Surfactin production from potato process effluent by Bacillus subtilis in a chemostat

The biosurfactant surfactin has potential to aid in the recovery of energy resources (oil recovery) or subsurface organic contaminants (environmental remediation). However, high medium and purification costs limit its use in these high-volume applications. In previous work, we showed that surfactin could be produced from an inexpensive low-solids potato process effluent with minimal amendments or pretreatments. Previous research has also shown that surfactin can be both produced in Bacillus subtilis cultures and recovered by foam fractionation in an airlift reactor. 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 hamper continuous surfactin production. The research reported here features the use of a chemostat operated in batch mode for producing surfactin with concomitant use of antifoam to prevent surfactant loss. The antifoam did not interfere with surfactin recovery by acid precipitation or its efficacy. Initial trials took about 48 h to produce 0.9 g/L of surfactin from potato process effluent. Increasing the oxygen mass transfer by increasing the stirring speed and adding a baffle decreased production time to 12–24 h and produced about 0.6 g/L of surfactin from two different potato-processing facilities.     

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.     

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.     

Production of L(+)-Lactic Acid Using Acid-Adapted Precultures of <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> in a Stirred Tank Reactor

Cultivations of filamentous fungi in stirred tank reactors (STRs) to produce metabolites are often limited by insufficient mixing and mass transfer because of the formation of mycelial clumps inside the reactors. This study developed an acid-adapted preculture approach to control the morphology of filamentous Rhizopus arrhizus in a STR, consequently to enhance the production yield and productivity of L(+)-lactic acid efficiently using waste potato starch as substrate. Using the acid-adapted precultures as inoculum, the morphology of R. arrhizus was maintained as large clumps, coalesced loose small pellets, and freely dispersed small pellets. The highest lactic acid concentration of 85.7 g/L with a yield of 86% was obtained in association with the formation of coalesced loose small pellets. The results indicate that the use of the acid-adapted precultures as inoculum is a promising approach for lactic acid production in STRs.     

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.     

Simultaneous Saccharification of Inulin and Starch Using Commercial Glucoamylase and the Subsequent Bioconversion to High Titer Sorbitol and Gluconic Acid

A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.     

Ethanol production from corn starch in a fluidized-bed bioreactor

The production of ethanol from industrial dry-milled corn starch was studied in a laboratory-scale fluidized-bed bioreactor using immobilized biocatalysts. Saccharification and fermentation were carried out either simultaneously or separately. Simultaneous saccharification and fermentation (SSF) experiments were performed using small, uniform κ-carrageenan beads (1.5–2.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. Dextrin feeds obtained by the hydrolysis of 15% drymilled corn starch were pumped through the bioreactor at residence times of 1.5–4h. Single-pass conversion of dextrins ranged from 54–89%, and ethanol concentrations of 23–36 g/L were obtained at volumetric productivities of 9–15 g/L-h. Very low levels of glucose were observed in the reactor, indicating that saccharification was the rate-limiting step. In separate hydrolysis and fermentation (SHF) experiments, dextrin feed solutions of 150–160 g/L were first pumped through an immobilized-glucoamylase packed column. At 55°C and a residence time of 1 h, greater than 95% conversion was obtained, giving product streams of 162–172 g glucose/L. These streams were then pumped through the fluidized-bed bioreactor containing immobilized Z. mobilis. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L were achieved, resulting in an overall process productivity of 23 g/L-h. Atresidence times of 1.5 and 1 h, conversions of 75 and 76%, ethanol concentrations of 49 and 47 g/L, and overall process productivities of 19 and 25 g/L-h, respectively, were achieved.     

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.     

