Maximum virginiamycin production by optimization of cultivation conditions in batch culture with autoregulator addition

A strategy for optimization of non-growth-associated production in batch culture employing an empirical approach was developed through the study of virginiamycin production. The strategy is formulated with two aims: attaining a high cell concentration at the beginning of the production phase without decrease in production activity; and enhancing the production activity during the production phase. As a practical example, the goal of a maximum virginiamycin (M and S) production in the batch culture of Streptomyces virginiae was set. To attain a high cell concentration in the production phase of the batch culture, that is, to extend the growth phase for as long as possible, the optimum composition and concentration of the complex medium, especially the yeast extract (YE) concentration, were first investigated. Dissolved oxygen (DO) concentration control was also a parameter considered in maintaining the production activity during the production phase. In addition, to enhance the production activity, an optimum addition strategy of an autoregulator, virginiae butanolide-C (VB-C), was investigated. Combining these measures, the optimum cultivation conditions were found to be an initial YE concentration in the complex medium of 45 g/L, the shot addition of 300 mug/L of VB-C 11.5 h after the start of the batch culture, and a DO concentration maintained above 2 mg/L. The maximum concentrations of virginiamycin M and S were about ninefold those obtained under nonoptimum cultivation conditions. Nonoptimum cultivation conditions consisted of an initial YE concentration one sixth (7.5 g/L) that of the optimum cultivation conditions, and no VB-C addition. These conditions were used as representative of the standard cultivation of virginiamycin in this study. The strategy developed here will be applicable to the production of other antibiotics, especially to the cultivation of Streptomyces species, in which a hormonelike signal material (an autoregulator) plays an important role in antibiotic production. (c) 1996 John Wiley & Sons, Inc.     

Comparative study on the influence of dissolved oxygen control approaches on the microbial hyaluronic acid production of <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

Three different dissolved oxygen (DO) control approaches were proposed to improve hyaluronic acid (HA) production: a three-stage agitation speed control approach, a two-stage DO control approach, and an oxygen vector perfluorodecalin (PFC) applied approach. In the three-stage agitation speed control approach, agitation speed was 200 rpm during 0–8 h, 400 rpm during 8–12 h, and 600 rpm during 12–20 h. In the two-stage DO control strategy, DO was controlled at above 10% during 0–8 h and at 5% during 8–20 h. In the PFC applied approach, PFC (3% v/v) was added at 8 h. HA production reached 5.5 g/L in the three-stage agitation speed control culture model, and 6.3 g/L in two-stage DO control culture model, and 6.6 g/L in the PFC applied culture model. Compared with the other two DO control approaches, the PFC applied approach had a lower shear stress and thus a higher HA production was achieved.     

Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

An oxygen supply strategy for the large-scale production of tissue plasminogen activator by microcarrier cell culture

An oxygen supply strategy involving agitation speed and aeration method for the large-scale production of tissue plasminogen activator (TPA) by a microcarrier cell culture was investigated by small-scale model experiments. A preliminary calculation indicated that diffusion limitation of dissolved oxygen (DO) could be caused in a microcarrier sedimentation layer more than 0.5 mm in thickness. Within an agitation speed range above 70 rpm, which was the critical speed for all of the microcarrier beads to remain suspended and thus for avoiding a deficiency of DO, the TPA productivity was higher at a lower agitation speed, while the cell concentration was not affected by the agitation speed. The addition of soluble starch to the culture medium prevented sedimentation of the microcarrier beads, even at the low agitation speed of 20 rpm, resulting in a TPA productivity higher than that at 70 rpm, which was the optimum speed without soluble starch. Use of an air spray system with an optimized air flow rate resulted in a k_La 2.35 times higher than that with simple surface aeration. Increasing the internal pressure of the culture from 0.2 kg/cm² (1209 hPa) to 1.5 kg/cm² (2483 hPa) had no effect on the cell growth but slightly increased the TPA production rates. However, based on the glucose consumption, both the cell and TPA yields were much improved by pressurization. As an optimum mixing and oxygen supply strategy for the production of TPA on a large scale, it is recommended that soluble starch be added to the culture medium to allow the microcarrier suspension to be maintained at a low agitation speed, while keeping a high oxygen transfer rate by means of an air spray system and pressurization.     

