A new strategy for lipid production by mix cultivation of Spirulina platensis and Rhodotorula glutinis

Mix cultivation of microalgae (Spirulina platensis) and yeast (Rhodotorula glutinis) for lipid production was studied. Mixing cultivation of the two microorganisms significantly increased the accumulation of total biomass and total lipid yield. Dissolved oxygen and medium components in the mixed fermentation medium were analyzed. Mix cultivation in monosodium glutamate wastewater was further studied. Result indicated 1,600 mg/L of biomass was obtained and 73% of COD were removed.     

Nutrient Removal and Biomass Production in an Outdoor Pilot-Scale Phototrophic Biofilm Reactor for Effluent Polishing

An innovative pilot-scale phototrophic biofilm reactor was evaluated over a 5-month period to determine its capacity to remove nitrogen and phosphorus from Dutch municipal wastewater effluents. The areal biomass production rate ranged between 2.7 and 4.5 g dry weight/m²/day. The areal nitrogen and phosphorus removal rates averaged 0.13 g N/m²/day and 0.023 g P/m²/day, which are low compared to removal rates achieved in laboratory biofilm reactors. Nutrient removal increased during the day, decreased with decreasing light intensity and no removal occurred during the night. Additional carbon dioxide supply was not requisite as the wastewater was comprised of enough inorganic carbon to sustain microalgal growth. The study was not conclusive for the limiting factor that caused the low nutrient removal rate, possibly the process was limited by light and temperature, in combination with pH increases above pH 9 during the daytime. This pilot-scale study demonstrated that the proposed phototrophic biofilm reactor is not a viable post-treatment of municipal wastewater effluents under Dutch climate conditions. However, the reactor performance may be improved when controlling the pH and the temperatures in the morning. With these adaptations, a phototrophic biofilm reactor could be feasible at lower latitudes with higher irradiance levels.     

Improved Carotenoid Productivity and COD Removal Efficiency by Co-culture of Rhodotorula glutinis and Chlorella vulgaris Using Starch Wastewaters as Raw Material

Utilization of low-cost raw materials for the bio-based chemical production, such as carotenoids, by the co-culture of Rhodotorula glutinis and Chlorella vulgaris has recently become an attractive option. In this study, the primary nutrients of starch wastewater were analyzed, which were used for carotenoid production by the co-culture strategy in a 5-L fermenter around 4000 Lux light intensity. Synergistic effect of gas utilization revealed that the two species could build up the beneficial balance on mutualism. The maximum carotenoid productivity and COD removal efficiency were 12.34 mg/L and 79.6%, respectively, which were higher than those of monoculture yeast (8.31 mg/L and 54.1%). The organic acids, amino acids, and sugar removal efficiencies were increased by 85%, 31%, and 44%, respectively, and more than three kinds of carotenoids were identified compared with those of monoculture yeast. The results demonstrated that the co-culture strategy of two different nutritional microorganisms could significantly improve carotenoid productivity and COD removal efficiency.

     

The Utilization of Post-chlorinated Municipal Domestic Wastewater for Biomass and Lipid Production by <Emphasis Type="Italic">Chlorella</Emphasis> spp. Under Batch Conditions

South Africa has a rich microalgal biodiversity which has the potential to be used for renewable bio-fuel production in the region. Bioprospecting for oleaginous microalgae in KwaZulu Natal Province, South Africa, resulted in the establishment of a microalgal culture collection system for alternative energy research in the country. A potential hyper-lipid-producing Chlorella spp. strain was isolated, purified, and cultured in supplemented post-chlorinated wastewater for biomass and lipid production at the laboratory scale under batch mode. The microalgal strain was cultivated in different strengths of BG-11 media supplemented with wastewater from a local municipal domestic wastewater treatment plant. The Chlorella spp. was grown using ambient dissolved carbon dioxide in shake flasks under photosynthetically active radiation (±120 μmolm⁻²s⁻¹). Microalgal biomass and lipid productivity were monitored at 24-h intervals in the batch mode. The microalgal biomass was analyzed by direct light microscopy and indirectly by spectrophotometry at 600 nm, and the lipids were extracted and quantified. The growth rate of the Chlorella spp. was enhanced in post-chlorinated wastewater supplemented with 5 mM NaNO₃ with maximal biomass productivity. A dramatic increase in lipid yield was achieved with the post-chlorinated wastewater supplemented with 25 mM NaNO₃. Low dosages of free chlorine were found to enhance microalgal growth. These findings serve as a basis for further scale-up trials using municipal wastewater as a medium for microalgal biomass and lipid production.     