Evaluation of Ethanol Production from Corncob Using Scheffersomyces (Pichia) stipitis CBS 6054 by Volumetric Scale-up

In scale-up, the potential of ethanol production by dilute sulfuric acid pretreatment using corncob was investigated. Pretreatments were performed at 170 °C with various acid concentrations ranging from 0% to 1.656% based on oven dry weight. Following pretreatment, pretreated biomass yield ranged from 59% to 67%. More than 90% of xylan was removed at 0.828% of sulfuric acid. At same pretreatment condition, the highest glucose yield obtained from pretreated biomass by enzymatic hydrolysis was about 76%, based on a glucan content of 37/100 g. In hydrolysate obtained by pretreatment, glucose concentration was low, while xylose concentration was significantly increased above 0.368% of sulfuric acid. At 1.656% of sulfuric acid, xylose and glucose concentration was highest. In subsequent, fermentation with hydrolysate, maximal ethanol yield was attained after 24 h with 0.368% of sulfuric acid. The fermentation efficiency of hydrolysate obtained by enzymatic hydrolysis reached a maximum of 75% at an acid charge of 0.368%.     

Production of lactic acid from food wastes

Conversion of food wastes into lactic acid by simultaneous saccharification and fermentation (SSF) was investigated. The process involves saccharification of the starch component in food wastes by a commercial amylolytic enzyme preparation (a mixture of amyloglucosidase, α-amylase, and protease) and fermentation by Lactobacillus delbrueckii. The highest observed overall yield of lactic acid in the SSF was 91% of theoretical. Lactic acid concentration as high as 80 g/L was attainable in 48 h of the SSF. The optimum operating conditions for the maximum productivity were found to be 42°C and pH 6.0. Without supplementation of nitrogen-containing nutrients, the lactic acid yield in the SSF decreased to 60%: 27 g/L of lactic acid from 60 g/L of food waste. The overall performance of the SSF, however, was not significantly affected by the elimination of mineral supplements.     

Synthesis of stable cadmium sulfide nanoparticles using surfactin produced by Bacillus amyloliquifaciens strain KSU-109

In this study, a surfactin was extracted from a novel surfactant producing bacterial strain Bacillus amyloliquifaciens KSU-109, isolated from rhizosphere of date palm (Phoenix dactylifera), and characterized based on 16Sr RNA and sfp genes using Blastn, Blastx and phylogenetic analyses. The study was performed to obtain a renewable bioresource for surfactin production, and its application in nanotechnology as a non-hazardous and environmentally compatible nanoparticle (NP) stabilizer. The strain KSU-109 produced the surfactin with an average yield of 160 mg/L with strong surfactant activity, reducing the surface tension of the medium from 72 mN/m to 29.3 mN/m. The surfactin preparation was used for synthesizing the cadmium sulfide nanoparticles (CdS-NPs) by mixing 0.005% surfactin with 1mM Cd(NO(3))(2) in 1:1 ratio (v/v) and 10mM Na(2)S solution at pH 7.2 and ambient temperature, which were stable up to 120 days. The surfactin stabilized CdS-NPs were characterized using XRD, TEM, and spectroscopic techniques. The data revealed a significant role of surfactin as a stabilizer and capping agent, which also causes phase transition to yield the cubic/hexagonal CdS-NPs of average size of 3-4 nm. The results elucidated the significance of biocompatible and biodegradable surfactin as an effective and inexpensive stabilizing agent for developing stable CdS nanoparticles.     

Optimization of Surfactin Production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Isolate BS5

Bacillus subtilis BS5 is a soil isolate that produces promising yield of surfactin biosurfactant in mineral salts medium (MSM). It was found that cellular growth and surfactin production in MSM were greatly affected by the environmental fermentation conditions and the medium components (carbon and nitrogen sources and minerals). Optimum environmental conditions for high surfactin production on the shake flask level were found to be a slightly acidic initial pH (6.5-6.8), an incubation temperature of 30 degrees C, a 90% volumetric aeration percentage, and an inoculum size of 2% v/v. For media components, it was found that the optimum carbon source was molasses (160 ml/l), whereas the optimum nitrogen source was NaNO(3) (5 g/l) and the optimum trace elements were ZnSO(4).7H(2)O (0.16 g/l), FeCl(3).6H(2)O (0.27 g/l), and MnSO(4).H(2)O (0.017 g/l). A modified MSM (molasses MSM), combining the optimum medium components, was formulated and resulted in threefold increase in surfactin productivity that reached 1.12 g/l. No plasmid could be detected in the tested isolate, revealing that biosurfactant production by B. subtilis isolate BS5 is chromosomally mediated but not plasmid-mediated.     

