Table 2.

Successive steps which should be taken into consideration during the design of biosurfactant-mediated bioremediation processes

Design step	Relevant step	Criteria
I. Initial characterization of the polluted area	1. Initial recognition of pollutants	Establishment of either single or multi-contaminant type pollution
	2. Assessment of the target pollutants concentration range	Determination of readily bioavailable, potentially bioavailable and unavailable pollutant fractions
	3. Analysis of relevant environmental factors	Range of temperature, pH, redox potential, moiety, soil properties, etc.
	4. Evaluation of nutrient levels	Potential limitation due to insufficient microelements, electron acceptors, etc.
	5. Analysis of autochthonous microflora	Screening for native microbial consortia with the ability to either remove or mobilize the pollutant by producing biosurfactants
II. Laboratory scale experiments	1. Selection of appropriate bioremediators for conducting the bioremediation process	Microorganisms or plants which exhibit high tolerance toward target pollutants and distinct remediation potential (relevant catabolic genes, hyperaccumulative properties, etc.)
	2. Selection of additional amendments	Nutrients, co-inoculants, plant growth promoting microorganisms, arbuscular mycorrhizal fungi, etc.
Laboratory scale feasibility studies for biosurfactant-supplementation, approach A: Addition of externally produced biosurfactants (ex situ methods)	1. Selection of a biosurfactant and biosurfactant-producing microorganisms	Previous studies related to the topic or the native habitat of biosurfactant-producing microorganisms
	2. Assessment of potential biosurfactant-induced toxicity	EC50 values for relevant bioremediators towards biosurfactant only as well as biosurfactant-pollutant combinations; Analysis of microbial community dynamics as a response to the presence of biosurfactants
	3. Evaluation of efficiency for biosurfactant-amended remediation	Increase in pollutant bioavailability, increased removal rate, short-term stimulation, enhanced biomass growth for the bioremediator
	4. Determination of biosurfactant degradability	Biosurfactant not preferentially utilized compared to target pollutant, efficient usefulness period for short-term stimulation, time for re-introduction
	5. Establishment of an optimal biosurfactant production method	Assessment of potential carbon sources for biosurfactant production (waste materials); optimization of the production process; Determination of whether crude biosurfactant-containing cultivation broth may be used or is purification necessary
Laboratory scale feasibility studies for biosurfactant-supplementation, approach B: Stimulation of biosurfactant production on-site (in situ methods)	1. Selection of appropriate biosurfactant-producers	Preferentially – selection of biosurfactant-producing isolates from native microflora (autochthonous soil/marine microbes, rhizobacteria, etc.); Alternatively – use of non-producing isolates which may be genetically modified to secrete biosurfactants or application of microbial consortia with high bioaugmentation potential (high similarity between consortium members and autochthonous microorganisms). Both alternative approaches are subject to additional regulations
	2. Evaluation of biocompatibility between biosurfactant producers and the biofactor relevant for the treatment process	Lack of antagonistic interactions, simultaneous growth, increase in pollutant bioavailability, enhanced removal rate
	3. Selection of an introduction method	Spraying of the whole cultivation broth with free-living cells or immobilization on appropriate carriers
	4. Initial bioaugmentation tests	Satisfactory performance in terms of adaptability and survivability of the introduced biosurfactant-producers, no apparent shifts in microbial community dynamics, lack of antagonistic interactions with native microflora
	5. Long-term ability to produce biosurfactants	Monitoring the level of biosurfactants upon bioaugmentation, the presence of relevant biosurfactant-associated genes after a certain period of time
III. Field scale feasibility study	1. Environmental response towards biosurfactants or biosurfactant-producers	Shifts in microbial populations; toxicity of biosurfactant to native organisms; adaptability and survivability of bioremediators and/or biosurfactant producers upon introduction; other potentially negative effects (i.e. uncontrolled mobilization of pollutants)
	2. Efficiency of treatment	Short-term and long-term removal of target pollutants in biosurfactant-amended treatment compared to control; duration
	3. Evaluation of treatment feasibility	Justification of each treatment step; Potential efficiency enhancement vs. additional costs associated with biosurfactant-supplementation