The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension

Shake-flask cultivation of T. lanuginosus strain SSBP on coarse corn cobs yielded β-xylanase levels of 56,500 nkat/ml at 50 °C, whereas other hemicellulases (β-xylosidase, β-glucosidase, and α-l-arabinofuranosidase) were produced at levels less than 7 nkat/ml. Cultivation on d-xylose yielded much lower levels of xylanase (350 nkat/ml), although other hemicellulase levels were similar to those produced on corn cobs. The influence of agitation rate and dissolved oxygen tension (DOT) on hemicellulase production was studied further in a bioreactor. On xylose, xylanase activities of 4,330 nkat/ml and 4,900 nkat/ml were obtained at stirrer speeds up to 1,400 rpm to control DOT. At a constant stirrer speed of 400 rpm, xylanase activities of 10,930 nkat/ml and 15,630 nkat/ml were obtained when cultivated on xylose and beechwood xylan respectively, despite DOT levels below 5% for the duration of fermentation. The results indicate that there is an interaction between agitation rate and DOT, impacting on xylanase and accessory enzyme production. Higher agitation rates favoured the production of xylosidase, arabinofuranosidase and glucosidase by T. lanuginosus strain SSBP, whereas the lower agitation rates favoured xylanase production. Rheological difficulties precluded cultivation on corn cobs in the bioreactor. Volumetric xylanase productivities of 1,060,000 nkat/l · h and 589,000 nkat/l · h obtained on beechwood xylan and xylose indicate that T. lanuginosus strain SSBP is a hyper-xylanase producer with considerable industrial potential. Received: 5 May 1999 / Received revision: 31 May 2000 / Accepted: 3 June 2000     

Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture?

  When Aureobasidium pullulans was grown at a number of agitation rates under batch conditions, exopolysaccharide yields were dramatically reduced at high rates i.e. at least 750 rpm. Investigations with gas blending, which allowed pO₂ manipulation and control independently of the agitation rate, showed that this yield reduction was due solely to the high pO₂ levels that occurred at these agitation rates. Thus, polysaccharide production at 1000 rpm could be elevated by maintaining the pO₂ at a low level during the initial phase of the fermentation. However, both the timing of the pO₂ decrease and the level at which it was maintained were crucial for obtaining yields at 1000 rpm, similar to those observed at low agitation rates. Received: 29 February 1996 / Received revision: 11 July 1996 / Accepted: 15 July 1996     

Alginate production by Azotobacter vinelandii DSM576 in batch fermentation

The kinetics of growth and alginate production from glucose in a nitrogen and phosphate-rich medium by Azotobacter vinelandii DSM576 were studied in a laboratory fermenter at pH 7 and 35°C. Batch fermentations were carried out both without control of dissolved oxygen concentration (DO) and at 1, 2, 5 and 10% DO. Although growth was faster at higher DO, maximum biomass concentration was lower. No alginate was produced at 10% DO. Alginate production was faster at 5 and 2% DO but higher alginate concentrations and yields were obtained without DO control. Alginate production was growth-associated at 5% DO, but significant amounts of alginate were produced after growth had stopped at lower DO values. In fermentations without DO control the molecular weight of the polymer reached a maximum (11–17.6 × 10⁴) when specific growth rate was between 0.02 and 0.04 h⁻¹ and residual concentration of ammoniacal nitrogen was between 0.01 and 0.02 g L⁻¹ and then sharply decreased. Received 15 August 1997/ Accepted in revised form 08 January 1998     

Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter vinelandii DSM576 in batch fermentation

Growth and alginate production by Azotobacter vinelandii DSM576 as a function of initial ammonium sulphate concentration (0.45–1.05 g l⁻¹) and agitation speed (300–700 rpm) were studied in batch fermentations at controlled pH. The time course of growth, alginate production and substrate consumption and the effect of nitrogen concentration and agitation speed on kinetic parameters and on maximum alginate molecular weight (MW) was modelled using empirical equations. The kinetics of growth, alginate production and polymerization were deeply affected by agitation speed and, to a lesser extent, by inorganic nitrogen concentration. Average and maximum specific growth rate and maximum alginate MW all increased with agitation speed, and were higher at intermediate ammonium sulphate concentration. Maximum alginate MW (>250,000) was obtained at high agitation speed (600–700 rpm) and alginate depolymerization was limited or did not occur at all when the agitation speed was higher than 500 rpm, while at 400 rpm depolymerization significantly reduced the alginate. However, alginate yield was negatively affected by increasing agitation speed. A good compromise between alginate yield (>2 g l⁻¹) and quality (MW>250,000) was obtained with agitation speed of 500–600 rpm and 0.75–0.90 g l⁻¹ of ammonium sulphate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 242–248. Received 23 February 2000/ Accepted in revised form 04 August 2000     

