solid, with 90% of cellulose, was then used as the substrate. A butanediol concentration of 25 g/L and an ethanol concentration of 7 g/L were produced byK. oxytoca from 80 g/L of corn cob cellulose with a cellulase dosage of 8.5 IFPU/g corn cob cellulose after 72 h of SSF. With only dilute acid hydrolysis, a butanediol production rate of 0.21 g/L/h was obtained that is much lower than the case in which corn cob was treated with ammonia steeping prior to acid hydrolysis. The butanediol production rate for the latter was 0.36 g/L/h.     

Optimization of biosurfactant lipopeptide production from Bacillus subtilis S499 by Plackett-Burman design

Bacillus subtilis S499 is well-known for its ability to produce two families of surfactant lipopeptides: Iturin A and Surfactin S1. Fermentation optimization for this strain was performed to amplify the surfactant production. Ten active variables were analyzed by two successive Plackett-Burman designs, consisting respectively of 12 and 16 experiments to give an optimized medium. The amount of biosurfactant lipopeptides in the supernatant of a culture carried out in this optimized medium was about five times higher than that obtained in nonoptimized rich medium. The analysis of the surfactant molecules produced in such optimized conditions has revealed the presence of a third family of lipopeptides: the fengycins. The time-dependent production of these three families of molecules in bioreactors showed that surfactin S1 is produced during the exponential phase and iturin A and fengycins during the stationary phase.     

Production of citric acid using immobilized conidia of Aspergillus niger

Conidia of Aspergillus niger were immobilized in calcium alginate gel for the production of citric acid. First, the type of the preactivation medium, together with the preactivation period, was investigated. It was found that A. niger requires a 2-d preactivation period at a 0.05 g/L NH₄NO₃ concentration. Second, preactivated cells were used to determine the effects of nitrogen concentration and the flow rate of oxygen and air on the production of citric acid. Maximum citric acid production was attained with medium containing 0.01 g/L of NH₄NO₃. The rate of citric acid production in the nitrogenous medium was 33% higher when oxygen was used instead of air during the production phase. This corresponds to an increase of 85% when compared to production when neither oxygen nor air was fed into the system. In the nonnitrogenous medium citric acid concentration remained similar regardless of the use of air or oxygen. However, in the nonnitrogenous production medium, citric acid production was not influenced considerably when oxygen was used instead of air. The advantage of using immobilized cells is that production is achieved easily in the continuous system. Therefore, citric acid production was also tested using a packed-bed bioreactor, and an increase in productivity by a factor of 22 was achieved compared to the batch system.     

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.     

Continuous production of butanol from starch-based packing peanuts

Acetone, butanol, ethanol (ABE, or solvents) were produced from starch-based packing peanuts in batch and continuous reactors. In a batch reactor, 18.9 g/L of total ABE was produced from 80 g/L packing peanuts in 110 h of fermentation. The initial and final starch concentrations were 69.6 and 11.1 g/L, respectively. In this fermentation, ABE yield and productivity of 0.32 and 0.17 g/(L·h) were obtained, respectively. Compared to the batch fermentation, continuous fermentation of 40 g/L of starch-based packing peanuts in P2 medium resulted in a maximum solvent production of 8.4 g/L at a dilution rate of 0.033 h⁻¹. This resulted in a productivity of 0.27 g/(L·h). However, the reactor was not stable and fermentation deteriorated with time. Continuous fermentation of 35 g/L of starch solution resulted in a similar performance. These studies were performed in a vertical column reactor using Clostridium beijerinckii BA101 and P2 medium. It is anticipated that prolonged exposure of culture to acrylamide, which is formed during boiling/autoclaving of starch, affects the fermentation negatively.