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.     

Gelatinization properties of native starches and their Näegeli dextrins

Cassava, potato, sweet potato, and Peruvian carrot starches were hydrolyzed with 15% v/v sulfuric acid solution for up to 30 days. Näegeli dextrins obtained from 1, 3, 6, 12, and 30 days were evaluated using differential scanning calorimeter (DSC) and scanning electron microscopy (SEM). Two phases of hydrolysis were found. The first phase was attributed to faster degradation of amorphous areas of granules, whereas the second phase corresponded to slower degradation of crystalline regions. Peruvian carrot starch was the most susceptible to acid, whereas potato and sweet potato starches were the most resistant. From DSC, it was observed a progressive reduction in peak height and a broadening of peaks with increasing hydrolysis time. The peaks shifted to higher temperatures. Onset temperature decreased on first day of hydrolysis for cassava and Peruvian carrot starches, and on third day for potato and sweet potato. Enthalpy decreased during first stage of hydrolysis in cassava and Peruvian carrot starches, and during second phase, it reduced in all starches. SEM showed that the granule surfaces were degraded by erosion on the first day of treatment, followed by degradation of amorphous areas. On third day, potato and sweet potato starches still displayed some granules almost intact, whereas cassava and Peruvian carrot starch granules were totally degraded, confirming their high susceptibility to acid attack. On sixth day of hydrolysis, starch granules had faceted structures, characteristic of crystalline material. The effect that acid hydrolysis had on thermal properties of starches depended on both hydrolysis stage and starch source.     

Effect of environmental factors and carbohydrate on gellan gum production

Submerged culture fermentation studies were carried out in batch mode for optimizing the environmental parameters and carbon source requirement by Pseudomonas elodea for the production of gellan gum. The maximum production of gellan gum was obtained with 16-h-old culture and 8% inoculum at 30°C and pH 7.0 after 52 h of incubation (6.0 g/L). Of the various carbon sources tested, 2% sucrose, glucose, and soluble starch yielded considerably high amounts of gellan. Studies on the concentration of various carbohydrates on gellan gum production indicated that the optimum concentration of glucose and starch was 3%, whereas for sucrose it was 4%. The addition of glucose in the medium above 3% had a detrimental effect on gellan yield. The investigation of intermediate two-step addition of glucose under identical conditions of fermentation showed an enhanced production of gellan (8.12 g/L) as compared with the control (6.0 g/L). To optimize the recovery of gellan from fermented broth, different solvents were tested for precipitation of gellan gum. Among the various solvents tested, tetrahydrofuran gave better recovery of gellan (82%) as compared with the conventional solvent isopropanol (49%).     

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200–250°C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4c/gal of ethanol. This work examined the growth and fermentation performance of xyloseutilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated asthe “adapted” strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30h, followed by CP4:pZB5 at 48h, with corresponding volumetric productivities of 1.4 and 0.89 g/(L·h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to fermentarabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.     

Regulation of β-amylase activity by alkylresorcinols

The effects of two alkylresorcinols, namely, methylresorcinol (MR) and hexylresorcinol (HR), which are chemical analogues of the natural anabiosis factors of some bacteria, on the catalytic activity of β-amylase has been investigated. When water-soluble potato starch is used as a substrate, MR in a concentration ranging from 0.8 to 12.9 mmol/L raises the activity of β-amylase to 170%. HR at a concentration ranging from 0.05 to 0.35 mmol/L stimulates a weak increase in the activity of β-amylase to 115%. A further increase in the concentration (0.75 mmol/L) leads to inhibition by 50%. The interaction of MR with β-amylase allows one to increase the general yield of the hydrolysis products of potato and malt starch by 1.5 to 1.6 times, to enhance the rate of hydrolysis by 4 to 5 times, and to extend the temperature and pH ranges of catalysis. Non-competitive activation in the presence of MR and non-competitive inhibition in the presence of HR are observed upon determining the kinetic parameters of the starch hydrolysis by β-amylase modified with alkylresorcinols.     

