Microalgae as bioreactors for bioplastic production

Being protein function a conformation-dependent issue, avoiding aggregation during production is a major challenge in biotechnological processes, what is often successfully addressed by convenient upstream, midstream or downstream approaches. Even when obtained in soluble forms, proteins tend to aggregate, especially if stored and manipulated at high concentrations, as is the case of protein drugs for human therapy. Post-production protein aggregation is then a major concern in the pharmaceutical industry, as protein stability, pharmacokinetics, bioavailability, immunogenicity and side effects are largely dependent on the extent of aggregates formation. Apart from acting at the formulation level, the recombinant nature of protein drugs allows intervening at upstream stages through protein engineering, to produce analogue protein versions with higher stability and enhanced therapeutic values.     

Microalgae as natural sources for antioxidative compounds

Antioxidants are substances that have the ability to reduce free, energized radicals. Thus, they prevent the oxidation of sensitive metabolites like lipids or amino acids and shield them from being destroyed by interrupting auto- or photooxidative chain reactions inside the cell. Antioxidants are also of industrial importance because they can be used as food, drug, or plastics additives. Ubiquinol, the reduced form of coenzyme Q₁₀, is one of the most effective antioxidants in human cells. This paper explores optimization strategies to increase Q₁₀ concentration in the biomass of Porphyridium purpureum, based on the variation of photosynthetic photon flux density. In addition, a cultivation process was performed in the 120-L scale followed by an automized extraction procedure (Accelerated Solvent Extraction?) resulting in an increase of the product recovery by a factor of 14 compared to the standard extraction method, hence reaching a specific coenzyme Q₁₀ concentration of 141?μg?g _{dry weight} ^?1 and a volumetric coenzyme Q₁₀ concentration of 1.96?mg?L^?1, respectively.     

Absorber Tower as a Photobioreactor for Microalgae

Green microalgae were grown under natural light in a photobioreactor similar to a transparent plate absorber. A proper temperature was maintained through the control of evaporation and the minimization of convective heat waste. Carbon dioxide desorption was lower in comparison to its level during cultivation in open or covered ponds. A yield of 1 g/l per day and over 100 g/m² projection area was achieved.     

Potato tubers as bioreactors for palatinose production

Palatinose (isomaltulose, 6-O-alpha-D-glucopyranosyl-D-fructose) is a structural isomer of sucrose with very similar physico-chemical properties. Due to its non-cariogenicity and low calorific value it is an ideal sugar substitute for use in food production. Palatinose is produced on an industrial scale from sucrose by an enzymatic rearrangement using immobilized bacterial cells. To explore the potential of transgenic plants as alternative production facilities for palatinose, a chimeric sucrose isomerase gene from Erwinia rhapontici under control of a tuber-specific promoter was introduced into potato plants. The enzyme catalyses the conversion of sucrose into palatinose. Expression of the palI gene within the apoplast of transgenic tubers led to a nearly quantitative conversion of sucrose into palatinose. Despite the soluble carbohydrates having been altered within the tubers, growth of palI expressing transgenic potato plants was indistinguishable from wild type plants. Therefore, expression of a bacterial sucrose isomerase provides a valid tool for high level palatinose production in storage tissues of transgenic crop plants.     

Microalgae as a raw material for biofuels production

Biofuels demand is unquestionable in order to reduce gaseous emissions (fossil CO₂, nitrogen and sulfur oxides) and their purported greenhouse, climatic changes and global warming effects, to face the frequent oil supply crises, as a way to help non-fossil fuel producer countries to reduce energy dependence, contributing to security of supply, promoting environmental sustainability and meeting the EU target of at least of 10% biofuels in the transport sector by 2020. Biodiesel is usually produced from oleaginous crops, such as rapeseed, soybean, sunflower and palm. However, the use of microalgae can be a suitable alternative feedstock for next generation biofuels because certain species contain high amounts of oil, which could be extracted, processed and refined into transportation fuels, using currently available technology; they have fast growth rate, permit the use of non-arable land and non-potable water, use far less water and do not displace food crops cultures; their production is not seasonal and they can be harvested daily. The screening of microalgae (Chlorella vulgaris, Spirulina maxima, Nannochloropsis sp., Neochloris oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) was done in order to choose the best one(s), in terms of quantity and quality as oil source for biofuel production. Neochloris oleabundans (fresh water microalga) and Nannochloropsis sp. (marine microalga) proved to be suitable as raw materials for biofuel production, due to their high oil content (29.0 and 28.7%, respectively). Both microalgae, when grown under nitrogen shortage, show a great increase (~50%) in oil quantity. If the purpose is to produce biodiesel only from one species, Scenedesmus obliquus presents the most adequate fatty acid profile, namely in terms of linolenic and other polyunsaturated fatty acids. However, the microalgae Neochloris oleabundans, Nannochloropsis sp. and Dunaliella tertiolecta can also be used if associated with other microalgal oils and/or vegetable oils.     

