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.     

The use of multi-parameter flow cytometry to study the impact of <Emphasis Type="Italic">n</Emphasis>-dodecane additions to marine dinoflagellate microalga <Emphasis Type="Italic">Crypthecodinium cohnii</Emphasis> batch fermentations and DHA production

The physiological response of Crypthecodinium cohnii batch cultivations and docosahexaenoic acid (DHA) production to n-dodecane additions were studied. Different n-dodecane concentrations [0, 0.5, 1, 2.5, 5, 10 and 20% (v/v)] were added to preliminary shake flask cultivations. The n-dodecane fraction that gave best results in terms of biomass and DHA production was 0.5% (v/v). The n-dodecane fractions of 2.5, 5, 10 and 20% (v/v) to C. cohnii preliminary shake flask cultures inhibited the microalgal growth and DHA production, although a high proportion of cells with intact cytoplasmic membrane was present in the end of these fermentations. After the addition of a pulse of n-dodecane (0.5% v/v) to C. cohnii exponential growing cells in a bioreactor, glucose uptake volumetric rate increased 2.5-fold, while biomass production volumetric rate increased 2.8-fold. The specific growth rate was increased 1.5-fold. The DHA % in biomass, DHA % of TFA and DHA concentration also increased (54, 22 and 58%, respectively), after the n-dodecane addition. At this n-dodecane fraction (0.5% v/v), multi-parameter flow cytometry demonstrated that C. cohnii cell membrane integrity was not affected. The results demonstrated that the addition of 0.5% of n-dodecane (v/v) to C. cohnii fermentations can be an easy and cheap way for enhancing the biomass and DHA production, avoiding the use of high speed rates (resulting in important power agitation costs) that affects the microalga proliferation and increases the bioprocess costs. A new strategy to improve the DHA production from this microalga in two-phase large-scale bioreactors is now in progress.     

Growth of Trichoderma reesei RUT C-30 in stirred tank and reciprocating plate bioreactors

A series of fed-batch experiments at different agitation speeds were performed using the industrially important strain Trichoderma reesei RUT C-30 in two different bioreactors to understand the close relationship that exists between the shear field within a bioreactor, the morphology of the microorganism, the rheology of cultivation broth, and the process performance. The two bioreactors, stirred tank bioreactor (STB) and reciprocating plate bioreactor (RPB), are characterized by a significantly different shear field to which microorganisms are exposed. Highest biomass concentration (ca. 15 g l⁻¹) was obtained at higher agitation rates in both bioreactors due to better oxygen supply. However, better filter paper activities per mg of protein were obtained at lower agitation in both bioreactors. In both bioreactors, young and healthier fungi in the batch phase were not affected by shear even at higher agitation rates. However, during the fed-batch phase, higher degree of fragmentation of clump morphology at high agitation intensity was confirmed by image analysis. Also, the rheological analysis showed an increase in apparent viscosity during the batch phase and early fed-batch phase due to the increase in the biomass concentration. During the late stages of cultivation, the apparent viscosity decreased due to cell lysis and spore formation.     

Reduction of ammonia and volatile organic compounds from food waste-composting facilities using a novel anti-clogging biofilter system

The performance of a pilot-scale anti-clogging biofilter system (ABS) was evaluated over a period of 125 days for treating ammonia and volatile organic compounds emitted from a full-scale food waste-composting facility. The pilot-scale ABS was designed to intermittently and automatically remove excess biomass using an agitator. When the pressure drop in the polyurethane filter bed was increased to a set point (50 mm H₂O m⁻¹), due to excess biomass acclimation, the agitator automatically worked by the differential pressure switch, without biofilter shutdown. A high removal efficiency (97-99%) was stably maintained for the 125 days after an acclimation period of 1 week, even thought the inlet gas concentrations fluctuated from 0.16 to 0.55 g m⁻³. Due the intermittent automatic agitation of the filter bed, the biomass concentration and pressure drop in the biofilter were maintained within the ranges of 1.1-2.0 g-DCW g PU⁻¹ and below 50 mm H₂O m⁻¹, respectively.     