The effect of lactic acid on fuel ethanol production byZymomonas

Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase the yield and to reduce the cost of producing fuel ethanol. During prehydrolysis (acid hydrolysis or autohydrolysis of hemicellulose), acetic acid is formed as a consequence of the deacetylation of the acetylated moiety of hemicellulose. Recombinant Escherichia coli B (ATCC 11303), carrying the plasmid pLO1297 with pyruvate decarboxylase and alcohol dehydrogenase II genes from Zymomonas mobilis (CP4), converts xylose to ethanol with a product yield that approaches theoretical maximum. Although other pentose-utilizing microorganisms are inhibited by acetic acid, the recombinant E. coli displays a high tolerance for acetic acid. In xylose fermentations with a synthetic medium (Luria broth), where the pH was controlled at 7, neither yield nor productivity was affected by the addition of 10.7 g/L acetic acid. Nutrient-supplemented, hardwood (aspen) hemicellulose hydrolysate (40.7 g/L xylose) was completely fermented to ethanol (16.3 g/L) in 98 h. When the acetic acid concentration was reduced from 5.6 to 0.8 g/L, the fermentation time decreased to 58 h. Overliming, with Ca(OH)₂ to pH 10, followed by neutralization to pH 7 with sulfuric acid and removal of insolubles, resulted in a twofold increase in volumetric productivity. The maximum productivity was 0.93 g/L/h. The xylose-to-ethanol conversion efficiency and productivity in Ca(OH)₂-treated hardwood prehydrolysate, fortified with only mineral salts, were 94% and 0.26 g/L/h, respectively. The recombinant E. coli exhibits a xylose-to-ethanol conversion efficiency that is superior to that of other pentose-utilizing yeasts currently being investigated for the production of fuel ethanol from lignocellulosic materials.     

Production of ethanol from starch by co-immobilizedZymomonas mobilis-glucoamylase in a fluidized-bed reactor

The production of ethanol from starch was studied in a fluidized-bed reactor (FBR) using co-immobilizedZymomonas mobilis and glucoamylase. The FBR was a glass column of 2.54 cm in diameter and 120 cm in length. TheZ. mobilis and glucoamylase were co-immobilized within small uniform beads (1.2-2.5 mm diameter) of κ-carrageenan. The substrate for ethanol production was a soluble starch. Light steep water was used as the complex nutrient source. The experiments were performed at 35κC and pH range of 4.0-5.5. The substrate concentrations ranged from 40 to 185 g/L, and the feed rates from 10 to 37 mL/min. Under relaxed sterility conditions, the FBR was successfully operated for a period of 22 d, during which no contamination or structural failure of the biocatalyst beads was observed. Volumetric productivity as high as 38 g ethanol/(Lh), which was 74% of the maximum expected value, was obtained. Typical ethanol volumetric productivity was in the range of 15-20 g/(Lh). The average yield was 0.49 g ethanol/g substrate consumed, which was 90% of the theoretical yield. Very low levels of glucose were observed in the reactor, indicating that starch hydrolysis was the rate-limiting step.     

Influence of the Medium Composition and the Culture Conditions on Surfactin Biosynthesis by a Native Bacillus subtilis natto BS19 Strain

An effective microbial synthesis of surfactin depends on the composition of the culture medium, the culture conditions and the genetic potential of the producer strain. The aim of this study was to evaluate the suitability of various medium components for the surfactin producing strain and to determine the impact of the culture conditions on the biosynthesis of surfactin isoforms by the newly isolated native strain Bacillus subtilis natto BS19. The efficiency of surfactin biosynthesis was determined by measuring the surface tension of the medium before and after submerged culture (SmF) and by qualitative and quantitative analysis of the obtained compound by high performance liquid chromatography. The highest efficiency of surfactin biosynthesis was achieved using starch as the carbon source and yeast extract as the nitrogen source at pH 7.0 and 37 °C. Potato peelings were selected as an effective waste substrate. It was shown that the increase in the percentage of peel extract in the culture medium enhanced the biosynthesis of surfactin (mg/L) (2–30.9%; 4–46.0% and 6–58.2%), while reducing surface tension of the medium by about 50%. The obtained results constitute a promising basis for further research on biosynthesis of surfactin using potato peelings as a cheap alternative to synthetic medium components.