Analysis of hormone antagonists in clinical and municipal wastewater by isotopic dilution liquid chromatography tandem mass spectrometry

A comprehensive method was developed for the simultaneous trace analysis of ten hormone antagonist pharmaceuticals (raloxifene, exemestane, letrozole, anastrozole, mifepristone, finastride, tamoxifen, N-desmethyltamoxifen, clomiphene, and toremifene) in municipal sewage and hospital wastewater samples. The target compounds were firstly extracted using an Oasis HLB cartridge, followed by purification by an aminopropyl cartridge, and were then analyzed by liquid chromatography electrospray ionization tandem mass spectrometry in positive ion mode. The recoveries for the analytes based on internal standard calibration in different test matrices ranged from 67.6 to 118.6% (with the exception of mifepristone in clinical wastewater samples), with relative standard deviations less than 20%. The method quantification limits of the ten pharmaceuticals were in the range 0.10–2.0 ng/L. Excluding exemestane and N-desmethyltamoxifen, eight drugs were detected at 0.20–195.0 ng/L in hospital wastewater and municipal wastewater samples from Beijing.     

Modified <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> biomass for sorption of <Superscript>241</Superscript>Am and other radionuclides

The improvement and the refinement of non-viable Rhizopus arrhizus biomass were investigated via immobilization. Immobilization was carried out by using sodium alginate/CaCl₂ solution and formaldehyde/HCl cross-linking with dead Rhizopus arrhizus biomass and were used for the sorption of radionuclides from low level effluent wastes. The sodium alginate/CaCl₂ immobilized biomass (ratio 1:2) showed about 86% sorption for ²⁴¹Am activity but due to its soft nature and tendency to undergo distortion in shape, is unsuitable for practical applications. The biomass cross-linked with 15% formaldehyde/0.1 M HCl solution has a relatively high mechanical strength and rigidity. It was showing a sorption of >99% for ²⁴¹Am activity and has the sorption capacity of ~65 mg/g for americium and uranium. Hence, it can be utilized for the removal of radionuclides from radioactive waste effluents.     

Tetracycline antibiotics in hospital and municipal wastewaters: a pilot study in Portugal

This study investigated the occurrence of tetracyclines (TCs), namely minocycline (MIN), TC, and its epimer epitetracycline (ETC), and doxycycline (DC), in four hospital wastewater effluents and its fate in municipal wastewater treatment plants (WWTPs), in Coimbra, Portugal. Analytical determination was carried out by solid-phase extraction followed by liquid chromatography with fluorescence detection. A gradient system with a mobile phase containing oxalic acid 0.02 M and acetonitrile was used. After postcolumn derivatization with magnesium reagent, TCs were detected at λ _exc 386 nm and λ _em 500 nm. The proposed method allowed good sensitivity, accuracy, and precision. LOQs were 0.5 μg l⁻¹ for ETC and TC and 15 and 5 μg l⁻¹ for MIN and DC, respectively. The recovery values ranged between 66.4% and 117.1%, and intraday and interday repeatability was lower than 6.8%. The method was successfully used to determine the presence of the above-mentioned TCs in 24 wastewater composite samples obtained from hospital effluents and from influent and effluent of the WWTP located in Coimbra, Portugal. MIN and TC were found in 41.7% of the samples; ETC and DC were found in 25% and 8.3% of the samples, respectively. The levels found ranged from 6 to 531.7 μg l⁻¹ in hospital effluents, while its concentrations in WWTP ranged from 95.8 to 915.3 μg l⁻¹. A seasonal influence in the concentrations found has also been observed, the levels found in samples collected during spring being higher than those observed in samples collected during autumn; however, these are only preliminary results. The WWTP removal rate ranged between 89.5% and 100%.     