Bacterial degradation of styrene in waste gases using a peat filter

A biofiltration process was developed for styrene-containing off-gases using peat as filter material. The average styrene reduction ratio after 190 days of operation was 70% (max. 98%) and the mean styrene elimination capacity was 12 g m⁻³ h⁻¹ (max. 30 g m⁻³ h⁻¹). Efficient styrene degradation required addition of nutrients to the peat, adjustment of the pH to a neutral level and efficient control of the humidity. Maintenance of the water balance was easier in a down-flow than in an up-flow process, the former consequently resulting in much better filtration efficiency. The optimum operation temperature was around 23 °C, but the styrene removal was still satisfactory at 12 °C. Seven different bacterial isolates belonging to the genera Tsukamurella, Pseudomonas, Sphingomonas, Xanthomonas and an unidentified genus in the γ group of the Proteobacteria isolated from the microflora of active peat filter material were capable of styrene degradation. The isolates differed in their capacity to decompose styrene to carbon dioxide and assimilate it to biomass. No toxic intermediate degradation products of styrene were detected in the filter outlet gas or in growing cultures of isolated bacteria. The use of these isolates in industrial biofilters is beneficial at low styrene concentrations and is safe from both the environmental and public health points of view. Received: 30 May 1997 / Received revision: 22 August 1997 / Accepted: 25 August 1997     

Development of a potent <Emphasis Type="Italic">in vitro</Emphasis> source of <Emphasis Type="Italic">Phylanthus amarus</Emphasis> roots with pronounced activity against surface antigen of the hepatitis B virus

Summary In vitro culture of hairy roots of Phyllanthus amarus induced by Agrobacterium rhizogenes was established. Their growth and ability for in vitro inactivation of hepatitis B virus surface antigen was studied and compared with adventitious roots grown in vitro. The selected hairy root clone HR-1 was capable of growing at a very fast rate, and an approximately 900-fold increase in weight of root biomass was achieved after 4 wk of culture in hormone-free quarter-strength liquid Murashige and Skoog medium with continuous agitation. Non-transformed roots cultured in the presence of 1.0 mg l⁻¹ (5.71 μM) indole-3-acetic acid increased by 330-fold. The immuno-inactive property of roots was maximal in the crude extract. The hairy roots were shown to possess 85% inhibition (in contrast to 15% in the control) in binding of hepatitis B surface antigen (HBsAg) to its antibody (anti-HBs) after 24 h of incubation with HbsAg-positive sera in vitro at 37°C. Out of three fractions selected on the basis of molecular weight components of the extract, the Fraction III containing comparatively lower molecular weight substances (≤3500) yielded the highest activity. The extract from non-transformed roots was found to possess similar efficiency (87% inhibition). The levels of activity in both types of in vitro-raised roots were higher than those of naturally occurring roots and leafy shoots. The ability of P. amarus hairy root cultures to yield high biomass with the anti-viral property at high levels may provide an alternative source of raw material for more detailed study in the field of pharmaceutical research.     

Analysis of the biomass quality and photosynthetic efficiency of a nitrogen-fixing cyanobacterium grown outdoors with two agitation systems

The efficiency of two different agitation systems (airlift and paddlewheel) in the biomass photoproduction of a nitrogen-fixing filamentous blue-green alga was evaluated outdoors, and the elemental and molecular composition of the cells grown with each system was analyzed. With the paddlewheel system, the productivity values achieved were over 30% higher than with the airlift system, both in summer and winter. In this last season, a conversion efficiency of total solar energy into stored biomass energy of 3.3% was estimated for the paddlewheel system. Moreover, the algal cells grown with this system exhibited a higher net protein (58.9% of dry weight) and nitrogen (11.3%) content than those grown with the airlift device, with an estimated nitrogen fixation rate of more than 2 g N m(-2) day(-1). These advantages of the paddlewheel system make this procedure more appropriate for the large-scale photoproduction of nitrogen-fixing blue-green algae outdoors.     

