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Evaluation of biofilm reactor solid support for mixed-culture lactic acid production

A combination of lactobacilli and biofilm-forming bacteria were evaluated in continuous fermentations for lactic acid production using various supports. Twelve different bacteria, including species of Bacillus, Pseudomonas, Streptomyces,
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  Appl Microbiol Biotechnol (1993) 38:728-733 pplied Fwrobiology Biotechnology © Springer-Verlag 1993 Evaluation of biofilm reactor solid support for mixed-culture lactic acid production Aii Demirei, Anthony L. Pometto IIl, Kenneth E. Johnson Department of Food Science and Human Nutrition, Center for Crops Utilization Research, Iowa State University, Ames, Iowa 50011, USA Received: 14 May 1992/Accepted: 8 October 1992 Abstract. A combination of lactobacilli and biofilm- forming bacteria were evaluated in continuous fermen- tations for lactic acid production using various sup- ports. Twelve different bacteria, including species of Ba- cillus, Pseudomonas, Streptomyces, Thermoactino- myces, and Thermomonospora were tested for biofilm- forming capabilities. Solid supports that were evaluated in either batch or continuous fermentations were pea gravels, 3M-macrolite ceramic spheres, and polypropy- lene mixed with 25 of various agricultural materials (e.g. corn starch, oat hulls) and extruded to form chips (pp-composite). Biofilm formation was evaluated by the extent of clumping of solid supports, weight gain and (in some instances) Gram stains of the supports after drying overnight at 70 ° C. The supports consistently producing the best biofilm were pp-composite chips followed by 3M-Macrolite spheres then by pea gravels. The best bio- film formation was observed with P. fragi (ATCC 4973), S. viridosporus T7A (ATCC 39115), and Ther- moactinomyces vulgaris (NRRL B-5790), grown opti- mally at 25, 37, and 45 ° C, respectively, on various pp- composite chips. Lactic acid bacteria used in the fer- mentations were Lactobacillus amylophilus (NRRL B- 4437), L. casei (ATCC 11443), and L. delbrueckii mu- tant DP3; these grow optimally at 25, 37, and 45 ° C, re- spectively. Lactic acid and biofilm bacteria with compa- tible temperature optima were inoculated into 50-ml reactors (void volume 25 ml) containing sterile pp-com- posite supports. Lactic acid production and glucose con- sumption were determined by HPLC at various flow rates from 0.06 to 1.92 ml/min. Generally, mixed-cul- ture biofilm reactors produced higher levels of lactic acid than lactic acid bacteria alone. S. viridosporus T7A and L. casei on pp-composite chips were the best combi- nation of those tested, and produced 13.0 g/1 lactic acid in the reactors without pH control. L. casei produced 10.3 g/1 lactic acid under similar conditions. * Journal paper no. J-14840 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project nos. 2889 and 0178 Correspondence to: A. L. Pometto Introduction Lactic acid is an organic acid (C3H603) han can be pro- duced both biologically and chemically (Holten et al. 1971). In 1985, 23 x 103 tons of lactic acid were pro- duced in the U.S. (Ohleyer et al. 1985). Lactic acid is used in the food industry as an acidulant and preserva- tive. Lactic acid and its derivatives also have potential industrial uses in the non-food industry (Holten et al. 1971). Polyesters of lactic acid form a plastic with good tensile strength, thermoplasticity, fabricability, and biodegradability (Lipinsky and Sinclair 1986). To pro- duce such polymers, extremely pure L- or D-lactic acid is required. Polylactics are currently used for resorbable sutures, prosthetic devices, and slow-release carriers of herbicides and pesticides. Lactic acid can also be used as a feedstock for the chemical and biological production of other organic acids, such as propionic, acrylic, acetic acids, propylene glycol, ethanol, and acetaldehyde. To meet a growing need for more lactic acid and to make the price of lactic acid more attractive, the effi- ciency of microbial fermentation and recovery must be improved. Methods used to increase cell density and rate of lactic acid production include hollow-fiber