APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1986, p. 985-989
`0099-2240/86/050985-05$02.00/0
`Copyright C 1986, American Society for Microbiology
`
`Vol. 51, No. 5
`
`Pilot Plant Production of Rhamnolipid Biosurfactant by
`Pseudomonas aeruginosa
`H. E. REILING,t U. THANEI-WYSS, L. H. GUERRA-SANTOS,t R. HIRT, 0. KAPPELI,* AND A. FIECHTER
`Department of Biotechnology, Swiss Federal Institute of Technology, Honggerberg, CH - 8093 Zurich, Switzerland
`
`Received 2 December 1985/Accepted 2 February 1986
`
`Rhamnolipid biosurfactants were continuously produced with Pseudomonas aeruginosa on the pilot plant
`scale. Production and downstream processing elaborated on the laboratory scale were adapted to the larger
`scale. Differences in performance resulting from the scale-up are discussed. A biosurfactant concentration of
`approximately 2.25 g liter-' was achieved. The biosurfactant yield on glucose was 77 mg g- h-1, and the
`productivity was 147 mg liter-' h-1, corresponding to a daily production of 80 g of biosurfactant. The first
`enrichment step consisted of an adsorption chromatography which was followed by an anion-exchange
`chromatography. The resulting product was 90% pure, and the overall recovery of active material was above
`60% with the downstream processing used.
`
`Pseudomonas aeruginosa produces rhamnolipids that ex-
`ert an important role when the cells grow at the expense of
`7). The active fraction contained two
`hydrocarbons (5,
`compounds, R-1 and R-2 (1, 6). They differed in rhamnose
`content, with R-1 having two rhamnose units and R-2 having
`3-hydroxydecanoic acid in both
`one. The lipid part was
`cases. Recently, Wagner and co-workers (11) reported that
`compounds with a variable length of the carbohydrate moi-
`ety are produced under appropriate conditions (resting
`cells).
`An increased interest for potential application of microbial
`surface active compounds is based on their broad range of
`functional properties which mainly comprise emulsification,
`phase separation, wetting, foaming, surface activity, and
`viscosity reduction of heavy crude oils (3, 8). Potential
`applications can be envisaged in several industries such as
`agriculture, food, textiles, cosmetics, petrochemical, and
`petroleum production.
`In view of wide-spread utilization, process development
`for large-scale production of biosurfactants is an obvious
`necessity. In a previous study we have shown the depen-
`dence of biosurfactant production on the medium composi-
`tion (4). It became obvious that environmental parameters
`considerably affect biosurfactant release. Here, we report on
`the production of biosurfactant on the pilot plant level and
`on the downstream processing adapted to the scale.
`MATERIALS AND METHODS
`Microorganism. A strain of Pseudomonas aeruginosa
`(DSM 2659) was used throughout this work which was
`originally isolated from soil samples in the vicinity of an oil
`refinery. The cells were maintained as frozen glycerol
`cultures (1:1 mixture of freshly grown cells and 30% [wt/vol]
`glycerol solution) at -70°C.
`Medium. The optimized medium 3M which was used for
`P. aeruginosa growth and biosurfactant production was the
`same as described previously (4), except that iron sulfate
`was omitted and phosphoric acid (84%) was added to a final
`concentration of 55 ,ul g-' of glucose supplied. The main
`
`* Corresponding author.
`t Present address: R. Maag AG, Agrobiology Research, CH-8157
`Dielsdorf, Switzerland.
`t Present address: Transgene S.A., F-67000 Strasbourg, France.
`
`characteristics of medium 3M are: carbon and phosphorus
`excess as well as nitrogen and iron limitation.
`Glucose supply normally amounted to 30 g liter-', but
`concentrations up to 80 g liter-1 were also tested. The
`medium for the continuous culture was prepared in 2,000-
`liter batches in an in situ sterilizable 3,000-liter stirred-
`tank bioreactor. Inocula were grown in a 2% (wt/vol) glucose
`medium 3M including iron sulfate but 4.1 g of
`Na2HPO4 - 2H20 liter-l plus 5.9 g of KH2PO4 liter-l were
`used as buffering components in place of phosphoric acid.
`Unless unavailable, all chemicals used were of technical
`grade. Because of the severe influence of trace elements on
`growth and production, deionized water was used through-
`out.
`Cultivation conditions. Medium optimization with respect
`to biosurfactant production in continuous culture was per-
`formed with the used organism on lab scale in a 5-liter
`compact loop reactor (COLOR; Braun, Melsungen, Federal
`Republic of Germany) (4). On the pilot plant scale continu-
`ous production was carried out in a 50-liter COLOR bioreac-
`tor which was described in detail elsewhere (2).