Toluene-styrene secondary acclimation improved the styrene removal ability of biotrickling filter

In this work, ceramic pellets were used as packing material to establish a biotrickling filter (BTF) with acclimated sludge being inoculated on the surface of the packing to purify waste gas containing styrene. A method of toluene-styrene secondary acclimation was applied to achieve rapid formation of biological films. Results showed that the total time of start-up was 48 days and the removal efficiency (RE) of styrene reached up to 95%. The suitable empty bed residence time (EBRT) was obtained that is 57 s for higher RE of styrene with the inlet loading rates of 6.7–271.6 g/m³/h. The pH and moisture content showed small effect on styrene removal indicating that the operation of BTF was stable. Biomass accumulation was normal and its rising velocity under the condition of short EBRT was faster than that of long EBRT.     

Biodegradation of gas-phase styrene in a high-performance biotrickling filter using porous polyurethane foam as a packing medium

A biotrickling filter (BTF) packed with porous polyurethane (PU) foam sheets was developed and operated for removal of gas-phase styrene. The specific surface area and void fraction of the PU foam sheet were determined to be 497 m²/m³ and 0.92, respectively, by using mathematical modeling and experimental measurement. The effects of gas flow direction (co-current and counter-current), styrene loading rate and empty bed residence time on the efficiency of the BTF were analyzed. The BTF achieved a high elimination capacity of 4.0 ∼ 5.0 kg styrene/m³ day due to the high specific surface area of the PU foam. The BTF could be operated repeatedly when excessively-grown biomass was periodically removed, using circulating NaOH solution for 2 h every four days.     

Biomass control in waste air biotrickling filters by protozoan predation

Two protozoan species as well as an uncharacterized protozoan consortium were added to a toluene-degrading biotrickling filter to investigate protozoan predation as a means of biomass control. Wet biomass formation in 23.6-L reactors over a 77-day period was reduced from 13.875 kg in a control biotrickling filter to 11.795 kg in a biotrickling filter enriched with protozoa. The average toluene vapor elimination capacity at 1 g/m3 toluene and 64 m3/(m3. h) was 31.1 g/(m3. h) in the control and 32.2 g/(m3. h) in the biotrickling filter enriched with protozoa. At higher toluene inlet concentrations, toluene degradation rates increased and were slightly higher in the biotrickling filter enriched with protozoa. The lower rate of biomass accumulation after the addition of protozoa was due to an increase of carbon mineralization (68% as compared to 61% in the control). Apparent biomass yield coefficients in the control and enriched trickling filter were 0.72 and 0.59 g dry biomass/g toluene, respectively. The results show that protozoan predation may be a useful tool to control biomass in biotrickling filters, however, further stimulation of predation of the biomass immobilized in the reactor is required to ensure long-term stability of biotrickling filters.     

Integrated Fertilization Regimes Boost Heavy Metals Accumulation and Biomass of <i>Sedum alfredii</i> Hance

The hyperaccumulator Sedum alfredii Hance (S. alfredii) may be employed for zinc (Zn) and cadmium (Cd)-polluted soil remediation. However, the low phytoremediation efficiency, related to the low biomass production, limits its use with that purpose. In this experiment, nitrogen (N), phosphorus (P), and potassium (K) fertilizers, and organic manure were applied to investigate the phytoremediation ability of S. alfredii. Hydroponic and pot experiments were conducted using Zn-Cd polluted soil. The hydroponic experiment indicated that appropriate fertilizer application could increase (p < 0.05) the amount of accumulated Zn and Cd in S. alfredii. When N supply ranged from 0.5 to 2.5 mmol L⁻¹, it could improve growth and accumulation of Zn and Cd in whole plants of S. alfredii. The 1 mmol L^-1 N was an optimal N dosage for shoot biomass production and Cd accumulation in shoots, while the 2.5 mmol L^-1 was an optimal N dosage for Zn accumulation in shoots. Both low (<0.05 mmol L^-1) and high (>0.8 mmol L^-1) P supply decreased growth, and Zn/Cd accumulation in whole plants of the studied species. The 0.1 mmol L^-1 P was an optimal dosage for S. alfredii biomass production and Zn/Cd accumulation in shoots. The supply levels within the range from 0.3 to 1 mmol L^-1 K could significantly improve the biomass production of S. alfredii and its capability to accumulate Zn and Cd in the biomass. The 0.5 mmol L^-1 K was an optimal dosage for the whole biomass production and Zn accumulation in shoots, while the 1 mmol L^-1 was an optimal K dosage for Zn accumulation in shoots, which was 17.2% higher than the control. Moreover, the soil pot experiment showed that the combination of organic (fermented manure) and inorganic fertilizers made significant effects on the Zn and Cd-polluted soil remediation by S. alfredii. These effects varied, however, with the application of different proportions of N, P, K and organic matter. The Zn accumulation by S. alfredii reached the highest efficiency ability under the highest fertilizer mixing rate (N: 50 mg kg^-1, P: 40 mg kg^-1, K: 100 mg kg^-1, organic matter: 1%). Even more, S. alfredii showed the strongest ability to accumulate Cd with a lower fertilizer mixing rate (N: 25mg kg^-1, P: 20mg kg^-1, K: 50 mg kg^-1, organic matter: 0.5%).

