The application of exogenous electromagnetic influences has been used for various commercial applications and an overview is given in Table 2.

The electric field pulses, or electroporation, have been traditionally implemented in metabolic engineering for gene transformation. Direct electroporation of a cyanobacterium Synechococcus elongates, introduced the enzyme Clostridial hydrogenase, which may lead to the development of a variety of hydrogenases for hydrogen production, coupled to pho­tosynthesis in cyanobacteria for bioenergy production [186]. In addition to membrane-permeabilizing effects, it can also induce biochemical and physiological changes in plant protoplasts, such as stimulating protein and DNA synthesis, and cell division and differentiation [187]. Alternatively, electroporation can also be used as a process for cell membrane modifi­cation for enhanced oil/lipid extraction from microalgae for biodiesel. A preliminary study found 20% increase in oil yield, shorter extraction time, and 2/3 less solvent used without affecting the composition of extracted fatty acids compared to chemical solvent alone [33].

Although most electrochemical and electromagnetic effects mentioned thus far have been focused on biological responses, an integrated biorefin­ing system also requires process engineering technologies for harvesting algae for instance. An electrochemical process using direct electric cur­rent, called electroflocculation, has a long history as a wastewater treat­ment technology for solid/liquid, and liquid/liquid separation [188]. This technology combines the use of a sacrificial electrode that dissolves to coagulate suspended particles (electrocoagulation) along with the use of electrolysis, which produces H2 microbubbles that float the aggregates or flocs to the surface (electroflotation) for easy removal from the water. Electroflocculation is a promising technology for harvesting microalgae biomass since it has several advantages over other conventional processes. The efficiency of particle/biomass separation in electroflocculation is over 90% and this technology does not require moving parts, and consumes relatively little energy (0.3 kWh m-3) with substantially lower capital costs [189]. In fact, a more recent study shows a 99.5% removal of total sus­pended solids (TSS) and chlorophyll a (algae) by applying 0.55 kWh m-3 for 15 minutes [190].

Bioelectrochemical denitrification is a novel technology being used for the treatment of ammonium and nitrate-containing wastewater by means of denitrifying bacteria and hydrogen gas produced on the cathode by the electrolysis of water. The denitrifying microorganisms are usually immo­bilized as a biofilm on graphite or a stainless steel cathode. A nitrate re­moval efficiency of 98% was observed at 20 mA when phosphate was used as a buffer. The studies suggested that the application of bioelectroreactors could be used for reduction and oxidation treatments of ammonium and nitrate-containing wastewaters [191-193].

In many cases, real-time monitoring of cultures is critical for produc­tive and efficient cultivation/fermentation in which the optical density, pH, and dissolved gas levels may not elucidate the underlying bioprocesses occurring, especially when evaluating electrochemical or electromagnetic interactions. Pulsed Amplitude Modulation Fluorometry or PAM fluorom — etry is a special method for measuring fluorescence from photosynthetic organisms for real-time culture monitoring of the photosynthetic appara­tus. It uses the characteristics of the fluorescence emitted by chlorophyll a as a probe for the biophysics and biochemical events occurring in the electron transport chain of Photosystem I & II. These measurements are a unique indicator of photosynthesis and provide information about the maximum photosynthetic efficiency (by a dark-adapted sample), the effec­tive photosynthetic efficiency (under constant illumination), and the non­photochemical quenching (heat dissipation). These parameters indicate what fraction of the photon energy absorbed by the organism is used for photochemistry, dissipated as heat, and re-emitted as fluorescence [194]. Papazi and his colleagues found that PAM Fluorometry in conjunction with traditional biomass analysis was able to show how extremely high CO2 concentrations impacted the photosynthetic apparatus, which stimu­lated intense biomass production in the microalgae, Chlorella minutissima [195].

The use of the biophotonic method of delayed luminescence (DL) has been used for quality control applications with fruits and vege s. A study with tomato fruits revealed marked changes due to different harvesting maturities. It was found that tomatoes exhibited DL measurements related to color and respiration as well as significant differences in soluble solids content and dry matter percentage. Therefore, DL values are directly re­lated to tomato harvest maturity. Qualitative traits can depend on harvest maturity, thus suggesting that delayed luminescence could be used as a nondestructive indicator of fruit quality [196].