reactors (Roy et al. 1982), cell-recycle reactors (Ohleyer et al. 1986), and immobilized-cell reactors (Zayed and Zahran 1991). In immobilized-cell reactors, entrapment is com- monly achieved with calcium alginate or K-carrageen. Stenrous et al. (1982) reported that lactic acid produc- tion rates that are growth-associated are low in immobil- ized reactors because entrapped cells grow slowly if at all. Also, long-term use of immobilized cells results in gel bead swelling and rupture (Demain and Solomon 1986). Biofilm is a natural form of cell immobilization that results from microbial attachment to solid supports (Characklis and Marshall 1990). In nature, mixed-cul- ture biofilms are commonly found on the surfaces of underwater rocks, buried metal surfaces and on decay- ing organic matter (Characklis and Marshall 1990). At- kinson and Davies (1972) reported that, in continuous stirred tank fermentors, biofilms maximized substrate  Table 1. The potential biofilm-forming and lactic acid bacteria, media and incu- bation temperatures used in the study 729 Microorganism Medium a Incubation tempera- ture (°C) Biofilm formers Bacillus licheniformis (NRRL NRS 1264) SSB B. stearothermophilus (NRRL B 1172) SSB Pseudomonas amyloderamosa (ATCC 21262) 3 P. fragi (ATCC 4973) 3 P. thermocarboxydovorans (ATCC 35961) 1492 Streptomyces badius 252 (ATCC 39117) 196 S. setonii 75Vi2 (ATCC 39116) 19 S. viridosporus T7A (ATCC 39115) 196 Thermomonospora curvata (ATCC 19995) 1489 T. fusca (ATCC 27730) 74 Thermoactinomyces vulgaris (ATCC 43649) 18 Lactic acid bacteria Lactobacillus amylophilus (NRRL B 4437) MRS L. casei (ATCC 11443) MRS L. delbrueckii DP3 MRS 30 45 30 25 45 37 37 37 45 45 45 25 37 45 SSB: soluble starch, 10 g/l; Difco Yeast Extract in nitrogen-free salt solution, 5 g/l; MRS: Lactobacillus MRS Broth, 55 g/l; medium 3: Beef extract, 3 g/l; Peptone, 5 g/l; medium 18: Trypticase Soy Broth, 30 g/l; medium 196: Yeast Extract in nitrogen-free salt solution, 6 g/l; medium 741: Tryptone, 3 g/l; Difco Yeast Extract, 3 g/l; glucose, 3 g/l; KzHPO4, 1 g/l; medium 1489: Dextrin, 10 g/l; Tryptone, 2 g/l; Beef Extract, 1 g/l; Difco Yeast Extract, 1 g/l; CoClz, 2 rag/l; medium 1492: pyruvate, 2 g/1 and mineral solution. All numbered media are from ATCC (1989) utilization at high flow rates and minimized the loss of microbial cells. Biofilm reactors have found application in waste-treatment plants, production of vinegar by the 'quick vinegar' process (Crueger and Crueger 1990), al- cohol production (Vega et al. 1988) and soy sauce pro- duction (Iwasaki et al. 1992). In this paper, we evaluate various solid supports and biofilm-forming bacteria and Lactobacillus spp. for lactic acid production in pure- and mixed-culture systems. Materials and methods Microorganisms. Bacteria were obtained from either the American Type Culture Collection (ATCC) (Rockville, Md., USA) or the Northern Regional Research Laboratory (NRRL) (Peoria, Ill., USA) (Table 1). L. delbrueckii DP3 is a mutant developed in our laboratory (Demirci and Pometto 1992). Solid supports. The supports evaluated were local pea gravels, 3M-macrolite ceramic spheres (aluminum oxide; Industrial Miner- al Products Division/3M, St. Paul, Minn., USA), and polypropy- lene (pp)-composite chips containing 25 (w/w) of the agricultur- al materials listed in Table 2. The pp-composite chips were pre- pared by high-temperature extrusion of the polypropylene (Quan- tum USI Division, Rolling Meadows, Ii1., USA) and various agri- cultural materials in a Brahender PL2000 twin-screw extruder (C. W. Brabender Instruments, South Hackensack, N.J., USA). The barrel temperatures were 200, 210, and 220 °C, die temperature was 220°C, and the screw speed was 20 rpm. The agricultural products used (Table 2) were: carhoxymethylcellulose (Sigma, St. Louis, Mo., USA), cellulose (Sigmacell, Type 20; Sigma), ground (20 mesh) oat hulls (National Oats Co., Cedar Rapids, Iowa, USA), ground (20 mesh) soybean hulls (ISU Center for Crops Uti- lization Research, Ames, Iowa, USA), soybean flour (Archer Daniels Midland Company, Decatur, Ill., USA), zein (Sigma), and xylan (from oat spelts containing approximately 10 arabinose, and 15 glucose residues; Sigma). Each materials