Spiros N. Agathos

Insect cell culture for industrial production of recombinant proteins.

Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.

Emerging pollutants in the environment: present and future challenges in biomonitoring, ecological risks and bioremediation

Emerging pollutants reach the environment from various anthropogenic sources and are distributed throughout environmental matrices. Although great advances have been made in the detection and analysis of trace pollutants during recent decades, due to the continued development and refinement of specific techniques, a wide array of undetected contaminants of emerging environmental concern need to be identified and quantified in various environmental components and biological tissues. These pollutants may be mobile and persistent in air, water, soil, sediments and ecological receptors even at low concentrations. Robust data on their fate and behaviour in the environment, as well as on threats to ecological and human health, are still lacking. Moreover, the ecotoxicological significance of some emerging micropollutants remains largely unknown, because satisfactory data to determine their risk often do not exist. This paper discusses the fate, behaviour, (bio)monitoring, environmental and health risks associated with emerging chemical (pharmaceuticals, endocrine disruptors, hormones, toxins, among others) and biological (bacteria, viruses) micropollutants in soils, sediments, groundwater, industrial and municipal wastewaters, aquaculture effluents, and freshwater and marine ecosystems, and highlights new horizons for their (bio)removal. Our study aims to demonstrate the imperative need to boost research and innovation for new and cost-effective treatment technologies, in line with the uptake, mode of action and consequences of each emerging contaminant. We also address the topic of innovative tools for the evaluation of the effects of toxicity on human health and for the prediction of microbial availability and degradation in the environment. Additionally, we consider the development of (bio)sensors to perform environmental monitoring in real-time mode. This needs to address multiple species, along with a more effective exploitation of specialised microbes or enzymes capable of degrading endocrine disruptors and other micropollutants. In practical terms, the outcomes of these activities will build up the knowledge base and develop solutions to fill the significant innovation gap faced worldwide.

Anaerobic Dechlorinating Bacteria

Anaerobic dehalogenation is attracting great interest since it opens new research horizons based on the novel biochemical mechanisms identified in this field such as halorespiration, i.e. the utilization of halogenated compounds as electron acceptors. Moreover, anaerobic bacteria seem to be more efficient than their aerobic counterparts in removing halogen atoms from polyhalogenated compounds. Thus, anaerobic dehalogenation can be considered as a promising means for bioremediation treatments of persistently polluted environments. In this line, identification of pure strains capable of dehalogenation will give important information about the diversity of organisms implicated in this process and also fundamental explanations of the diverse biochemical mechanisms involved. In light of these considerations, we chose to focus this review on the physiological descriptions, dechlorination activities, phylogenetic diversity, and potential biotechnological applications of these pure anaerobic strains capable of dehalogenation.

Utilization of fungi for biotreatment of raw wastewaters

Fungal biomasses are capable of treating metal-contaminated effluents with efficiencies several orders of magnitude superior to activated carbon (F-400) or the industrial resin Dowex-50. Additionally, fungal biomasses are susceptible to engineering improvements and regeneration of their capabilities. With regard to organic pollutants, excessive nutrients and dyes, fungi can remove them from wastewaters, leading to a decrease in their toxicities. However, the detoxification rates seem to be dependent on media and culture conditions. The postreatement by anaerobic bioprocesses of effluents that have been pretreated with fungi can lead to higher biogas than the original effluents. In addition to the degradation of organic pollutants, fungi produce added-value products such as enzymes (LiP, MnP, Lacc, amylase, etc.) and single-cell protein (SCP). Most research on fungal capacities to purify polluted effluents has been performed on a laboratory scale, hence there is a need to extend such research to pilot scale and to apply it to industrial processes.

Environmental genomics: exploring the unmined richness of microbes to degrade xenobiotics.

Increasing pollution of water and soils by xenobiotic compounds has led in the last few decades to an acute need for understanding the impact of toxic compounds on microbial populations, the catabolic degradation pathways of xenobiotics and the set-up and improvement of bioremediation processes. Recent advances in molecular techniques, including high-throughput approaches such as microarrays and metagenomics, have opened up new perspectives and pointed towards new opportunities in pollution abatement and environmental management. Compared with traditional molecular techniques dependent on the isolation of pure cultures in the laboratory, microarrays and metagenomics allow specific environmental questions to be answered by exploring and using the phenomenal resources of uncultivable and uncharacterized micro-organisms. This paper reviews the current potential of microarrays and metagenomics to investigate the genetic diversity of environmentally relevant micro-organisms and identify new functional genes involved in the catabolism of xenobiotics.

Biodegradation: Updating the Concepts of Control for Microbial Cleanup in Contaminated Aquifers

Biodegradation is one of the most favored and sustainable means of removing organic pollutants from contaminated aquifers but the major steering factors are still surprisingly poorly understood. Growing evidence questions some of the established concepts for control of biodegradation. Here, we critically discuss classical concepts such as the thermodynamic redox zonation, or the use of steady state transport scenarios for assessing biodegradation rates. Furthermore, we discuss if the absence of specific degrader populations can explain poor biodegradation. We propose updated perspectives on the controls of biodegradation in contaminant plumes. These include the plume fringe concept, transport limitations, and transient conditions as currently underestimated processes affecting biodegradation.