In addition to fluorescent measurements, the fast, non-invasive mea­surement of biological cells by dielectric spectroscopy, or impedance spectroscopy, is currently being utilized to determine cellular parameters, such as living cell volume, cell number distribution over cell cycle phase, cell length, internal structure, complex permittivity, and intracellular and extracellular media and morphological factors. The electrical and

morphological properties of the cell membrane are assumed to represent sensitive parameters of the cellular state [197]. It has been demonstrated to be a powerful method for dielectric monitoring of biomass and cell growth in ethanol fermentation and the extension of the scanning dielectric micro­scope is a promising tool for dielectric imaging of biological cells [198]. The real-time monitoring of yeast cell division by measuring the dielectric dispersion can enable to tracking of cell cycle progression using an elec­tromagnetic induction method [199]. Recently, online monitoring of lipid storage in microorganisms (yeasts) was conducted which found that us­ing dielectric spectroscopy data, the change in capacitance divided by the characteristic frequency being used showed a clear shift from the growth phase to the lipid accumulation phase, which could be of use for technical control of intracellular biopolymer or oil accumulation, as well as enzyme overproduction [200]. Moreover, it has been established that there exists a connection between D. L. and impedance spectroscopic parameters, which explore related structures and mechanisms in living samples [201]. By applying the knowledge, gained from biophotonic and bioelectromagnet­ic experiments, it may be possible to detect, interpret and interact with the endogenous coherent electromagnetic signals that are correlated with regulation, communication, and organization of biological systems since oscillation dynamics are of essential importance in intercellular and intra­cellular signal transmission and cellular differentiation [70]. These signals may initially give us real-time insight into the internal dynamics of an organism or culture, which may precede the physically/chemically observ­able events.

Induction of specific cellular response to biophotonic signals could perhaps be achieved to stimulate a desired biological effect such as en­hancement of lipid or enzyme synthesis or metabolite modulation using electromagnetic fields instead of an external stress or a biochemical ini­tiator. Electromagnetic bioprocesses such as electroflocculation and elec­troporation can be used for algal harvesting and biomass processing. The use of static and oscillating electromagnetic fields has a potential for the enhancement of cell proliferation, metabolite production and cell cultiva­tion for biomass production. After extraction, fermentation of the algae feedstock, using applied electric field parameters, can be designed for en­hanced substrate utilization and higher ethanol/butanol yields. Any residual biomass may then be used for enhanced production of methane from an­aerobic digestion using specific frequencies of microwaves reported by Banik et al. [5], who showed how the EM exposure parameters could be used for potential bioenergy/biofuel applications.

The application of electromagnetic coupling to electrochemical bio­logical pathways, which have been studied and commercialized for bio­medical applications can be introduced into bioengineering. Here inves­tigations into the electrochemical impedance properties for triggering biochemical cascades of desired signaling pathways in microorganisms for bioenergy applications deserve significant attention. The application of exogenous EMF influences may synergistically couple with endogenous electric fields for enhancing directed mass transport in cells. It is conceiv­able that any cell could be stimulated, inhibited, or made to exhibit passive response, depending upon the appropriate choice of frequencies and am­plitudes of the excitation signals employed [62]. The induction of mitosis for cell proliferation, as well as the stimulation of enzymatic pathways as­sociated with energy metabolism and storage such as lipid accumulation, needs modeling and more experimentation. Such electrochemical process­es may also be relevant for accelerating enzymes, such as Rubisco, in the carboxylation pathway of photosynthesis to enhance specific binding of CO2 and limiting photorespiration to enhance overall system efficiency in microalgae or plants. The greatest challenge may be the evaluation of the proper dosimetry for modulation of the desired biochemical cascade [1].