was vacuum- dried for 48 h at 110 ° C. Polypropylene chips were compounded with different levels and blends of agricultural materials extruded as a 3-mm diameter rod, air cooled, then pelletized into a 2-3-ram long chips. All chips compounded with protein were difficult to produce and charred by the high temperatures employed. Further- more, any free materials associated with pp-composite chips were removed by sifting prior to use. Biofilm formation in batch reactors. Solid supports were weighed, place in 20x150mm test tubes fitted with bubbler tube units (Pometto and Crawford 1986) and 10 ml culture medium, as indi- cated in Table 1, was added to each tube. The culture apparatus Table 2. Polypropylene (pp) composite support formulation a pp-Composite chip Agricultural Minor product agricultural ( ) product (5%) Polypropylene NA b NA Carboxymethyl cellulose 25 NA Cellulose 25 NA Cellulose-soy flour 20 Soy flour Cellulose-zein 20 Zein Oat hulls 25 NA Oat hulls-soy flour 20 Soy flour Oat hulls-zein 20 Zein Soy flour 25 NA Soy hulls 25 NA Soy hulls-soy flour 20 Soy flour Soy hulls-zein 20 Zein Zein 25 NA Xylan 25 NA a pp-Composite chips consisted of 75°7o polypropylene (w/w) b NA, not added  730 Feed stock Air Pump Air filter Manifold Air blank H2 2MNaOH 1MH2SO~ Inoculation port Aseptic Water bath Effluent collector Fig. 1. Schematic diagram of the biofilm reactor was sterilized at 121°C for 15 min, and aseptically inoculated with 0.1 ml of a 24-h culture. Incubation was at the optimum tempera- ture of the bacteria (Table 1) with continuous aeration using COz- free air. Corresponding control tubes were prepared and incu- bated in the same manner but were not inoculated. After 1 week of incubation, the culture medium was drained and the solid sup- ports were rinsed with deionized water, placed into a preweighed 250-ml flask, and dried at 70°C overnight. After cooling in a de- siccator, the flasks were reweighed. Before and after drying, the supports were visually evaluated for cell mass accumulation and clumping of the supports. Biofilm formation in continuous reactors. Fifty milliliters of solid supports were weighed, and placed in a 50-ml plastic syringe fitted with a silicone stopper. A 10-1 Carboy containing 4 1 of the corre- sponding culture medium (Table 1) was connected to a T-shaped tubing connector (Fig. 1). One arm of the T was connected by sil- icone tubing to a syringe at the hypodermic needle port, and the second arm to an air line containing a cotton plug to supply filter- sterilized, CO2-free air. The barrel-mouth of the syringe was fitted with a silicone stopper that was penetrated by two glass connect- ing tubes. One tube was covered with a septum for inoculation. The other tube was used as a medium exit line. The complete sys- tem was sterilized in an autoclave at 121 ° C for 1 h. After cooling, the reactors were placed in water baths set at appropriate temper- atures (Table 1). Culture medium was pumped into the reactor to fill it, and the reactor was inoculated with a 24-h broth culture. The reactors were incubated as batch cultures for 24 h with contin- uous aeration. Culture medium was then continuously pumped into the reactor (working volume 20-25 ml) at a flow rate of 0.06 ml/min for 6 weeks. The supports were evaluated as before, and by Gram-staining of pp-composite chips only. For Gram-staining, small portions of the pp-composite chips were taken before and after the fermentation, then placed in a test tube and Gram- stained by submersion. The chips were washed with alcohol and water until all excess color was removed, dried at 70 ° C overnight, and then visually evaluated for increased blue color. Continuous lactic acM fermentation. The biofilm-forming bacte- ria selected for mixed-culture fermentations were Pseudomonas fragi (ATCC 4973), Streptomyces viridosporus T7A (ATCC 39115), and Thermoactinomyces vulgaris (NRRL B-5790), which grow optimally at 25, 37 and 45 ° C, respectively. For both pure- and mixed-culture lactic acid fermentations, the lactic acid bacte- ria used were L. amylophilus (NRRL B-4437) producing L( + )-lac- tic acid, L. easei (ATCC 11443) producing L( + )-lactic acid, and L. delbrueckii mutant DP3 (Demirci and Pometto 