`The working volume was 23 liters, growth temperature
`was 33°C, aeration was at 0.5 volume of air per volume of
`liquid per min (vvm), stirrer speed was 1,000 rpm, and the
`pH was maintained at 6.2 with 6 N H3PO4 plus 6 N KOH.
`The bioreactor was equipped and operated with a centrifuge-
`type mechanical foam separator operated at 1,000 rpm. In
`addition, a foam recycling system was connected to the air
`outlet for security reasons. The outlet air was fed into a
`separation funnel from which the air escaped on top, and the
`liquid accumulating at the bottom was recycled to the
`bioreactor. The system was operated at a dilution rate of
`0.065 h-1 during the production process. A constant liquid
`volume was maintained in the bioreactor by a weight con-
`trol. Inocula were grown in several baffled 1-liter shake
`flasks with 200 ml of medium on a rotary shaker at 33°C.
`Late-exponential-phase cells (1 liter) served as the inoculum
`for the start-up batch cultivation in the bioreactor. When the
`glucose was exhausted, the culture was switched from batch
`to continuous operation.
`Analytical methods. Determination of biomass, residual
`glucose concentration, surface and interfacial tension, and
`Fcmc (an indirect measure for biosurfactant concentration)
`values has been described elsewhere (4). Nitrate and nitrite
`
`985
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`986
`
`REILING ET AL.
`
`APPL. ENVIRON. MICROBIOL.
`
`Biosurfactont product
`FIG. 1. Flow sheet for the pilot plant production and downstream processing of rhamnolipid biosurfactants from P. aeruginosa.
`
`were measured semiquantitatively with Merckoquant test
`sticks (E. Merck AG, Darmstadt, Federal Republic of Ger-
`many). Oxygen partial pressure and the composition of the
`exit air were recorded continuously as reported earlier
`(4, 9).
`Downstream processing. The general procedure for down-
`stream processing of the produced biosurfactant was devel-
`oped for laboratory-scale investigations but was principally
`transferable to pilot plant scale. A scheme of the whole
`downstream process is given in Fig. 1. The outlet of the
`continuous production in the bioreactor was collected in a
`cooled reservoir tank (300 liters). Periodically the cells were
`separated by centrifugation (Westfalia, type SA14-47-476),
`and the cell-free culture liquid was stored in a second
`reservoir where 0.2% (vol/vol) formaldehyde was added to
`prevent microbial growth. All chromatographic steps were
`installed in duplicates. In this way, loading and regeneration
`of the columns could be done in parallel.
`Loading of the columns. A supply line was installed to the
`columns which ended in a special device consisting of five
`stainless steel tubings arranged spiderlike to feed the liquid
`evenly to the surface of the resin. A sieve (mesh size, 0.2
`mm) was placed on top of the resin to protect it from solids
`still in the liquid and from being whirled up. Flow to the
`
`columns was induced by peristaltic pumps (type 5025;
`Watson-Marlow, Falmouth, United Kingdom).
`The liquid level in the column was monitored by a capac-
`itive probe (NTK 20; Visolux, Baden-Baden, Federal Re-
`public of Germany) attached to the outer surface of the
`column. The supply pump controlled by this probe, main-
`tained a constant liquid level in the column, which allowed a
`continuous unattended loading of the columns.
`Adsorption chromatography. The primary enrichment of
`the rhamnolipids was achieved by adsorption chromatogra-
`phy on a polystyrene resin (Amberlite XAD-2; Rohm and
`Haas, Philadelphia, Pa.). The resin (7 kg) was placed in a
`glass column (650 by 200 mm), yielding a bed height of
`approximately 330 mm and corresponding to a bed volume of
`about 10.4 liters. The column was equilibrated with 0.1 M
`potassium phosphate buffer (pH 6.1), and then the cell-free
`culture liquid was applied at a flow rate of approximately 20
`liters h-' at the beginning and 10 liters h-1 towards the end
`of the loading procedure. The adsorption of the active
`compounds was assayed by measuring the surface tension at
`the column outlet. Adsorption chromatography was termi-
`nated when the surface tension dropped below 35 mN m-
`The column was then washed with 2 to 3 bed volumes of
`distilled water. The elution of the biosurfactant was subse-
`
`PETITIONERS
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`EXHIBIT NO. 1030 Page 2 of 5
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`

`

`VOL. 51, 1986
`
`PILOT PLANT BIOSURFACTANT PRODUCTION
`
`987
`
`TABLE 1. Comparison of continuous growth and biosurfactant production of P. aeruginosa on laboratory and pilot plant scales
`Yx/s
`Yp,s
`Biomass concn
`rp
`Biosurfactant concn
`Residual glucose concn
`Initial glucose concn
`Bioreactor size,
`(mg liter-' h-')
`(mg g-')
`(g liter-')a
`(g liter-')
`(g liter-')
`(g liter-')
`(mg g-1)
`dilution rate (h-1)
`5 liters
`2.5
`1.7
`0.2
`18.2
`0.05
`2.6
`3.8
`0.6
`36.5
`0.12
`2.25
`2.5
`0.7
`50 liters (0.065)
`30.0
`a Calculated from an average rhamnose content on the laboratory scale (4) and from Fcmc on the pilot plant scale.