Rhamnolipid Production by <Emphasis Type="Italic">Pseudomonas Aeruginosa</Emphasis> GIM 32 Using Different Substrates Including Molasses Distillery Wastewater

A rhamnolipid production strain newly isolated from oil-contaminated soil was identified as Pseudomonas aeruginosa GIM32 by its morphology and 16S rDNA sequence analysis. The effect of carbon source and carbon to nitrogen (C/N) ratio on rhamnolipids production was investigated. Palm oil was favorable as a carbon source for rhamnolipid production. The maximum biomass and rhamnolipid concentration were 8.24 g/L and 30.4 g/L, respectively, with an optimization medium containing 50 g/L palm oil and 5 g/L sodium nitrate. Molasses distillery wastewater as an unconventional substrate for rhamnolipid production was investigated. It was found that 2.6 g/L of rhamnolipids was produced; this amount was higher than that of past reports using wastewater as a substrate. In addition, 44% of the chemical oxygen demand of wastewater was removed at the same time under the optimization condition. Eleven kinds of different molecular weight rhamnolipid homologues were identified in the rhamnolipids obtained from molasses distillery wastewater by P. aeruginosa GIM32 by LC–MS analysis.     

Date pits waste as a solid phase extraction sorbent for the analysis of lead in wastewater and for use in manufacturing brick: An eco-friendly waste management approach

Lead (Pb(II)), an extremely hazardous heavy metal that has been shown to have detrimental effects on both the environment and humans, mostly gets into the ecosystem through industrial activities. In this work, a new solid-phase extraction (SPE) based on treated date pits bio-sorbent and iCAP Q inductively-coupled plasma mass spectrometry (iCAP Q ICP/MS) method has been optimized for the trace determination of Pb(II) in various industrial wastewater effluents. A cost-effective biomass material was prepared from date pits (DP), and chemically modified with H₂O₂ and successively used as SPE bio-sorbent for Pb(II) extraction. Extracting solutions for instance H₂SO₄, HNO₃ and HCl at various concentrations (1–5 mM) were optimized, and best extraction of Pb(II) was obtained by HCl (1 mM). The optimized SPE and iCAP Q ICP/MS method has offered excellent validation conditions in terms of coefficient of determination (CoD, R² > 0.999), detection limit (DL, 0.011 µg/L), quantification limit (QL, 0.034 µg/L), and run-to-run and day-to-day precision (RSD < 6 %). The optimized procedure was practically applied in the determination of Pb(II) in industrial wastewater comprising iron and steel, textile, printing and battery industries. Among the analyzed samples, the battery industry produced higher amounts of Pb(II) (18.55 µg/L) followed by iron and steel (14.65 µg/L), petroleum (12.38 µg/L) printing (5.78 µg/L) and textile (3.76 µg/L) industries. The recovery values were achieved between 95 % and 99 %. The obtained results have established the appropriateness of the offered technique as a new useful method for the routine examination of Pb(II) in industrial wastes. In addition, the current method could be expansively used in the proficient removal and identification of other heavy metals contaminants from similar matrices. Further, the metal ions saturated bio-sorbents were used in the preparations of bricks and it was found to be a successful approach for heavy metals and agricultural waste management.     

Heterotrophic Culture of <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> in Various Nitrogen Sources for Lipid Production

The influences of urea, yeast extract, and nitrate as the nitrogen source on heterotrophic growth of four strains of Chlorella protothecoides were investigated in 9-day feed-batch cultures. Biomass dry weight concentration (DWC) and lipid yield (LY) of the four strains in all media were compared. The highest LY in 9 days was 654 mg/L/day by UTEX 255 in 2.4 g/L KNO₃ medium with a biomass DWC of 11.7 g/L and lipid content of 50.5%. Using green autotrophic seeds instead of yellow heterotrophic seeds improved the biomass DWC (13.1 vs. 11.7 g/L), LY (850 vs. 654 mg/L/day), and lipid to glucose consumption ratio (0.607 vs. 0.162). Moreover, 17.0 g/L DWC and 489 mg/L/day LY were obtained from the sequentially mixed-nitrogen medium, and the lipid to glucose consumption ratio was improved to 0.197 from 0.162 in 2.4 g/L nitrate medium and from 0.108 in 4.2 g/L yeast extract medium in the first batch.     