Microalgae as cell factories producing recombinant commercial proteins

Extraction of natural substances and chemical synthesis are the main sources of pharmaceutical molecules. When possible, one may transfer the gene of the molecule in living cells creating individual factories producing on demand and in a safe way the requested molecule. Today, bacteria, yeast, mammalian cells and plants constitute the main plate-forms for various commercial products. Microalgae present numerous advantages and could offer a powerful tool for the production of commercial molecules in a near future.     

Soybeans as bioreactors for biopharmaceuticals and industrial proteins

Plants present various advantages for the production of biomolecules, including low risk of contamination with prions, viruses and other pathogens, scalability, low production costs, and available agronomical systems. Plants are also versatile vehicles for the production of recombinant molecules because they allow protein expression in various organs, such as tubers and seeds, which naturally accumulate large amounts of protein. Among crop plants, soybean is an excellent protein producer. Soybean plants are also a good source of abundant and cheap biomass and can be cultivated under controlled greenhouse conditions. Under containment, the plant cycle can be manipulated and the final seed yield can be maximized for large-scale protein production within a small and controlled area. Exploitation of specific regulatory sequences capable of directing and accumulating recombinant proteins in protein storage vacuoles in soybean seeds, associated with recently developed biological research tools and purification systems, has great potential to accelerate preliminary characterization of plant-derived biopharmaceuticals and industrial macromolecules. This is an important step in the development of genetically engineered products that are inexpensive and safe for medicinal, food and other uses.     

Plant seeds as bioreactors for recombinant protein production

Plants are attractive expression systems for the economic production of recombinant proteins. Among the different plant-based systems, plant seed is the leading platform and holds several advantages such as high protein yields and stable storage of target proteins. Significant advances in using seeds as bioreactors have occurred in the past decade, which include the first commercialized plant-derived recombinant protein. Here we review the current progress on seeds as bioreactors, with focus on the different food crops as production platforms and comprehensive strategies in optimizing recombinant protein production in seeds.     

Microalgae as a source of stable isotopically labeled compounds

Microalgae have the ability to convert inorganic compounds into organic compounds. When they are cultured in the presence of stable (non-radioactive) isotopes (i.e.¹³CO₂,¹⁵NO ₃ ^– ,²H₂O) their biomass becomes labeled with the stable isotopes, and a variety of stable isotopically-labeled compounds can be extracted and purified from that biomass.Two applications for stable isotopically-labeled compounds are as cell culture nutrients and as breath test diagnostics. Bacteria that are cultured with labeled nutrients will produce bacterial products that are labeled with stable isotopes. The presence of these isotopes in the bacterial products, along with recent developments in NMR technology, greatly reduces the time and effort required to determine the three-dimensional structure of macromolecules and the interaction of proteins with ligands. As breath test diagnostics, compounds labeled with¹³C are used to measure the metabolism of particular organs and thus diagnose various disease conditions. These tests are based on the principle that a particular compound is metabolized primarily by a single organ, and when that compound is labeled with¹³C, the appearance of¹³CO₂ in exhaled breath provides information about the metabolic activity of the target organ. Tests of this type are simple to perform, non-invasive, and less expensive than many conventional diagnostic procedures.The commercialization of stable isotopically labeled compounds requires that these compounds be produced in a cost-effective manner. Our approach is to identify microalgal overproducers of the desired compounds, maximize the product content of those organisms, and purify the resulting products.     

微藻生物质制备燃料乙醇关键技术研究进展

燃料乙醇作为一种优良的可再生液体燃料,其开发利用受到了人们的广泛关注。微藻是一种高光合、高产生物量的生物质资源,很多的藻体细胞中含有大量的淀粉、纤维素（Iα型）等多糖物质,是制备燃料乙醇的优良原料。发展利用微藻制备燃料乙醇技术工艺,对于缓解我国目前日益短缺的能源问题,减少温室气体排放和环境污染等具有很好的应用前景。综述了国内外利用微藻生物质制备燃料乙醇中所用到的关键技术、存在的问题以及今后的发展前景等。     

Microalgae and cyanobacteria as a source of glycosidase inhibitors

Culture filtrates and organic solvent extracts of over 500 freshwater and marine eukaryotic microalgae and cyanobacteria were screened for the presence of glycosidase inhibitors. Rapid colorimetric assays were used to detect inhibitors of alpha-glucosidase, alpha-amylase and beta-galactosidase. Inhibitors were found from 38 species. The results suggest that microalgae and cyanobacteria have potential as a source of glycosidase inhibitors which may have clinical applications.     