     

供磷水平对米老排苗木生长及养分状况的影响

为探讨供磷(P)对米老排(Mytilaria laosensis)生长和养分状况的影响,采用土培的方法,研究了不同供磷水平下米老排苗木的生长、养分含量、养分累积量和P吸收效率。结果表明,随着供P水平的提高,米老排苗木的苗高、生物量和养分累积量均呈先上升后下降的趋势;地径呈先增加后稳定的趋势;叶片和全株中的养分含量变化一致,氮(N)含量变化不明显,钾(K)含量呈先降低后升高的趋势,而P含量明显提高;P吸收效率呈现降低-升高-降低的趋势。在单株供P为30 mg时,米老排苗木的叶生物量、根生物量、叶片中N累积量及P吸收效率最大。当供P水平达到45 mg时,米老排苗木的苗高、茎生物量、总生物量、叶片中的P、K累积量和全株中的N、P、K累积量均达到最大值。而供P水平达60 mg时,米老排苗木苗高、生物量、养分累积量和P吸收效率均明显下降。这说明适合米老排苗木生长的供P水平为每株30~45 mg。     

Effects of agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum

The effect of mechanical agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum was investigated in aerated continuous cultures with and without the added shear protectant Pluronic F68. Damage to cells was quantified through a decrease in the steady state concentration of the biomass in the photobioreactor. For a given aeration rate, the steady state biomass concentration rose with increasing rate of mechanical agitation until an upper limit on agitation speed was reached. This maximum tolerable agitation speed depended on the microalgal species. Further increase in agitation speed caused a decline in the steady state concentration of the biomass. An impeller tip speed of >1.56 m s^–1 damaged P. tricornutum in aerated culture. In contrast, the damage threshold tip speed for P. cruentum was between 2.45 and 2.89 m s^–1. Mechanical agitation was not the direct cause of cell damage. Damage occurred because of the rupture of small gas bubbles at the surface of the culture, but mechanical agitation was instrumental in generating the bubbles that ultimately damaged the cells. Pluronic F68 protected the cells against damage and increased the steady state concentration of the biomass relative to operation without the additive. The protective effect of Pluronic was concentration-dependent over the concentration range of 0.01–0.10% w/v.     

Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli arcA</Emphasis> mutant in fed-batch microaerobic cultures

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the 'one-factor-at-a-time' technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett-Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box-Wilson design. Under such optimized conditions (22.02 g l(-1) glycerol, 1.78 g l(-1) CAS, and 1.83 g l(-1) inoculum) microaerobic batch cultures gave rise to 8.37 g l(-1) CDW and 3.52 g l(-1) PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l(-1). After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l(-1), respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures.     

Biomass and lutein productivity of <Emphasis Type="Italic">Scenedesmus almeriensis</Emphasis>: influence of irradiance,dilution rate and temperature