Characterization of Biosorption Process of Acid Orange 7 on Waste Brewery’s Yeast

The use of cheap, high-efficiency, and ecofriendly adsorbent has been studied as an alternative way for the removal of dyes from wastewater. This paper investigated the use of waste brewery’s yeast for the removal of acid orange 7 from aqueous solution. The optimum removal of acid orange 7 was found to be 3.561 mg/g at pH 2.0, 10 mg/L initial concentration and 303 K. The kinetic studies indicated that the biosorption process of acid orange 7 agreed well with the pseudo-second-order model. The external diffusion is the rate-controlling step of the initial fast adsorption (<20 min) and then the intraparticle diffusion dominated the mass transfer process. Langmuir, Freundlich, and Dubinin–Radushkevich models were applied to describe the biosorption isotherm of acid orange 7 by waste brewery’s yeast. Langmuir isotherm model fits the equilibrium data, at all the studied temperatures, better than the other isotherm models which indicates monolayer dye biosorption process. The highest monolayer biosorption capacity was found to be 2.27 × 10⁻³ mol/g at 303 K. The calculated thermodynamic parameters (ΔG, ΔS, ΔH) showed the biosorption process to be spontaneous and exothermic in nature. Amine or amino, amide, carboxyl, phosphate groups are responsible for the dyes biosorption based on the result of Fourier transform infrared analysis.     

Anabolic, doping, and lifestyle drugs, and selected metabolites in wastewater—detection, quantification, and behaviour monitored by high-resolution MS and MS n before and after sewage treatment

Municipal wastewater has been examined for steroids, β₂-agonists, stimulants, diuretics, and phosphodiesterase type V inhibitors (PDE type V inhibitors), which are “dual-use-drugs” applied either as anabolic, doping, and lifestyle drugs or for treatment of diverse diseases. To identify their origin, fitness centre discharges under suspicion of being point sources and sewage-treatment plant feed and effluents were sampled and concentrations determined. Sensitive and selective methods for determination and quantification based on solid-phase extraction (SPE) followed by high-performance liquid chromatography–high resolution mass and tandem mass spectrometry (HPLC–(HR)MS and HPLC–MS–MS) were developed and established for analysis of these compounds in wastewater and to assess their effect on the environment. The methods developed enabled quantification at trace concentrations (limit of quantification (LOQ): 5 ng L⁻¹). Of the steroids and stimulants under investigation, testosterone, methyltestosterone, and boldenone or ephedrine, amphetamine, and MDMA (3,4-methylendioxy-N-methylamphetamine) were observed at up to 5 μg L⁻¹ (ephedrine). Of the β₂-agonists salbutamol only, and of the diuretics furosemide and hydrochlorothiazide were confirmed in the extracts. Quite high concentrations of the PDE type V inhibitors sildenafil, tadalafil, and vardenafil and their metabolites were confirmed in fitness centre discharges (sildenafil: 1,945 ng L⁻¹) whereas their concentrations in municipal wastewater did not exceed 35 ng L⁻¹. This study identified anabolic and doping drugs in wastewater for the first time. Results obtained from wastewater treatment plant effluents proved that these “dual-use-drugs”, with the exception of hydrochlorothiazide, were mostly eliminated.     

Combined biological and chemical assessment of estrogenic activities in wastewater treatment plant effluents

Five wastewater treatment plant effluents were analyzed for known endocrine disrupters and estrogenicity. Estrogenicity was determined by using the yeast estrogen screen (YES) and by measuring the blood plasma vitellogenin (VTG) concentrations in exposed male rainbow trout (Oncorhynchus mykiss). While all wastewater treatment plant effluents contained measurable concentrations of estrogens and gave a positive response with the YES, only at two sites did the male fish have significantly increased VTG blood plasma concentrations after the exposure, compared to pre-exposure concentrations. Estrone (E1) concentrations ranged up to 51 ng L^–1, estradiol (E2) up to 6 ng L^–1, and ethinylestradiol (EE2) up to 2 ng L^–1 in the 90 samples analyzed. Alkylphenols, alkylphenolmonoethoxylates and alkylphenoldiethoxylates, even though found at µg L^–1 concentrations in effluents from wastewater treatment plants with a significant industrial content, did not contribute much to the overall estrogenicity of the samples taken due to their low relative potency. Expected estrogenicities were calculated from the chemical data for each sample by using the principle of concentration additivity and relative potencies of the various chemicals as determined with the yeast estrogen screen. Measured and calculated estradiol equivalents gave the same order of magnitude and correlated rather well (R ²=0.6).An erratum to this article can be found at     