`
`88
`309
`147
`
`140
`140
`85
`
`97
`72
`77
`
`quently effected with methanol. Fractions of 1 to 2 liters
`were collected and analyzed for biosurfactant content by
`measuring interfacial tension and Fcmc values. Fractions
`with Fcmc values above 10 were pooled, and the solvent was
`evaporated (Rotavapor R 151; Buchi, Flawil, Switzerland).
`The adsorbent was regenerated by eluting the column with
`2 to 3 bed volumes of 1 N NaOH in methanol. An additional
`flushing of the column with 20% acetone in 1 N NaOH was
`performed after five adsorption-desorption cycles and then
`the column was rinsed with distilled water until the pH of the
`outlet was neutral. Subsequently 2 to 3 bed volumes of 1 N
`H2SO4 were applied, followed by a distilled water rinse.
`Finally, the column was equilibrated with potassium phos-
`phate buffer (pH 6.1).
`Ion-exchange chromatography. Further purification of sur-
`face-active compounds was achieved by ion-exchange chro-
`matography on DEAE-Sepharose CL 6B (Pharmacia, Up-
`psala, Sweden). DEAE-Sepharose (6.5 liters) was packed
`into a column (400 by 200 mm), yielding a bed volume of
`approximately 6.2 liters and a bed height of 200 mm.
`The column was equilibrated with 10 mM Tris hydrochlo-
`ride buffer (pH 8) containing 10% (vol/vol) ethanol. The
`concentrated residue from the adsorption column was di-
`luted 10-fold with the same buffer containing 20% (vol/vol)
`ethanol and applied to the column after filtration through a
`cotton filter. The flow rate was 6 liters h-1. The effluent was
`again assayed for surface-active compounds to survey the
`charging of the column.
`The column was washed with 2 to 3 bed volumes of 0.1 M
`NaCl in 10 mM Tris hydrochloride buffer containing 10%
`(vol/vol) ethanol. The biosurfactants were then released by
`0.8 M NaCl in the same buffer. Fractions (1 to 2 liters) were
`collected. The fractions with low surface tension (below 35
`mN m-1) were pooled and subjected to a second adsorption
`chromatography on XAD-2 resin. The methanol eluate was
`evaporated in vacuo, and the residue was lyophilized.
`Regeneration of the affinity column was carried out with 2
`M NaCl in Tris hydrochloride buffer (pH 8) containing 20%
`ethanol. After 5 cycles, the column was additionally treated
`with 0.2 M NaOH containing 20% methanol. This removed a
`reddish pigment that also accumulated in the ion-exchange
`column.
`
`RESULTS AND DISCUSSION
`Biosurfactant production. The best conditions for bio-
`surfactant production in the pilot plant were obtained with
`the slightly modified medium 3M of Guerra-Santos et al. (4).
`With a dilution rate of 0.065 h-1 and a 3% glucose concen-
`tration, steady states were maintained for several weeks.
`Residual glucose concentration was approximately 0.7 g
`liter-1, and oxygen partial pressure was always above 50%
`of air saturation.
`Biosurfactant concentration stood at an Fcmc of about 150.
`When an average critical micelle concentration of 15 mg
`
`liter-1 (10) was used, the product concentration p amounted
`to 2.25 g liter-' (Table 1), corresponding to a product yield
`on glucose (Yp,s) of 77 mg of biosurfactant per g of glucose.
`The productivity of the system rp thus amounted to 0.146 g
`liter-' h-1. It follows that a daily production of 80.6 g of
`biosurfactant resulted.
`The biomass concentration in the bioreactor (X) was 2.5 g
`liter-' (Table 1), giving a yield of biomass on glucose (Yx/s)
`of 85 mg of biomass per g of glucose. This is a low yield but
`indicates that the process is not designed for biomass pro-
`duction. Growth is limited by iron and nitrogen (4).