In this paper, the biomass and lutein productivity of the lutein-rich new strain Scenedesmus almeriensis is modelled versus irradiance and temperature. The results demonstrate that S. almeriensis is a mesophile microorganism with an optimal growth temperature of 35 degrees C, and capable of withstanding up to 48 degrees C, which caused culture death. This strain is also tolerant to high irradiances, showing no signs of photoinhibition even at the maximum irradiance essayed of 1625 microE m(-2) s(-1) accumulating up to 0.55% dry weight (d.wt.) of lutein. The optimal conditions that maximise the biomass productivity also favour the lutein productivity, lutein being a primary metabolite. Maximal biomass and lutein productivities of 0.87 g l(-1) day(-1) and 4.77 mg l(-1) day(-1), respectively, were measured. The analysis of light availability inside the cultures, quantified as average irradiance, demonstrates that the cultures were mainly photo-limited, although photosaturation also took place at high external irradiances. The effect of temperature was also investigated finding that the specific maximal growth rate is modified by the temperature according to the Arrhenius equation. The influence of both light availability and temperature was included in an overall growth model, which showed, as a result, capable of fitting the whole set of experimental data. An overall lutein accumulation rate model was also proposed and used in a regression analysis. Simulations performed using the proposed models show that under outdoor conditions a biomass productivity of 0.95 g l(-1) day(-1) can be expected, with a lutein productivity up to 5.31 mg l(-1) day(-1). These models may be useful to assist the design and operation optimisation of outdoor cultures of this strain.     

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).     

Effect of various process parameters on morphology, rheology, and polygalacturonase production by Aspergillus sojae in a batch bioreactor

The effects of pH, agitation speed, and dissolved oxygen tension (DOT), significant in common fungal fermentations, on the production of polygalacturonase (PG) enzyme and their relation to morphology and broth rheology were investigated using Aspergillus sojae in a batch bioreactor. All three factors were effective on the response parameters under study. An uncontrolled pH increased biomass and PG activity by 27% and 38%, respectively, compared to controlled pH (pH 6) with an average pellet size of 1.69 +/- 0.48 mm. pH did not significantly affect the broth rheology but created an impact on the pellet morphology. Similarly, at constant agitation speed the maximum biomass obtained at 500 rpm and at 30 h was 3.27 and 3.67 times more than at 200 and 350 rpm, respectively, with an average pellet size of 1.08 +/- 0.42 mm. The maximum enzyme productivity of 0.149 U mL-1 h-1 was obtained at 200 rpm with an average pellet size of 0.71 +/- 0.35 mm. Non-Newtonian and pseudoplastic broth rheology was observed at 500 rpm agitation speed, broth rheology exhibited dilatant behavior at the lower agitation rate (200 rpm), and at the medium agitation speed (350 rpm) the broth was close to Newtonian. Furthermore, a DOT range of 30-50% was essential for maximum biomass formation, whereas only 10% DOT was required for maximum PG synthesis. Non-Newtonian shear thickening behavior (n > 1.0) was depicted at DOT levels of 10% and 30%, whereas non-Newtonian shear thinning behavior (n < 1.0) was dominant at 50% DOT. The overall fermentation duration (50-70 h) was considerably shorter compared to common fungal fermentations, revealing the economic feasibility of this particular process. As a result this study not only introduced a new strain with a potential of producing a highly commercially significant enzyme but also provided certain parameters significant in the design and mathematical modeling of fungal bioprocesses.     

Increased Hepatitis B surface antigen production by recombinant <Emphasis Type="BoldItalic">Aspergillus niger</Emphasis> through the optimization of agitation and dissolved oxygen concentration

The capacity of the filamentous fungi Aspergillus niger to produce and assemble complex immunogenic viral proteins into virus-like particles (VLPs) in batch culture was enhanced by optimizing the bioprocessing parameters, agitation intensity and dissolved oxygen (dO₂) concentration. Response surface methodology (RSM) and a two-factor-two-level central composite rotatable design (CCRD) were employed to evaluate the interactive response pattern between parameters and their optimum combination. The recombinant hepatitis B surface antigen (HBsAg) was used as a model VLP system to determine the effect of these parameters on biomass yield, fungal morphology, HBsAg production and bioreactor kinetics. The response surface model predicted optimum cultivation conditions at an agitation of rate of 100 rpm and a dO₂ concentration of 25%, obtaining highest intracellular membrane-associated HBsAg levels of . HBsAg production levels were increased tenfold compared to yields obtained in shake flask cultivation. Although hepatitis B VLPs mostly accumulated intracellularly, optimal bioreactor conditions resulted in significant HBsAg release in culture supernatant. These results compare favourably with other recombinant VLP systems in batch culture, and therefore, indicate a substantial potential for further engineering of the A. niger production system for the high level of intracellular and extracellular VLP production.