Minerals and Organic Nitrogen Present in Grape Marc Hydrolyzates Enhance Xylose Consumption by <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis>

This work deals with the nutritional evaluation of grape marc hydrolyzates as fermentation medium for Lactobacillus pentosus. Usually, the fermentation of xylose and arabinose in the presence of glucose remains a primary obstacle for economical biomass conversion. The few microorganisms that can grow simultaneously on both pentose and hexose sugars contained in lignocellulosic feedstocks typically grow slowly and demonstrate marginal yields and productivities. Moreover, lignocellulosic hydrolyzates contain phenolic compounds and other components originated by the degradation of sugars that can inhibit lactic acid fermentation. However, in this case, grape marc hydrolyzates not only did not need a detoxification stage, but it also improved the xylose consumption by Lactobacillus pentosus with a faster and more efficient conversion of hemicellulosic sugars compared with synthetic media. After analysis of grape marc hydrolyzates, it was observed that minerals such as K (2,707 mg/L), Ca (3,681 mg/L), and Mg (198.5 mg/L) are present in higher concentration than those found in the general medium of Lactobacillus (1,705 mg/L of K, 58.3 mg/L of Ca, and 27.0 mg/L of Mg). Moreover, grape marc hydrolyzates contain an additional source of nitrogen (9.2 g/L) which, together with their elevated mineral concentration, improved lactic acid fermentation compared with synthetic media.     

<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Growth Under Increased Air Pressure: Influence on Enzyme Production

Improvement of microbial cell cultures oxygenation can be achieved by the increase of total air pressure, which increases oxygen solubility in the medium. In this work, a pressurized bioreactor was used for Yarrowia lipolytica batch cultivation under increased air pressure from 1 to 6 bar. Cell growth was strongly enhanced by the pressure rise. Fivefold and 3.4-fold increases in the biomass production and in specific growth rate, respectively, were observed under 6 bar. The increase of oxygen availability caused the induction of the antioxidant enzyme superoxide dismutase, which indicates that the defensive mechanisms of the cells against oxidative stress were effective and cells could cope with increased pressure. The pregrowth of Y. lipolytica under increased pressure conditions did not affect the lipase production ability of the cells. Moreover, the extracellular lipase activity increased 96% using a 5-bar air pressure instead of air at 1-bar pressure during the enzyme production phase. Thus, air pressure increase in bioreactors is an effective mean of cell mass and enzyme productivity enhancement in bioprocess based in Y. lipolytica cultures.     

Industrial Waste Utilization for Low-Cost Production of Raw Material Oil Through Microbial Fermentation

In view of ever-growing demand of biodiesel, there is an urgent need to look for inexpensive and promising renewable raw material oils for its production. In this context, the aim of this study was to evaluate the potential use of industrial wastes for low-cost production of oils through microbial fermentation. Among the strains tested, Yarrowia lipolytica grew best and produced highest lipid when grown on decanter effluent from palm oil mill. When crude glycerol by-product from a biodiesel plant was added into the effluent as a co-substrate, Y. lipolytica produced a higher biomass of 3.21 g/L and a higher amount of lipid of 2.21 g/L which was 68 % of the dry weight. The scale up and process improvement in a 5-L bioreactor increased the biomass and lipid up to 5.53 and 2.81 g/L, respectively. A semi-continuous mode of operation was an effective mode for biomass enhancement while a fed-batch mode was effective for lipid enhancement. These yeast lipids have potential to be used as biodiesel feedstocks because of their similar fatty acid composition to that of plant oil.     

Analysis of free estrogens and their conjugates in sewage and river waters by solid-phase extraction then liquid chromatography-electrospray-tandem mass spectrometry

Summary ‘Free’ steroidal estrogens have been identified as compounds possibly responsible for endocrine-disruption of aquatic fauna populating rivers in which municipal sewage-treatment plants (STP) discharge their effluents. Natural and synthetic estrogens are excreted, as glucuronides and sulfates, by man, in the urine but these are bioconverted back to the unconjugated forms in wastewater discharges. For this reason we have developed a sensitive analytical procedure, without derivatization, for identification and quantitation of conjugated and free estrogens in surface and waste waters. The hormones were extracted and fractionated, by use of Carbograph cartridges, into neutral and acid fractions which were then analyzed by liquid chromatography-tandem mass spectrometry. Recoveries were between 66 and 100% and limits of detection (LOD) between 15.0 and 0.003 ng L⁻¹, depending on the compound and the water matrix. When this methodology was applied to real sewage and river water we could measure the main free estrogens at ng L⁻¹ levels. Among the conjugates we always observed the presence of estrone 3-sulfate (at levels between 8.0 and 0.5 ng L⁻¹).     