Denitrification with methane as electron donor in oxygen-limited bioreactors

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10⁷ cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10⁷ cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10⁴ cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. “In vivo”¹³C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification. Received: 19 October 1999 / Received revision: 11 January 2000 / Accepted: 14 January 2000     

Plants as bioreactors for the production of vaccine antigens

Plants have been identified as promising expression systems for commercial production of vaccine antigens. In phase I clinical trials several plant-derived vaccine antigens have been found to be safe and induce sufficiently high immune response. Thus, transgenic plants, including edible plant parts are suggested as excellent alternatives for the production of vaccines and economic scale-up through cultivation. Improved understanding of plant molecular biology and consequent refinement in the genetic engineering techniques have led to designing approaches for high level expression of vaccine antigens in plants. During the last decade, several efficient plant-based expression systems have been examined and more than 100 recombinant proteins including plant-derived vaccine antigens have been expressed in different plant tissues. Estimates suggest that it may become possible to obtain antigen sufficient for vaccinating millions of individuals from one acre crop by expressing the antigen in seeds of an edible legume, like peanut or soybean. In the near future, a plethora of protein products, developed through ‘naturalized bioreactors’ may reach market. Efforts for further improvements in these technologies need to be directed mainly towards validation and applicability of plant-based standardized mucosal and edible vaccines, regulatory pharmacology, formulations and the development of commercially viable GLP protocols. This article reviews the current status of developments in the area of use of plants for the development of vaccine antigens.     

Microalgae biotechnology

Microalgae mass cultures combine attributes of both agriculture (plant photosynthesis) and industrial fermentations (suspended culture growth). However this combination is not necessarily favorable. Microbial fermentations provide greatly superior volumetric productivities (100 times higher or more) and cell concentrations (over 10-fold) than the large volume ponds used for algal cultivation. Agriculture is superior to microalgal cultivation because of much lower capital costs and its ability to derive CO₂ from the atmosphere. On the other hand, microalgae can use poor drainage soils and saline or low quality waters, generally unsuitable for agriculture and sites exist where climate, topography, water and (most importantly) low cost (⋍$100/metric tonne) CO₂ are available for microalgae production. In addition, microalgal production systems must find niches of unique or specialized products that neither agriculture nor industrial microbiology can produce efficiently.     

Nutritional Evaluation of Australian Microalgae as Potential Human Health Supplements

This study investigated the biochemical suitability of Australian native microalgal species Scenedesmus sp., Nannochloropsis sp., Dunaliella sp., and a chlorophytic polyculture as nutritional supplements for human health. The four microalgal cultures were harvested during exponential growth, lyophilized, and analysed for proximate composition (moisture, ash, lipid, carbohydrates, and protein), pigments, and amino acid and fatty acid profiles. The resulting nutritional value, based on biochemical composition, was compared to commercial Spirulina and Chlorella products. The Australian native microalgae exhibited similar, and in several cases superior, organic nutritional properties relative to the assessed commercial products, with biochemical profiles rich in high-quality protein, nutritious polyunsaturated fats (such as α-linolenic acid, arachidonic acid, and eicosapentaenoic acid), and antioxidant pigments. These findings indicate that the microalgae assessed have great potential as multi-nutrient human health supplements.     

Microalgae as sources of pharmaceuticals and other biologically active compounds

In the last decade the screening of microalgae, especially the cyanobacteria (blue-green algae), for antibiotics and pharmacologically active compounds has received ever increasing interest. A large number of antibiotic compounds, many with novel structures, have been isolated and characterised. Similarly many cyanobacteria have been shown to produce antiviral and antineoplastic compounds. A range of pharmacological activities have also been observed with extracts of microalgae, however the active principles are as yet unknown in most cases. Several of the bioactive compounds may find application in human or veterinary medicine or in agriculture. Others should find application as research tools or as structural models for the development of new drugs. The microalgae are particularly attractive as natural sources of bioactive molecules since these algae have the potential to produce these compounds in culture which enables the production of structurally complex molecules which are difficult or impossible to produce by chemical synthesis.     

Glass microspargers as effective frit spargers in single use bioreactors

For multiple-use bench scale and larger bioreactors, sintered stainless steel frit spargers are commonly used as microspargers. For bench-scale single-use bioreactors (SUBs), existing microspargers are sintered plastics, such as polyethylene. However, though plastics are readily sterilized by irradiation making them convenient for single use, these designs overlook surface energy properties of the materials of construction. For these sintered plastic spargers, forces at the water-air-surface interface cause bubble coalescence, leading to lower effective mass transfer, higher gas flow rates, and differing pCO₂ profiles in cell culture. Alternative materials of construction were evaluated based on contact angle information and bubble formation observations. Sintered glass was chosen over thermoplastic polymers for higher surface wettability as described in the glass/water contact angle, its history as a commonly sintered material, and availability at costs suitable for single use applications. Glass sintered spargers and traditional stainless steel frit spargers were compared by porosity, bubble size, and k_La studies. Mass transfer (k_La) and cell culture performance equal or greater than a standard 20 μm stainless steel microsparger mass transfer efficiency was achieved by a glass frit sparger, of international porosity standard “P40” according to ISO 4793-80, which corresponds to a range of 16–40 μm.