FORTIFICATION OF A PROTEIN-FREE CELL CULTURE MEDIUM WITH PLANT PEPTONES IMPROVES CULTIVATION AND PRODUCTIVITY OF AN INTERFERON-γ–PRODUCING CHO CELL LINE

SummaryA strong tendency is currently emerging to remove not only serum but also any product of animal origin from animal cell culture media during production of recombinant proteins. This should facilitate downstream processing and improve biosafety. One way consists in the fortification of protein-free nutritive media with plant protein hydrolysates. To investigate the effects of plant peptones on mammalian cell cultivation and productivity, CHO 320 cells, a clone of CHO K1 cells genetically modified to secrete human interferon-γ (IFN-γ), were first adapted to cultivation in suspension in a protein-free medium. Both cell growth and IFN-γ secretion were found to be equivalent to those reached in serum-containing medium. Eight plant peptones, selected on the basis of their content in free amino acids and oligopeptides, as well as molecular weight distribution of oligopeptides, were tested for their ability to improve culture parameters. These were improved in the presence of three peptones, all having an important fraction of oligopeptides ranging from 1 to 10 kDa and a small proportion of peptides higher than 10 kDa. These peptones do not seem to add significantly to the nutritive potential to basal protein-free nutritive medium. Nevertheless, supplementation of an oligopeptide-enriched wheat peptone improved cell growth by up to 30% and IFN-γ production by up to 60% in shake-flask experiments. These results suggest that the use of plant peptones with potential growth factor-like or antiapoptotic bioactivities could improve mammalian cell cultivation in protein-free media while increasing the product biosafety.

Transformation and mineralization of 2,4,6-trinitrotoluene (TNT) by manganese peroxidase from the white-rot basidiomycete Phlebia radiata.

The degradation of the nitroaromatic pollutant 2,4,6-trinitrotoluene (TNT) by the manganese-dependent peroxidase (MnP) of the white-rot fungus Phlebia radiata and the main reduction products formed were investigated. In the presence of small amounts of reduced glutathione (10 mM), a concentrated cell-free preparation of MnP from P. radiata exhibiting an activity of 36 nkat/ml (36 nmol Mn(II) oxidized per sec and per ml) transformed 10 mg/l of TNT within three days. The same preparation was capable of completely transforming the reduced derivatives of TNT. When present at 10 mg/l, the aminodinitrotoluenes were transformed in less than two days and the diaminonitrotoluenes in less than three hours. Experiments with 14C-U-ring labeled TNT and 2-amino-4,6-dinitrotoluene showed that these compounds were mineralized by 22% and 76%, respectively, within 5 days. Higher concentrations of reduced glutathione (50 mM) led to a severe inhibition of the degradation process. It is concluded that Phlebia radiata is a good candidate for the biodegradation of TNT as well as its reduction metabolites.

Combined cross-linked enzyme aggregates from versatile peroxidase and glucose oxidase: Production, partial characterization and application for the elimination of endocrine disruptors

Versatile peroxidase (VP) from Bjerkandera adusta was insolubilized in the form of cross-linked enzyme aggregates (CLEA®s). Of the initially applied activity 67% was recovered as CLEA®s. Co-aggregation of VP with glucose oxidase from Aspergillus niger led to an increased activity recovery of 89%. The combined CLEA®s showed higher stability against H(2)O(2) and exerted VP activity upon glucose addition. The elimination of the endocrine disrupting chemicals bisphenol A, nonylphenol, triclosan, 17α-ethinylestradiol and the hormone 17β-estradiol (10 mg L(-1) each) and the removal of their estrogenic activity by combined CLEA®s were tested in batch experiments. Within 10 min, the combined CLEA®s were able to remove all the endocrine disruptors except triclosan (residual concentration 74%). The removal of the estrogenic activity was higher than 55% for all compounds, except triclosan. A membrane reactor continuously operated with combined CLEA®s could almost completely remove bisphenol A (10 mg L(-1)) for 43 h.

Insect cells as factories for biomanufacturing

Insect cells (IC) and particularly lepidopteran cells are an attractive alternative to mammalian cells for biomanufacturing. Insect cell culture, coupled with the lytic expression capacity of baculovirus expression vector systems (BEVS), constitutes a powerful platform, IC-BEVS, for the abundant and versatile formation of heterologous gene products, including proteins, vaccines and vectors for gene therapy. Such products can be manufactured on a large scale thanks to the development of efficient and scaleable production processes involving the integration of a cell growth stage and a stage of cell infection with the recombinant baculovirus vector. Insect cells can produce multimeric proteins functionally equivalent to the natural ones and engineered vectors can be used for efficient expression. Insect cells can be cultivated easily in serum- and protein-free media. A growing number of companies are currently developing an interest in producing therapeutics using IC-BEVS, and many products are today in clinical trials and on the market for veterinary and human applications. This review summarizes current knowledge on insect cell metabolism, culture conditions and applications.