Chlorella vulgaris cultivation using ricotta cheese whey as substrate for biomass production

Journal of Applied Phycology - The batch production of Chlorella vulgaris and its potential to profit the remnant nutritional components from ricotta cheese whey (RCW) were evaluated. From a first...     

Microalgal cultivation in porous substrate bioreactor for extracellular polysaccharide production

Netrium digitus is a representative of the species-rich class Zygnematophyceae (Streptophyta). Its intensive extracellular polysaccharide (EPS) production makes this alga interesting for biotechnological applications with a focus on cosmetics and food additives. Quantitative data on growth and EPS production in suspension and, for the first time, in immobilized culture using lab-scale porous substrate bioreactors, so-called Twin-Layer (TL) systems, is presented. It is shown that the cell as well as the EPS dry weight content is increased at least sixfold in immobilized compared to suspension culture. Due to the high amount of EPS, the biofilms reach a thickness of more than 8 mm after 27 days at 70 μmol photons m^?2 s^?1 and with 1.5% CO₂ supply. Frequent exchange of the growth medium results in a linear cell biomass increase of 2.02?±?0.09 g m^?2 growth area day^?1 compared to 2.99?±?0.09 g m^?2 day^?1, when the medium is not exchanged. Under this mode of cultivation, the EPS production is lower and a final concentration of 12.18?±?1.25 g m^?2 compared to 20.76?±?0.85 g m^?2, when medium was exchanged, is reached. It is clearly demonstrated that the relatively slow growing, but excessively EPS producing, microalgal species N. digitus can be grown in porous substrate bioreactors and that this culturing technique is a promising alternative to suspension culture for the Zygnematophyceae.     

Mathematical modeling of a horizontal tubular loop bioreactor for biomass production from natural gas

In this study, mathematical modeling of a horizontal tubular loop bioreactor (HTLB) was considered for biomass production from natural gas. Gas inlet segments, static mixers, gas–liquid separator, and liquid pump of the HTLB were mathematically modeled according to the ideal stirred reactors, and the horizontal parts, riser, and down-comer sections were modeled in line with the dispersed plug-flow reactors as well. The set of ordinary and partial differential equations were coupled to calculate the oxygen and methane concentrations in the liquid through the length of bioreactor and time. Moreover, the tuned kinetic and hydrodynamic parameters of SCP process in the HTLB were determined based on the mathematical model at various operational conditions. The model was validated by considering experimental dissolved oxygen, methane, and biomass concentrations in liquid at different ratios of air to methane and liquid flow rates. The results showed satisfactory agreement between the developed model and the experimental data.     

Biological hydrogen production using a membrane bioreactor

A cross-flow membrane was coupled to a chemostat to create an anaerobic membrane bioreactor (MBR) for biological hydrogen production. The reactor was fed glucose (10,000 mg/L) and inoculated with a soil inoculum heat-treated to kill non-spore-forming methanogens. Hydrogen gas was consistently produced at a concentration of 57-60% in the headspace under all conditions. When operated in chemostat mode (no flow through the membrane) at a hydraulic retention time (HRT) of 3.3 h, 90% of the glucose was removed, producing 2200 mg/L of cells and 500 mL/h of biogas. When operated in MBR mode, the solids retention time (SRT) was increased to SRT = 12 h producing a solids concentration in the reactor of 5800 mg/L. This SRT increased the overall glucose utilization (98%), the biogas production rate (640 mL/h), and the conversion efficiency of glucose-to-hydrogen from 22% (no MBR) to 25% (based on a maximum of 4 mol-H(2)/mol-glucose). When the SRT was increased from 5 h to 48 h, glucose utilization (99%) and biomass concentrations (8,800 +/- 600 mg/L) both increased. However, the biogas production decreased (310 +/- 40 mL/h) and the glucose-to-hydrogen conversion efficiency decreased from 37 +/- 4% to 18 +/- 3%. Sustained permeate flows through the membrane were in the range of 57 to 60 L/m(2) h for three different membrane pore sizes (0.3, 0.5, and 0.8 microm). Most (93.7% to 99.3%) of the membrane resistance was due to internal fouling and the reversible cake resistance, and not the membrane itself. Regular backpulsing was essential for maintaining permeate flux through the membrane. Analysis of DNA sequences using ribosomal intergenic spacer analysis indicated bacteria were most closely related to members of Clostridiaceae and Flexibacteraceae, including Clostridium acidisoli CAC237756 (97%), Linmingia china AF481148 (97%), and Cytophaga sp. MDA2507 AF238333 (99%). No PCR amplification of 16s rRNA genes was obtained when archaea-specific primers were used.     

