Get some direction in your lab

Microbes are involved in many industrial biological processes, and improving them could increase output and decrease waste – but how can this be possible? One company think that a spot of directed evolution is the answer

Eco-Solution, a French company founded in 1999, combines knowledge in the fields of fundamental and applied microbiology, instrumentation and process engineering and aims to develop and improve industrial biological processes.

Eco-Solution concentrates its efforts on providing its clients with viable operational solutions for technical and economical improvements in the performances of their industrial biological processes. The spectrum of applications is broad, ranging from environment protection (degradation of persistent organic pollutants contained in water, soils or air), to renewable energy production (biogas and biofuel) and bio-molecules production (therapeutical, technical, agro-food, etc).

The solutions developed by Eco-Solution are based on the extension of microbial diversity by In Vivo Directed Evolution (IVDE) techniques.

The genome of living organisms changes continuously as a result of various factors and events such as mutations, genetic recombination and gene transfer, as well as other factors. In microbial cultures, these genomic transformations occur randomly at a given frequency and are either propagated or eliminated from a population as a result of the Darwinian natural selection mechanisms. If a modification leads to a better adaptation with regard to the culture environment constraints, this modification will be favoured and the corresponding organism will be multiplied. Conversely, if a modification is detrimental to one cellular characteristic that is submitted to a culture stress, the modification will be counter-selected and the corresponding organism will be progressively eliminated from the population.

This natural genome plasticity is the key enabling the IVDE process, which is used to direct or guide the natural evolution of a microorganism toward a desired activity or character.

This method is a non-GMO alternative to microorganism’s improvement. By definition, IVDE is different from genetic engineering in that it is based on the exploitation of the natural evolution of microbial genomes submitted to selective pressures.

IVDE does not require a detailed and full preliminary knowledge of the genomic sequences involved in a particular trait that needs to be improved, as opposed to genetic engineering. Rather, IVDE is applied on the phenotypic characters of a strain as a whole, without knowing the intervening genomic sequences, the number and the nature of all the genes involved in a trait, or what the complex regulation structure of this characteristic is.

However, IVDE can be applied to any type of microorganisms, such as pure microorganism cultures, co-cultured strains, natural populations (consortia).

IVDE is conducted using special culture automatons designed to set-up and to control the selective culture conditions, where the microbial culture is constantly diluted with “fresh” medium, thus making it possible to maintain multiplying cells over long periods of time under selective pressures.

Such processes can be used either to maintain the culture at a constant cell density, or to keep constant the concentration of some components.

Finally, the ultimate steps of the IVDE process consist in isolating “improved cells” from the microbial population, measuring their new performance, characterising these new variant cells and then integrating them into appropriate industrial systems (such as fermentors, pilots and production processes).

Eco-Solution proposes the development of turnkey solutions for the regulatory and economic optimisation of treatment plants in the chemistry and pharmacy industries. These solutions are based on the identification and isolation of effluent streams which pose problems for the system’s performance level and on the development of specific microflora that are adapted for the upstream biological treatment of such streams.

Strengthening the metabolism of microflora issued from existing collections by IVDE provides Eco-Solution with the means to develop the biological processes for treating these streams that contain non-biodegradable molecules and are generally incinerated. These on-site solutions do not require complex or expensive technologies, are easy to implement, generate savings as compared to outside elimination systems, and also allow a reduction in environmental risks related to the transport of hazardous wastes.

On 21 February this year, Eco-Solution applied for a new patent portfolio describing a new generation of the IVDE technological system, allowing the high throughput screening of bacterial variants and fitted with automated functions that facilitate its management, the control of cultures, and experimentation.

This system allows the positioning of a large number of simultaneous cultures, as well as an increase in the capacity of the automaton to analyse and cultivate greater volumes of a given microbial culture.

This last development provides Eco-Solution with a means to access new markets associated with the improvement of the cultural properties of microorganisms with low rates of cells growth, such as the production of methane (CH4) from agricultural sub-products using anaerobic microflora or of biofuels using photosynthetic microorganism, like microalgae.

The development of agricultural biofuels has some major drawbacks such as deforestation, risk of environmental degradation, overexposure of farmlands to phytosanitary products, competition over land use for human and animal food crops, geographic separation between the origin of the needs and the agricultural resource (needs in the north, resource in the south).

In this context, algal biofuels constitute an alternative route which has many advantages compared to agricultural biofuels. Not only is the production of algal biodiesel not based on the conversion of agricultural raw materials or on any other vegetal raw material, it does not use up land areas meant for food crop production.

On the technical front, the tremendous oil productivity of micro algae cultures per hectare compare to agricultural feedstock (more than 350 hectolitres of oil per year and per ha for micro algae and 12 for rapeseed) and the technological simplicity of the production systems justify this sector’s rapid development.

Oil productivity, however, which is the mass of oil produced per unit volume of the microalgal broth per day, depends both on the algal growth rate and on the oil content of cells.

One of the main targets of Eco-Solution’s R&D program is to select naturally occurring microalgae strains that already present an initial potential to accumulate oil and to improve by IVDE their growth rate in effective process conditions, in order to obtain a higher level of oil productivity (higher oil content, faster oil accumulation).

Over the last 10 years, waste conversion into biogas has been successfully applied to the treatment of effluents from various industries and from the agricultural sector. The technology is developing rapidly in its application to the conversion of various organic sub products into biogas.

Anaerobic digestion (or methanisation) is the biological transformation, in the absence of oxygen, of organic wastes into biogas, mainly composed of methane and carbon dioxide. The global anaerobic digestion process is mainly limited by the inhibitory effect of acetogenetic products (dihydrogen and, to a lesser extent, acetate), and by the low rates of growth of methanogenic microorganisms. In addition, the hydrolysis phase which converts biological macromolecules into smaller molecules (oligosaccharides, amino acids, fatty acids, and so on) may be limited by some substrates such as wastewater sludge or “green” ligno-cellulosic wastes which are not readily biodegradable.
 
The hydrolysis of these substrates has received a great deal of attention over the last few years because they represent a considerable pool of energy worldwide, one which remains rather unexploited.

Eco-Solution plans to improve anaerobic digestion using two independent but complementary approaches, using IVDE on some enzyme-producing micro-organisms. The first one aims to increase the digestibility and rate of hydrolysis of difficult substrates, opening up new markets (Ligno-cellulosic “green” wastes, water treatment sludge, domestic wastes), whereas the objective of the other is to increase the rate of methanogenesis in order to improve the applicable load and the robustness of methanisers, irrespective of the substrate being treated.

By Jean Barthomeuf. Jean is general manager of EcoSolutions. He has an extensive background in the management and supervision of processes associated with the development of technological products and services.