Analytical approaches for Hg determination in wastewater samples by means of total reflection X-ray fluorescence spectrometry

At present, there is a considerable interest in Hg monitoring in wastewater samples due to its widespread occurrence and the high toxicity of most of its compounds. Hg determination in water samples by means of total reflection X-ray fluorescence spectrometry (TXRF) entails some difficulties due to the high vapor pressure and low boiling point of this element that produce evaporation and loss of Hg from the surface of the reflector during the drying process, commonly used for sample preparation in TXRF analysis.The main goal of the present research was to develop a fast and simple chemical strategy to avoid Hg volatilization during the analysis of wastewater samples by TXRF spectrometry. Three different analytical procedures were tested for this purpose: (i) increasing the viscosity of the wastewater sample by adding a non-ionic surfactant (Triton^® X-114), (ii) Hg immobilization on the quartz reflectors using the extractant tri-isobutylphosphine (Cyanex 471X) and (iii) formation of a stable and non-volatile Hg complex into the wastewater sample. The best analytical strategy was found to be the formation of a Hg complex with thiourea (pH = 10) before the deposition of 10 μL of sample on the reflector for following TXRF analysis. Analytical figures of merit such as linearity, limits of detection, accuracy and precision were carefully evaluated. Finally, the developed methodology was applied for the determination of Hg in different types of wastewater samples (industrial effluents, municipal effluents from conventional systems and municipal effluents from constructed wetlands).     

Evaluation of toxic metals in the industrial effluents and their segregation through peanut husk fence for pollution abatement

The industrial pollution is exponentially growing in the developing countries due to the discharge of untreated effluents from the industries in the open atmosphere. This may cause severe health hazards in the general public. To reduce this effect, it is essential to remove the toxic and heavy metals from the effluents before their disposal into the biosphere. In this context, samples of the effluents were collected from the textile/yarn, ceramics and pulp/paper industries and the concentrations of the toxic metal ions were determined using neutron activation analysis (NAA) technique. The observed concentration values of the As, Cr and Fe ions, in the unprocessed industrial effluents, were 4.91 ± 0.8, 9.67 ± 0.7 and 9.71 ± 0.8 mg/L, respectively which was well above the standard recommended limits (i.e. 1.0, 1.0 and 2.0 mg/L, respectively). In order to remove the toxic metal ions from the effluents, the samples were treated with pea nut husk fence. After this treatment, 91.5% arsenic, 81.9% chromium and 66.5% iron metal ions were successfully removed from the effluents. Then the treated effluents contained concerned toxic metal ions concentrations within the permissible limits as recommended by the national environmental quality standards (NEQS).     

Bioprocess Optimization of Furanocoumarin Elicitation by Medium Renewal and Re-elicitation: A Perfusion-Based Approach

Effect of various abiotic (methyl jasmonate, salicylic acid) and biotic (yeast extract, Aspergillus niger) elicitors on furanocoumarin production and in situ product removal was studied using shoot cultures of Ruta graveolens L. Elicitation by yeast extract (1% w/v) on day 15 was most effective. It led to 7.8-fold higher furanocoumarin production that was attained 24 h after elicitation and 43% of the product was released into the medium. Changes in the relative concentration of furanocoumarins produced depend on the elicitor used. Molar ratio of bergapten increased to 93% in response to yeast extract. With the perspective of developing a commercially feasible process, an approach for preserving viability of biomass and its reuse needs to be developed. For this, medium renewal strategy was investigated. Removal of the spent medium 48 h after elicitation allowed in situ product removal and proved effective in revival of cultures, allowing reuse of biomass. A week after medium renewal, the revived biomass was re-elicited and a second furanocoumarin production peak was obtained. A perfusion-based bioprocess optimization approach, employing elicitation coupled with medium renewal with subsequent re-elicitation, as a new strategy for improved furanocoumarin production, has been suggested.