`The surface tension of the culture liquid was below 30 mN
`m-1, and the interfacial tension measured against a mixture
`of aliphatic hydrocarbons (chain length, C14 to C18) was
`below 0.5 mN m-1.
`A comparison of the data from the pilot plant with that of
`the laboratory scale is given in Table 1. It becomes apparent
`that the scale-up of the production process did not exces-
`sively change the performance of the system. On the labo-
`ratory scale, higher steady-state dilution rates were possible
`at which the organism still produced biosurfactants. On the
`pilot plant scale, steady states for biosurfactant and biomass
`production could be established up to a dilution rate of 0.08
`h-1. At higher dilution rates, the system was unstable, and
`washout of the culture occurred. This may be a consequence
`of the lower biomass yields observed in the pilot plant.
`Considering the differences between the cultivations on
`laboratory and pilot plant scales, iron content of the medium
`seems to be the most likely reason for this. On the laboratory
`scale, iron needed to be added to obtain steady-state condi-
`tions (4). Due to the fact that in the pilot plant all contain-
`ments were made of stainless steel (glass was used in the
`laboratory), iron could be omitted completely from the
`medium. Actually, iron introduced from the equipment and
`with the technical-grade chemicals seemed to be inhibitory
`for the cells in relation to the other medium components.
`With additional iron added, operation of the system at the
`dilution rate used here (0.065 h-1) was not possible. Washout
`of the culture occurred.
`Our data confirm that biosurfactant production is related
`to slowly growing cells (4). Wagner et al. (11) used resting or
`immobilized cells of P. aeruginosa for rhamnolipid produc-
`in accordance with the view that the
`tion. This is
`rhamnolipids are derived from the cell surface and have to be
`considered as secondary metabolites. Wagner et al. (11)
`reported somewhat higher product yields (Yp/s) when glyc-
`erol was the carbon source (approximately 100 mg g-1 of
`glycerol) and considerably higher product yields when n-
`alkanes served as the substrate in experiments with resting
`cells (230 mg g-1 alkanes). However, the productivity of the
`system was substantially lower when the long incubation
`periods (several days) are taken into account. The produc-
`tion of biosurfactants in continuous culture represents a
`most practicable system to control environmental aspects of
`
`PETITIONERS
`
`EXHIBIT NO. 1030 Page 3 of 5
`
`

`

`988
`
`REILING ET AL.
`
`APPL. ENVIRON. MICROBIOL.
`
`EzE
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`
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`Bed volumes
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`FIG. 2. Release of surface-active material from DEAE-Sepharose CL 6B after the first loading of the column (A) and after several
`regenerations (B).
`
`0
`
`4
`
`8
`
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`
`biosurfactant production and obviously allows efficient ex-
`ploitation of the potential of the cells.
`A problem of the continuous production process is the
`excessive foaming of the culture. The amount of foam is
`dependent on the initial glucose concentration and on the pH
`of the culture. It increases with both increasing glucose
`concentrations and increasing pH values. In the pilot plant,
`it was not possible to apply glucose concentrations higher
`than 30 g liter-', whereas, on the lab scale, glucose concen-
`trations of up to 70 g liter-' were used. Generally, the culture
`in the 50-liter bioreactor was more labile than the one in the
`5-liter bioreactor, most probably because the chemicals were
`of different purity and the materials for the containments
`were different from those used in the pilot plant. As a
`consequence, disturbances from excess foaming resulted in
`a decrease of biosurfactant production. Only with the con-
`ditions described and properly functioning process control
`could steady states be maintained for extended periods of
`time.
`Foam formation was decreased at pH values below 6,
`whereas the optimum for biosurfactant production was
`found to be at pH 6.25. Again, reliable pH control was
`necessary for avoiding disturbances of the steady-state pro-
`duction.
`Downstream processing. The adsorption on Amberlite
`XAD-2 proved to be the best method for the initial enrich-
`
`ment of the rhamnolipid biosurfactants on both laboratory
`and pilot plant scales. The flow rate to the column had to be
`adjusted according to the biosurfactant concentration and
`the degree of loading of the column. It varied between 10 and
`20 liters h-1. Clogging of the column during the loading
`procedure probably resulted from the fact that the cell-free
`culture liquid contained polysaccharide material which was
`also formed by the cells.
`The capacity of the resin for biosurfactant was calculated
`to be 60 g of surface-active material kg-' of XAD-2. The
`methanol eluate from this enrichment step contained on the
`average 60% pure active material. The elution volume of the
`active fractions amounted to 1 bed volume which corre-
`sponded to approximately 10 liters. Recovery of surface-
`active compounds on the pilot plant level from the adsorp-
`tion column was above 75%. The adsorption column was
`regenerated 15 to 20 times without losing efficiency or
`capacity.