Solid substrate fermentation for cellulase production using palm kernel cake as a renewable lignocellulosic source in packed-bed bioreactor

Palm kernel cake (PKC), is an agro-industrial residue created in the palm oil industry, and large quantities of PKC are produced in Malaysia. Sustainable development of the palm oil industry in Malaysia demands an economical technology for the environmentally friendly utilization of PKC in industrial utility systems. This research was carried out to evaluate the use of PKC in the production of cellulase by the cultivation of Aspergillus niger FTCC 5003 in a laboratory packed-bed bioreactor for seven days. A central composite design was used to perform eighteen trials of solid substrate fermentation under selected conditions of incubation temperature, initial moisture content of substrate, and airflow rate. Experimental results showed that a cellulase yield of 244.53 U/g of dry PKC was obtained when 100 g of PKC was hydrolyzed at an incubation temperature of 32.5°C, an initial moisture level of 60%, and an aeration rate of 1.5 L/min/g PKC. An empirical second-order polynomial model was adjusted to the experimental data to evaluate the effects of the studied operating variables on cellulase production. The statistical model revealed that the quadratic term for initial moisture content had a significant effect on the production of cellulase (P < 0.01). The regression model also indicated that the quadratic terms for incubation temperature and interaction effects between initial moisture content and aeration rate significantly influenced cellulase production (P < 0.05). The empirical model determined that the optimum conditions for cellulase production were an incubation temperature of 31.0°C, an initial moisture content of 59.0% and an airflow rate of 1.55 L/min/g PKC.     

Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides

Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l(-1) 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l(-1) gibberellic acid (GA(3)). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3l balloon type bubble bioreactor (BTBB) containing 2l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials.     

Use of waste Chinese cabbage as a substrate for yeast biomass production

The possibility of using waste Chinese cabbage (Brassica campestris) as a substrate for microbial biomass production was investigated. The juice from waste Chinese cabbage contains relatively high amounts of reducing sugars suitable for yeast culture. The cell mass and protein content of four species of yeast, Candida utilis, Pichia stipitis, Kluyveromyces marxianus, and Saccharomyces cerevisiae, were determined when cultured in juice extracted from cabbage waste. Compared to YM broth containing the same level of sugar, all the strains except C. utilis showed higher total protein production in cabbage juice medium (CJM).     

Ethanol production from olive cake biomass substrate

The inexpensive production of sugars from lignocellulose is an essential step for the use of biomass to produce fuel ethanol. Olive cake is an abundant by-product of the olive oil industry and represents a potentially significant lignocellulosic source for bioethanol production in the Mediterranean basin. Furthermore, converting olive cake to ethanol could add further value to olive production. In the present study, olive cake was evaluated as a feedstock for ethanol production. To this end, the lignocellulosic component of the olive cake was dilute-acid pretreated at a 13.5% olive-cake loading with 1.75% (w/v) sulfuric acid and heating at 160°C for 10 min. This was followed by chemical elimination of fermentation inhibitors. Soluble sugars resulting from the pretreatment process were fermented using E. coli FBR5, a strain engineered to selectively produce ethanol. 8.1 g of ethanol/L was obtained from hydrolysates containing 18.1 g of soluble sugars. Increasing the pretreatment temperature to 180°C resulted in failed fermentations, presumably due to inhibitory by-products released during pretreatment.     

Inulinase production in a batch bioreactor using agroindustrial residues as the substrate: experimental data and modeling

The production of inulinase employing agroindustrial residues as the substrate is a good alternative to reduce production costs and to minimize the environmental impact of disposing these residues in the environment. This study focused on the use of a phenomenological model and an artificial neural network (ANN) to simulate the inulinase production during the batch cultivation of the yeast Kluyveromyces marxianus NRRL Y-7571, employing a medium containing agroindustrial residues such as molasses, corn steep liquor and yeast extract. It was concluded that due to the complexity of the medium composition it was rather difficult to use a phenomenological model with sufficient accuracy. For this reason, an alternative and more cost-effective methodology based on ANN was adopted. The predictive capacity of the ANN was superior to that of the phenomenological model, indicating that the neural network approach could be used as an alternative in the predictive modeling of complex batch cultivations.     