Use of virginiamycin to control the growth of lactic acid bacteria during alcohol fermentation

The antibiotic virginiamycin was investigated for its effects on growth and lactic acid production by seven strains of lactobacilli during the alcoholic fermentation of wheat mash by yeast. The lowest concentration of virginiamycin tested (0.5 mg Lactrol ^TMkg⁻¹ mash), was effective against most of the lactic acid bacteria under study, but Lactobacillus plantarum was not significantly inhibited at this concentration. The use of virginiamycin prevented or reduced potential yield losses of up to 11% of the produced ethanol due to the growth and metabolism of lactobacilli. However, when the same concentration of virginiamycin was added to mash not inoculated with yeast, Lactobacillus rhamnosus and L. paracasei grew after an extensive lag of 48 h and L. plantarum grew after a similar lag even in the presence of 2 mg virginiamycin kg⁻¹ mash. Results showed a variation in sensitivity to virginiamycin between the different strains tested and also a possible reduction in effectiveness of virginiamycin over prolonged incubation in wheat mash, especially in the absence of yeast. Received 05 August 1996/ Accepted in revised form 18 December 1996     

Production of rosmarinic acid by<Emphasis Type="Italic">Lavandula vera</Emphasis> MM cell suspension in bioreactor: effect of dissolved oxygen concentration and agitation

The relationship between dissolved oxygen (DO) concentration, agitation rate and growth of Lavandula vera MM and rosmarinic acid biosynthesis was investigated in 3 l laboratory bioreactor. Lavandula vera MM cell suspension accumulated the highest amounts of biomass (34.8 g/l) and rosmarinic acid (1870.6 mg/l) on day 12 of cultivation at 50% dissolved oxygen and agitation speed 100 rpm and at 30% dissolved oxygen and agitation speed 300 rpm, respectively.     

Heterotrophic cultivation of Euglena gracilis Z for efficient production of α-tocopherol

Effects of hydrodynamic stress, dissolved oxygen (DO) concentration and carbon sources on heterotrophic α-tocopherol production by Euglena gracilis were investigated. In a jar fermentor without baffle plates, increasing the agitation speed up to 500 rpm had no significant effect on cell growth and α-tocopherol production. However, in a jar fermentor equipped with baffle plates, both the cell growth and α-tocopherol production were highly suppressed at 500 rpm. At high hydrodynamic stress, the cells secreted nucleic acid-related substances to the culture broth and the shape of the cells shifted from elongated toward spherical. High DO concentration had adverse effects on both cell growth and α-tocopherol production, the optimum DO concentration being below 0.8 ppm. In comparison with glucose, the growth rate was lower but the α-tocopherol content of the cells was almost four times higher when ethanol was used as the organic carbon source. In a fed-batch culture with ethanol, a very high cell concentration of 39.5 g L^-1 was obtained with α-tocopherol content of 1200 μg g-cell^-1. This α-tocopherol content is very close to the values reported for photoautotrophic and photoheterotrophic cultures. A very high α-tocopherol productivity of 102 μg L^-1 h^-1 was obtained, indicating that heterotrophic cultivation of E. gracilis has a very high potential as a substitute for the current method of extraction from vegetable oils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of 2,3-butanediol by newly isolated Enterobacter cloacae

Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for conversion of l-arabinose to fuel ethanol. The major product of fermentation by the bacterium was meso-2,3-butanediol (2,3-BD). In a typical fermentation, a BD yield of 0.4 g/g arabinose was obtained with a corresponding productivity of 0.63 g/l per hour at an initial arabinose concentration of 50 g/l. The effects of initial arabinose concentration, temperature, pH, agitation, various monosaccharides, and multiple sugar mixtures on 2,3-BD production were investigated. BD productivity, yield, and byproduct formation were influenced significantly within these parameters. The bacterium utilized sugars from acid plus enzyme saccharified corn fiber and produced BD (0.35 g/g available sugars). It also produced BD from dilute acid pretreated corn fiber by simultaneous saccharification and fermentation (0.34 g/g theoretical sugars). Received: 17 December 1998 / Revision received: 9 March 1999 / Accepted: 20 March 1999     

Enhancement of ribitol production during fermentation of <Emphasis Type="Italic">Trichosporonoides oedocephalis</Emphasis> ATCC 16958 by optimizing the medium and altering agitation strategies

To maximize the productivity of ribitol, which is an important starting material for the production of one expensive rare sugar, L-ribose, the effects of culture medium and agitation speed on cell growth as well as on the productivity of ribitol were thoroughly investigated in a 7 L fermentor. The maximum volumetric productivity, 0.322 g/L/h of ribitol, were obtained at an initial glucose concentration of 200 g/L in a batch culture. Based on the optimum glucose concentration, the ribitol yield conversed from glucose was up to 0.193 g/g when 1% yeast extract was used as a nitrogen source. When the agitation speed was maintained at 200 rpm, the ribitol concentration of 38.60 g/L was collected after 120 h of cultivation time. Additionally, the scheme of two-phase agitation and glucose infusion was employed. To begin, in the first 24 h of fermentation, a high agitation rate at 350 rpm and the initial glucose concentration of 50 g/L were applied, and the biomass concentration of 25.50 g/L was achieved at 36 h of incubation; whereas this value was observed until 60 h in the former batch fermentation methods. Then, in the second phase, with the agitation speed reduced to 150 rpm and the infusion amount of glucose controlled at 150 g/L, the yield of ribitol reached to 65.00 g/L in two-phase agitation fermentation and was 1.68 fold of that obtained in one-stage batch fermentation. To our knowledge, this study first demonstrates its significant effectiveness in improving ribitol production with the application of Trichosporonoides oedocephalis ATCC 16958.     

Optimum autoregulator addition strategy for maximum virginiamycin production in batch culture of Streptomyces virginiae

Virginiae butanolides (VBs) are autoregulators of Streptomyces virginiae, which induce virginiamycin biosynthesis. Generally, autoregulators are synthesized by the microorganism itself during culture. Addition of chemically synthesized virginiae butanolide-C (VB-C), which is one of the VBs, can also control the induction time and the amount of virginiamycin production. The optimum concentration and shot-feeding time of VB-C for the maximum production of virginiamycins M and S were investigated in flasks and jar-fermentor batch cultures. VB-C addition later than 8 h from the start of culture induced not only virginiamycin M and S synthesis but also VB synthesis. Virginiamycin M and S production increased with the decrease of total VBs (produced VBs and added VB-C) concentration. That is, although VBs are needed to induce virginiamycin M and S synthesis, the amount of VB-C added should be such that as small an amount as possible of VBs is synthesized to achieve the maximum production of virginiamycins M and S. However, the VB-C addition earlier than 8 h from the start of culture showed no clear relationship between the amounts of VBs and virginiamycins M and S produced. In conclusion, the maximum production of virginiamycins M and S was attained by the shot addition of 5 mug/L VB-C at 8 h from the start of culture. The maximum value was about twofold that without VB-C addition. The optimum addition strategy of VB-C was confirmed by the jar-fermentor experiments. (c) 1995 John Wiley & Sons, Inc.     

Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy

The effect of agitation speeds on the performance of producing pyruvate by a multi-vitamin auxotrophic yeast, Torulopsis glabrata, was investigated in batch fermentation. High pyruvate yield on glucose (0.797 g g(-1)) was achieved under high agitation speed (700 rpm), but the glucose consumption rate was rather low (1.14 g l(-1) h(-1)). Glucose consumption was enhanced under low agitation speed (500 rpm), but the pyruvate yield on glucose decreased to 0.483 g g(-1). Glycerol production was observed under low agitation speed and decreased with increasing agitation speed. Based on process analysis and carbon flux distribution calculation, a two-stage oxygen supply control strategy was proposed, in which the agitation speed was controlled at 700 rpm in the first 16 h and then switched to 500 rpm. This was experimentally proven to be successful. Relatively high concentration of pyruvate (69.4 g l(-1)), high pyruvate yield on glucose (0.636 g g(-1)), and high glucose consumption rate (1.95 g l(-1)h(-1)) were achieved by applying this strategy. The productivity (1.24 g l(-1) h(-1)) was improved by 36%, 23% and 31%, respectively, compared with fermentations in which agitation speeds were kept constant at 700 rpm, 600 rpm, and 500 rpm. Experimental results indicate that the difference between the performances for producing pyruvate under a favorable state of oxygen supply (dissolved oxygen concentration >50%) was caused by the different regeneration pathways of NADH generated from glycolysis.     