1992) produc- ing D( -- )-lactic acid, which have matching optimal temperatures with the biofilm formers, respectively. Mixed- and pure-culture fermentations were performed on various pp-composite chips de- scribed in Table 3. In mixed-culture fermentations, biofilm form- Table 3. Select biofilm-forming bacteria, the best pp-composite support, and the optimum incubation time for biofllm for- mation Biofilm-forming bacteria (medium and incubation temperature) Best Biofilm formation a polypropylene support 4 days 8 days 15 days P. fragi (0.8% nutrient broth at 25 ° C) 20% oat hulls plus 5% zein 20% oat hulls plus 5% soy flour ++ ++ S. viridosporus T7A (0.6% yeast extract broth at 37 ° C) 20% soy hulls + + + + + + plus 5% zein 20% soy hulls + + + + + plus 5% soy flour T. vulgar& 25% oat hulls + + + + + (3% Trypticase Soy Broth at 45 ° C) 20% oat hulls + + + + plus 5% zein 25% zein + + + + + Values are based on visual clumping of chips and color intensity after Gram staining. -, no detectable biofllm; +, the strength of biofilm observed  ers were used to capture the latic acid bacteria, which do not form biofilms. Therefore, biofilm-forming bacteria were initially grown in the continuous reactor in their corresponding culture me- dium (Table 1) with a flow rate of 0.06 ml/min for 15 days. Then heat-sterilized MRS Lactobacillus broth was added, and specific lactic acid bacteria were inoculated. In pure-culture fermenta- tions, lactic acid bacteria were inoculated without a biofilm form- er. Medium was pumped at various flow rates (0.06, 0.12, 0.24, 0.48, 0.96, 1.92 ml/min). Each was held constant for 24 h. Sam- ples were taken every 4 or 5 h. The pH, optical density (620 nm), and percentages of lactic acid and glucose in the effluents were analyzed using a pH meter, Spectronic 20 spectrophotometer (Mil- ton Roy Co, Rochester, N.Y., USA), and a Water's high perform- ance liquid chromatograph (HPLC) (Milford, Mass., USA), equipped with Water's Model 401 refractive index detector, re- spectively. The HPLC separation of lactic acid, glucose and other broth constituents was achieved on a Bio-Rad Aminex HPX-87H column (300 x7.8 mm) (Bio-Rad Chemical Division, Richmond, Calif., USA) using a 20-gl injection loop and 0.006 M H2SO4 as a mobile phase at a flow rate of 0.8 ml/min at 65 ° C. Results and discussion Pea gravels and 3M-macrolite spheres in batch fermentations Some bacteria formed biofilms on both pea gravels and 3M-macrolite spheres (Table 4). P. fragi and P. amylod- eramosa formed films on pea gravels and macrolite spheres, respectively. The three filamentous bacteria on pea gravels and six on macrolite spheres formed detecta- ble films. No detectable weight gain was observed on either support. Pea gravels are a mixture of different stones making their continued use unpredictable. The 3M-macrolite spheres were unpredictable, and the 3M- spheres floated and plugged the exit tubes. 731 Table 4. Biofilm formation in batch fermentation on different sol- id supports in batch fermentation Microorganisms Pea 3M- gravels Macrolite Spheres B. licheniformis nd - B. stearothermophilus nd - L. amylophilus nd - P. amyloderamosa nd + P. fragi + nd P. thermocarboxydovorans - - S. badius 252 - + S. setonii 75Vi2 + + S. viridosporus T7A + + Thermomonospora curvata - T. fusca Thermoactinomyces vulgaris - nd, not determined; -, no biofilm; +, biofilm present as deter- mined by clumping of the supports rus T7A, T. vulgaris, and P. fragi. This would be ex- pected because of biodegradation or leaching of the ag- ricultural materials from the chips. However, this biode- gradation of leaching did not change the physical shape of the chips. Clumping of the pp-composite chips was observed during the fermentation and after the chips were harvested, washed, and dried (Table 5). Gram stains of the chips also confirmed that biofilms were formed because significant color differences existed be- tween zero-time chips and those after incubation with cultures. The best biofilm formers were P. fragi, S. viri- dosporus T7A, and T. vulgaris on pp-composite chips (Table 3). Polypropylene composite chips in continuous fermentations Some weight loss occurred for all the pp-composite chips evaluated for