Metarhizium anisopliae conidia production in packed-bed bioreactor using rice as substrate in successive cultivations

This paper presents a study on scale-up and cost reduction of the production of spores of Metarhizium anisopliae IBCB 425, entomopathogenic fungus used in sugarcane crops. Rice was mixed with sugarcane bagasse (9:1 w/w) for substrate composition, assuring adequate physical structure for cultivation in packed-beds. Spores yield from only rice in bench-scale packed-bed bioreactor was 56 % of the one obtained from the mixture 9:1 w/w. In comparison to plastic packages used in bioindustries, equivalent spores yields per gram of substrate have been achieved in bench and pilot-scale bioreactors built by cylindrical jacketed modules, that provide better control of operational and environmental variables, attested by little variability among replicates. Although non-negligible temperature rise (5 °C above the ideal) occurred within the pilot-scale bioreactor, spores production was not harmed in comparison to bench-scale. By reusing rice up to three successive cultivations, a 2.5-fold increase of spores yields was achieved in comparison to single use. Temperatures and CO₂ profiles corroborates the fungus adapted differently to substrate at each usage. Such results are valuable for industrial producers of commercial formulations of the fungus spores, allowing process modernization by using packed-bed bioreactors and production costs and rice demand reduction by recycling the substrate.     

Cytotoxin production by a merineLyngbya strain (cyanobacterium) in a large-scale laboratory bioreactor

A cytotoxic compound was produced by the marine cyanobacteriumLyngbya sp. Pearl strain in large laboratory-scale batch cultures. Adsorption and fractionation of methanol extracts with reverse phase (C-18) cartridges provided a rapid method for removal of bioassay interference from salts, biopolymers and pigments and concentration of the cytotoxic principles. Cytotoxicity to the murine leukemia cell line P-388 was produced in two cycles coinciding with the initiation of exponential growth and again during the late exponential growth phase. Antiviral activity against influenza virus PR8 was found in extracts prepared from early exponential growth phase cells but antiviral activity was not detected in extracts of mid-log or late-log growth phase cells. These differences in bioactivity suggests that the cytotoxic principles produced during early and late exponential growth may be different compounds. Cytotoxicity assays using murine P-388 leukemia indicates that the semi-pure compound has an IC₅₀ of < 0.25 μg ml⁻¹ to this cell line. P-388 cytotoxicity in cell extracts increased during the late exponential growth phase and the specific yield was estimated at approximately 0.14 mg g⁻¹ (dry cells).     

Monoclonal antibody production using a new supermacroporous cryogel bioreactor

A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (approximately 20 days), the hybridoma cell line consumed 0.75 mM day-1 glucose, produced 2.48 mM day-1 lactic acid, and produced 6.5 microg mL-1 day-1 mAb during the exponential phase. The mAb concentration reached 130 microg mL-1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.     

Cheese whey as a renewable substrate for microbial lipid and biomass production by Zygomycetes

Three Zygomycetes, Mortierella isabellina, Thamnidium elegans and Mucor sp., were tested for their ability of producing biomass and lipid‐containing γ‐linolenic acid (GLA) during their cultivation on cheese whey. M. isabellina consumed all of the available lactose and a significant amount of the available protein. On the contrary, the two other fungi seemed incapable of consuming lactose after protein exhaustion. In the second series of experiments, for M. isabellina a supplementary quantity of lactose was added into the medium in order to increase the C/N ratio and hence to increase the production of fat. In the case of T. elegans and Mucor sp., a supplementary quantity of ammonium sulfate was added in order to favor the consumption of lactose and the production of biomass. Indeed, enhancement of lipid production was observed for M. isabellina and biomass production for T. elegans and Mucor sp.. Fatty acid analysis of the microbial lipid showed a composition that presented non‐negligible changes in relation with the age of the culture and the C/N molar ratio of the medium. Further analysis of the fat showed that the quantity of neutral lipids was the more abundant. The fatty acid composition of neutral lipids resembled to that of total lipids. Phospholipids were the more unsaturated fraction for Mucor sp. and M. isabellina. GLA was synthesized in all trials but its concentration presented differences related with the utilized strains and the fermentation time. Growth of M. isabellina on lactose‐supplemented whey resulted in a maximum GLA production of 301 mg/L.     

Fly-ash products from biomass co-combustion for VOC control

Experiments were conducted in a continuous flow reactor at room temperature to evaluate the elimination of low-concentration toluene in the gas phase to verify if fly-ash products from biomass combustion in an ozonation system could be used in the removal of volatile organic compounds. The fly-ash products from pure biomass combustion (Ash₁₀₀) demonstrated the highest ozonation activities upon the removal of low-concentration toluene (1.5 ppmv), followed by the fly-ash products from co-combustion (Ash₃₀) and the coal combustion (Ash₀). Kinetic experiments showed that the activation energy of the toluene elimination process was substantially reduced with the use of ozone and the reaction intermediates, such as formic acids, aldehydes, etc. Results also showed that the intermediates were reduced with increasing humidity level. The combined use of fly-ash products and zeolite 13X enhanced the removal of toluene to above 90% and suppressed the release of residual ozone and intermediates by holding them in the adsorbed phase.     