Colonisation of phase II compost by biotypes of Trichoderma harzianum and their effect on mushroom yield and quality

Colonisation assessments confirmed that Trichoderma harzianum biotypes Th1, Th2a, Th2b and Th3 inoculated into two distinct compost types at spawning became established by first flush assessment; the extension rate of two Th2 isolates was over 1000 times that of Th1 and Th3. Results subsequently confirmed that while Th1 and Th3 did not significantly affect yield, Th2 could reduce mushroom quality and productivity by as much as 80%. Analysis of compost type also indicated that the speed and magnitude of T. harzianum colonisation was influenced by key compost characteristics, most notably, moisture, ash content and degree of fermentation. This study has shown that compost parameters which have a positive influence on Agaricus growth and productivity also resulted in increased compost colonisation by T. harzianum. Commercially acceptable yields obtained from uninoculated compost confirmed that production of a high quality, productive substrate does not confer inherent immunity to colonisation by T. harzianum. Received: 25 September 1998 / Received revision: 30 November 1998 / Accepted: 5 December 1998     

Enhanced welan gum production using a two-stage agitation speed control strategy in Alcaligenes sp. CGMCC2428

Batch fermentative production of welan gum by Alcaligenes sp. CGMCC2428 was investigated under various oxygen supply conditions using regulating agitation speed. Based on a three kinetic parameters analysis that includes specific cell growth rate (μ), specific glucose consumption rate (q _s), and specific welan formation rate (q _p), a two-stage agitation speed control strategy was proposed to achieve high concentration, high yield, and high viscosity of welan. During the first 22 h, the agitation speed in 7.5 L fermenter was controlled at 800 rpm to maintain high μ for cell growth. The agitation was then reduced step-wise to 600 rpm to maintain a changing profile with stable dissolved oxygen levels and obtain high qp for high welan accumulation. Finally, the maximum concentration of welan was reached at 26.3 ± 0.89 g L⁻¹ with a yield of 0.53 ± 0.003 g g⁻¹ and the welan gum viscosity of 3.05 ± 0.10 Pa s, which increased by an average of 15.4, 15.2, and 20.1% over the best results controlled by constant agitation speeds.     

Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45–60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD⁺, NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20–50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process.     

Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture. Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998     

Optimisation of the operational conditions of trichloroethylene degradation using Trametes versicolor under quinone redox cycling conditions using central composite design methodology

Extracellular radicals produced by Trametes versicolor under quinone redox cycling conditions can degrade a large variety of pollutant compounds, including trichloroethylene (TCE). This study investigated the effect of the agitation speed and the gas–liquid phase volume ratio on TCE degradation using central composite design (CCD) methodology for a future scale-up to a reactor system. The agitation speed ranged from 90 to 200 rpm, and the volume ratio ranged from 0.5 to 4.4. The results demonstrated the important and positive effect of the agitation speed and an interaction between the two factors on TCE degradation. Although the volume ratio did not have a significant effect if the agitation speed value was between 160 and 200 rpm, at lower speed values, the specific pollutant degradation was clearly more extensive at low volume ratios than at high volume ratios. The fitted response surface was validated by performing an experiment using the parameter combination in the model that maximised TCE degradation. The results of the experiments carried out using different biomass concentrations demonstrated that the biomass concentration had a positive effect on pollutant degradation if the amount of biomass present was lower than 1.6 g dry weight l⁻¹. The results show that the maximum TCE degradation was obtained at the highest speed (200 rpm), gas–liquid phase volume ratio (4.4), and a biomass concentration of 1.6 g dry weight l⁻¹.