biofilm formation with S. viridospo- Optimization of biofilm formation on selected supports The time required for biofilm formation on the various pp-composite chips was determined for the three best bacteria by harvesting after 4, 8, and 15 days of contin- Table 5. Biofilm formation on pp-compos- ite chips as determined by clumping after a 6-week continuous fermentation pp-Composite chips Biofilm formation P. fragi S. viridosporus T. vulgaris T7A Carboxylmethyl cellulose nd - nd Cellulose - + + Cellulose-soy flour + + + + + + + Cellulose-zein + + + + + Oat hulls + + + + + + + Oat hulls-soy flour + + + + + + Oat hulls-zein + + + + + + Soy flour nd nd - Soy hulls - + + + + Soy hulls-soy flour + + + + + Soy hulls-zein + + + + Zein + + + + + Xylan nd - nd nd, not determined; -, no biofilm formation; +, strength of clumping  732 uous fermentation (Table 3). Substantial biofilms were formed by all three bacteria after 15 days of incuba- tion. Mixed- and pure-culture lactic acid fermentations 13 12 11 1 9 8 70 ~ 60 ~: 50 ~0 30 20 10 0 P. fragi, S. viridosporus T7A, and T. vulgaris had tem- perature optima of 25, 37, and 45 ° C, respectively, per- mitting evaluation of lactic acid bacteria with similar temperature optima in mixed-culture fermentations. Fermentations were evaluated in bioreactors containing selected pp-composite chips with mixed cultures (biofilm formers and lactic acid bacteria) and pure cultures (lac- ~3o 120 tic acid bacteria alone). Lactic acid and glucose concen- ~10 trations in the effluent were analyzed every 4 or 5 h for ~oo 24 h to determine the steady-state values. After 10 h of 90 continuous fermentation, lactic acid and glucose con- ~ 80 70 centrations were steady. Therefore, the lactic acid con- -~ 6o centration and glucose measured at 24 h of each flow ; ~0 rate represented 14-h steady-state values for each flow ~0 rate. 30 20 10 0 . fragi and L. amylophilus (25 ° C). The mixed-culture and pure-culture (L. amylophilus alone) fermentations were similar in lactic acid production when pp-compos- ite chips containing oat hulls-zein and oat hulls-soy t . fragi MIXED S. vC~dosorus T. uul~zris - . ~rr~ylcrphil'us L. o as~,/ PURE L. delbr'~ecki~ DPS L, a~,ylophilus L. case4 L. delbrueck£t DP3 ~ o o o m o o o gl 6 MIXED 1/, 12 10 o ~ 8 u 6 o, 16 P. pag£ S. vCri, dosorus L. amylopMlus L 0¢~ T. ~vAgt~r/~ L. ~elbr~evki£ DP3 PURE 12 10 8 .o 6 -J /4 L u~ylopA~lus L. cc~e~, L. d~ibrueci~i PP3 0.06 ml/min 0.i2 ml/min [~]] 0.413 ml/min 0.96 ml/min 1.9~ml/min rn N N m 3 I .~ ~ ~ ~ N EO r~ Fig. 2. Lactic acid production in mixed- and pure-culture fermen- tations on various polypropylene (pp)-composite chips Fig. 3. Percentage yields in mixed- and pure-culture fermentations on various pp-composite chips, defined as percentage lactic acid produced (g/l) per glucose consumed (g/l) flour were used (Fig. 2). The best lactic acid productions were observed at flow rates of 0.06-0.48 ml/min; 2.28 to 3.97 g lactic acid/1 was produced by the pure culture on oat hull-zein and 2.92 to 4.82 g lactic acid/1 was pro- duced by the mixed culture on oat hull-soy flour. Stable lactic acid production was observed over a broader dilu- tion rate for the mixed culture than the pure culture (Fig. 2). Figure 3 shows the percentage yields at various dilu- tion rates, which were defined as the percentage lactic acid produced (g/l) per glucose consumed (g/l). At the highest lactic acid concentrations, the percentage yields were consistently lower for the mixed-culture than for the pure-culture fermentations. For example, at the same flow rate (0.48 ml/min), the effluent lactic acid concentration was about the same for pure- and mixed- culture fermentations (3.97 g/1 on oat hulls-soy flour and 3.59 g/1 on oat hulls-zein, respectively), whereas the percentage yields were 100% and 81%, respectively. Ap- proximately twice the level of cell mass in the effluent was observed by absorbance for mixed-culture fermen- tation on oat hulls-zein compared to oat hulls-soy flour. The former produced the highest lactic acid concentra- tion. S. viridosporus T7A and L. casei (37 ° C). pp-Composite chips made with both soy hulls-zein and soy hulls-soy
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