The introduction of the complex topology of multipolar electromagnet­ic fields may provide an enhanced coupling effect to complex, interdepen­dent biological systems. Such systems may be tailored to uniquely control endogenous electromagnetic processes and communication for cellular functioning and organization. Furthermore, the bioproducts, generated by engineered multipolar hybrid biosystems have additional properties. For example biofuel/bioenergy production processes potentially can have higher productivities through better substrate utilization and conversion and shorter processing times.

The use of electrochemical/electromagnetic triggering of specific metabolic pathways could be coupled with biophotonic analysis, where rapid screening and fine tuning of a desired effect could be devised. Such bioelectromagnetic and biophotonic monitoring could also be of significant

Algae

Harvesting

m ethane production

КО со

FIGURE 7: Bioengineering of algae cultivation.

interest to metabolic and genetic engineering, by incorporating and corre­lating electrochemical and endogenous electromagnetic signals with gene expression and enzymatic activity. The pervasive utilization of water in the cultivation of microorganisms particularly with algae, suggests pos­sible application of the principles discovered in ultra-high dilution and activation studies to enhance and modulate biological responses. Water used in the growth medium for cultivation may be imprinted by various methods (electromagnetic information transfer probably being the most convenient) with specific information on relevant organic and inorganic nutrients as well as biochemical growth promoters to enhance growth characteristics, while decreasing demand for the potentially large amounts of the donor substance.

The combination of these separate disciplines, could blossom into a new integrative bioengineering approach that incorporates the diverse specializations of molecular biology, biochemistry, electrochemistry, bio­physics, and quantum physics that could open up significant biotechno­logical progress of engineering of living systems for bioprocessing, bio­conversion, biofuel and bioenergy applications (Figures 6 and 7).

6.6 CONCLUSIONS

Traditional cultivation and manipulation of biological systems have con­sisted of natural selection and genetic engineering modalities. Recently metabolic engineering and synthetic biology are gaining wide attention from the scientific community due to their immense potential in altering the metabolism in living systems especially microbes for medical, agri­cultural, industrial and environmental applications. However, genetic ma­nipulation of microbes and living systems for agricultural and environ­mental applications may affect the ecosystem adversely as the changes in the species are permanent and inherited. In case of bioelectromagnetic stimulation, system reacts more in a transient fashion. The changes even if inherited are not sustained by the species for long thus they might be safer over genetic manipulation.

This review provided a broad spectrum of potentially useful bioef­fects on microorganisms that are currently or potentially valuable in biotechnology and bioenergy. At this point it is difficult to ascertain ex­actly how economically feasible these emerging methods and potential technologies will be due to a variety of unknown factors from the nature and scalability of the bioeffects to the electronic design and efficiency for large-scale implementation. However, it is the aim to stimulate interest in the field and invite scientists with new ideas into the long standing disci­pline of bioelectromagnetics that modern biology is only recently starting to understand. In the new horizon of biologically derived fuels and mate­rials, advancements in the area of biostimulation could impact the direc­tion of biotechnology towards an energetic approach that may boost the potential for emerging biotechnologies such as microalgae based biofuel and biomass production.

Biofuels, bioenergy and carbon capture are considered to be the cur­rent priorities for the entire global community. The International Energy Agency (IEA) has reported that the world’s primary energy need is pro­jected to grow by 55% between 2005 and 2030, at an average annual rate of 1.8% per year. Fossil fuels are the main source of primary energy and if the governments around the world stick to current policies, the world will need almost 60% more energy in 2030 than today. Transportation is one of the fastest growing sectors using 27% of the primary energy. At the pres­ent staggering rates of consumption, the world’s fossil oil reserve will be exhausted in less than 45 years. Considering the negative impacts of utiliz­ing fossil fuel energy sources, many countries have already mandated the use of biofuels and set the targets to replace significant quantities of fossil derived fuels. Second and third generation biofuels such as lignocellulosic ethanol and algae biofuels are considered to be the viable alternatives as they do not compete with food needs. Bioelectromagnetic stimulation of microbes particularly with microalgae provides a new extended domain of disciplines and methodologies for cultivation, harvesting and processing of biomass for production of biofuels, bioenergy and added value bio­products. Though this technology is promising, lots of research efforts are needed in future to exploit its commercial potential for biotechnology and biofuel applications.