`Anion-exchange chromatography on DEAE-Sepharose
`CL 6B was chosen as a further purification step. A specific
`enrichment is possible by this type of exchanger because
`rhamnolipids contain a carboxylic acid group. The elution of
`the surface-active compounds was elaborated on the labora-
`tory scale (Fig. 2). The material was released from the matrix
`with 0.6 M NaCl in a volume corresponding to approxi-
`mately 2 bed volumes. Regeneration of the column did not
`
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`EXHIBIT NO. 1030 Page 4 of 5
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`

`

`VOL. 51, 1986
`
`PILOT PLANT BIOSURFACTANT PRODUCTION
`
`989
`
`change the characteristics of the exchanger. On the pilot
`plant scale it was found that, with 0.6 M NaCl, too many bed
`volumes were required to release the active compounds.
`With the 0.8 M NaCI solution, the active material was
`released in 8 to 10 bed volumes. This increase in elution
`volume on the pilot plant scale was probably due to changing
`flow characteristics from the small to the larger scale.
`Furthermore, the capacity of the exchanger was more ex-
`hausted before elution in the pilot plant than in the labora-
`tory.
`Before elution, the column was flushed with 0.1 M NaCl to
`remove weakly bound products. Ion-exchange chromatogra-
`phy yielded an about 90% enriched product which was
`passed once more over an adsorption column (Amberlite
`XAD-2), and the biosurfactant was released with methanol.
`The solvent was evaporated, and the highly viscous residue
`was lyophilized. A slightly brownish powder resulted which
`was stored in a dessicator because the biosurfactants were
`highly hygroscopic.
`The anion-exchange chromatography proved to be the
`best method for further enrichment of the biosurfactants. It
`was, for example, superior to acid precipitation of the
`biosurfactant, which gave rise to higher losses. The recovery
`of the products from ion-exchange chromatography was
`over 90%. The binding capacity of the DEAE-Sepharose CL
`6B was approximately 50 g of biosurfactant per liter of gel.
`The disadvantages of ion-exchange chromatography con-
`cern the cost of the exchanger and the relatively low flow
`rates of about 6 liters h-' applicable. Further improvement
`should be possible by changing the design and packing of the
`column. On the laboratory scale, we also tried other ion-
`exchange materials (e.g., DEAE-Sephadex), but none of
`them was as well suited as DEAE-Sepharose with respect to
`elution and regeneration.
`The overall recovery of the surface-active compound by
`the methods described (centrifugation and three chromatog-
`raphy steps) was approximately 60%. This value also con-
`tains inaccuracies originating from the exact amount of the
`two rhamnolipids present in culture liquid. The calculations
`reported were based on rhamnose content and the relative
`amount of both rhamnolipids, which was determined in the
`early stages of the work to be of R-1 (one rhamnose unit) and
`of R-2 (two rhamnose units). The exact composition may
`have changed slightly, depending on the cultivation condi-
`tions and therefore, makes the significance of the calculated
`values only relative. Nevertheless, the methods elaborated
`
`proved to be applicable on the pilot plant level, and it was
`possible to demonstrate that large-scale extraction can be
`avoided in a production process for biosurfactants.
`
`ACKNOWLEDGMENT
`This work was financially supported by Petrotec Systems AG.
`
`LITERATURE CITED
`1. Edwards, J. R., and J. A. Hayashy. 1965. Structure of a
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`2. Einsele, A., and D. Karrer. 1980. Design and characterization of
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`3. Finnerty, W. R., and M. E. Singer. 1983. Microbial enhance-
`ment of oil recovery. Bio-technology 1:47-54.
`4. Guerra-Santos, L. H., 0. Kappeli, and A. Fiechter. 1984. Pseu-
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`culture with glucose as carbon source. Appl. Environ. Micro-
`biol. 48:301-305.
`5. Hisatsuka, K., T. Nakahara, N. Sano, and K. Yamada. 1971.
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`function in hydrocarbon fermentation. Agr. Biol. Chem.
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`7. Itoh, S., and T. Suzuki. 1972. Effect of rhamnolipids on growth
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`public of Germany.
`9. Reiling, H. E., H. Laurila, and A. Fiechter. 1985. Mass culture of
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`Neue Verfahren zur mikrobiellen Herstellung grenz-
`flachenaktiver anionischer Glykolipide. Biotechnol. Forum
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`11. Wagner, F., J.-S. Kim, S. Long, Z.-Y. Li, G. Merwede, U.
`Matulovic, E. Ristau, and C. Syldatk. 1984. Production of
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