Large scale production of phage antibody libraries using a bioreactor

One of the limitations of the use of phage antibody libraries in high throughput selections is the production of sufficient phage antibody library at the appropriate quality. Here, we successfully adapt a bioreactor-based protocol for the production of phage peptide libraries to the production of phage antibody libraries. The titers obtained in the stirred-tank bioreactor are 4 to 5 times higher than in a standard shake flask procedure, and the quality of the phage antibody library produced is indistinguishable to that produced using standard procedures as assessed by Western blotting and functional selections. Availability of this protocol will facilitate the use of phage antibody libraries in high-throughput scale selections.     

The generation of high biomass from chlororespiring bacteria using a continuous fed-batch bioreactor

A continuous fed-batch reactor system was developed to rapidly obtain dense chlororespiring cultures of Anaeromyxobacter dehalogenans strain 2CP-C. A syringe pump continuously delivered concentrated 2,6-dichlorophenol (50–150 mM) to an anaerobic reactor vessel at a rate that sustained linear growth but prevented the substrate toxicity of chlorophenol. Dechlorination was not significantly inhibited by end product phenol up to 8 mM. A cell density of 76.8 mg protein l^–1 was obtained in 24 h. Specific growth rates averaged 0.033 h^–1at 50% substrate limitation, which was in agreement with the maximum specific growth rate of 0.068 h^–1. This reactor system provides an efficient, cost-effective, and convenient method to rapidly obtain dense dechlorinating biomass and is promising to accelerate investigations of enzymes involved in chlororespiration.     

Bioethanol production from the hydrolysate of rape stem in a surface-aerated fermentor

In this study, we investigated the feasibility of producing bioethanol from the hydrolysate of rape stem. Specifically, the most ideal yeast strain was screened, and the microaeration was performed by surface aeration on a liquid medium surface. Among the yeast strains examined, Pichia stipitis CBS 7126 displayed the best performance in bioethanol production during the surface-aerated fermentor culture. Pichia stipitis CBS 7126 produced maximally 9.56 g/l of bioethanol from the initial total reducing sugars (about 28 g/l). The bioethanol yield was 0.397 (by the DNS method). Furthermore, this controlled surface aeration method holds promise for use in the bioethanol production from the xylose-containing lignocellulosic hydrolysate of biomass.     

Large scale production of phage antibody libraries using a bioreactor

One of the limitations of the use of phage antibody libraries in high throughput selections is the production of sufficient phage antibody library at the appropriate quality. Here, we successfully adapt a bioreactor-based protocol for the production of phage peptide libraries to the production of phage antibody libraries. The titers obtained in the stirred-tank bioreactor are 4 to 5 times higher than in a standard shake flask procedure, and the quality of the phage antibody library produced is indistinguishable to that produced using standard procedures as assessed by Western blotting and functional selections. Availability of this protocol will facilitate the use of phage antibody libraries in high-throughput scale selections.     

Characterization of tannery wastewater and biomass in a membrane bioreactor using respirometric analysis

Respirometric techniques and an activated sludge model (ASM) were applied for the characterization of tannery wastewater and biomass in a pilot plant membrane bioreactor (MBR) operating at high sludge age. The traditional respirometric tests and the IWA-ASM1 were modified to take into account the specific operating conditions, the solid-liquid separation technology and the wastewater complexity. As a result the wastewater biodegradable COD was fractionated into four components: readily biodegradable, rapidly hydrolysable, slowly hydrolysable and inorganic (due to the presence of reduced sulphur compounds). The kinetic and stoichiometric parameters of the biomass (heterotrophic and nitrifying) were estimated through the integration of model simulations and respirometric tests results. In particular the ammonium and nitrite-oxidizing biomasses were separately characterized: the growth kinetics of ammonium and nitrite-oxidizing bacteria resulted noticeably lower than the traditional reference values (mu(max,AOB)=0.25d(-1)e mu(max,NOB)=0.23d(-1) at 20 degrees C, respectively). The ASM was finally used to confirm that the results of the wastewater and biomass characterization allow to properly simulate the mixed liquor suspended solids in the MBR pilot plant and the COD concentration in the effluent.     

Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin

Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79 x 10(-2) percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale.