Rouke Bosma

Harvesting of microalgae by bio-flocculation

The high-energy input for harvesting biomass makes current commercial microalgal biodiesel production economically unfeasible. A novel harvesting method is presented as a cost and energy efficient alternative: the bio-flocculation by using one flocculating microalga to concentrate the non-flocculating microalga of interest. Three flocculating microalgae, tested for harvesting of microalgae from different habitats, improved the sedimentation rate of the accompanying microalga and increased the recovery of biomass. The advantages of this method are that no addition of chemical flocculants is required and that similar cultivation conditions can be used for the flocculating microalgae as for the microalgae of interest that accumulate lipids. This method is as easy and effective as chemical flocculation which is applied at industrial scale, however in contrast it is sustainable and cost-effective as no costs are involved for pre-treatment of the biomass for oil extraction and for pre-treatment of the medium before it can be re-used.

Ultrasound, a new separation technique to harvest microalgae

In this article it is proven that ultrasound can be used to harvest microalgae. The separation process is based on gentle acoustically induced aggregation followed by enhanced sedimentation. In this paper, the efficiency of harvesting and the concentration factor of the ingoing biomass concentration are optimized and the relevance of this process compared to other harvesting processes is determined. For the optimisation, five parameters were modeled simultaneously by the use of an experimental design. An experimental design was chosen, because of possible interaction effects between the different parameters. The efficiency of the process was modeled with a R-squared of 0.88. The ingoing flow rate and the biomass concentration had a lot of influence on the efficiency of the process. Efficiencies higher than 90% were reached at high biomass concentrations and flow rates of 4–6 L day−1. At most, 92% of the organisms could be harvested and a concentration factor of 11 could be achieved at these settings. It was not possible to harvest this microalga with higher efficiencies due to its small size and its small density difference with water. The concentration factor of the process was modeled with a R-squared of 0.75. The ingoing flow rate, biomass concentration and ratio between harvest flow and ingoing flow rate had a significant effect on the concentration factor. Highest concentration factors, up to 20, could be reached at low biomass concentrations and low harvest flows. On industrial scale, centrifuges can better be used to harvest microalgae, because of lower power consumption, better efficiencies and higher concentration factors. On lab- or pilot-plant scale, an ultrasonic harvesting process has the advantages that it can be operated continuously, it evokes no shear stress and the occupation space is very small. Also, when the algae excrete a soluble high valued product this system can be used as a biofilter.

Towards industrial products from microalgae

Microalgae show an enormous potential as sustainable feedstock for numerous bioproducts. The current work analyzes the feasibility of business cases for different markets of products from microalgae. We perform a techno-economic evaluation of the whole process chain including cultivation, biorefinery and market exploitation for a 100 hectares facility in six locations. Our projections show a current cost per unit of dry biomass of 3.4 € kg−1 for microalgae cultivation in Spain (excluding biorefining products), with an expected reduction to 0.5 € kg−1 in ten years. A sensitivity analysis reveals the roadmap to achieve this. Production of high-value products (e.g. pigments) would be currently profitable, with a net present value of 657 M€ in 15 years. Markets aimed at food and chemical commodities require further cost reductions for cost competitiveness, reachable in the next decade.

Thermodynamically controlled synthesis of β-lactam antibiotics. Equilibrium concentrations and side-chain properties

For the enzymatic synthesis of the antibiotic cephalexin, an activated acyl donor is generally used as one of the substrates (kinetically controlled approach); however, the thermodynamically controlled approach might be of interest since there is no need for activation of the acyl donor and less waste is produced. If the synthesis reaction can be combined with an effective product removal step, the thermodynamic approach can be beneficial. The thermodynamically controlled synthesis of cephalexin was studied at various pH values, solvent concentrations, and temperatures. With direct synthesis in water, only small amounts of cephalexin were formed (0.1 mm from 20 mm starting material by the Xanthomonas citri enzyme). Addition of water-miscible organic solvent had a positive effect on synthesis (by the Escherichia coli enzyme); the equilibrium concentration of cephalexin, however, was at best increased by a factor of 2-3 (in methanol and triglyme). The equilibrium antibiotic concentrations reported in this study were notably lower than the values reported in the literature. These differences originate from the improved analytical methods that are available nowadays. Low product concentrations were also found for other side-chains with an amino group at the α-position. Side-chains without this group can be coupled and give acceptable product concentrations. For these antibiotics, a thermodynamically controlled process may be an alternative to kinetically controlled coupling. Copyright (C) 1999 Elsevier Science Inc. All rights reserved. | For the enzymatic synthesis of the antibiotic cephalexin, an activated acyl donor is generally used as one of the substrates (kinetically controlled approach); however, the thermodynamically controlled approach might be of interest since there is no need for activation of the acyl donor and less waste is produced. If the synthesis reaction can be combined with an effective product removal step, the thermodynamic approach can be beneficial. The thermodynamically controlled synthesis of cephalexin was studied at various pH values, solvent concentrations, and temperatures. With direct synthesis in water, only small amounts of cephalexin were formed (0.1 mM from 20 mM starting material by the Xanthomonas citri enzyme). Addition of water-miscible organic solvent had a positive effect on synthesis (by the Escherichia coli enzyme); the equilibrium concentration of cephalexin, however, was at best increased by a factor of 2-3 (in methanol and triglyme). The equilibrium antibiotic concentrations reported in this study were notably lower than the values reported in the literature. These differences originate from the improved analytical methods that are available nowadays. Low product concentrations were also found for other side-chains with an amino group at the α-position. Side-chains without this group can be coupled and give acceptable product concentrations. For these antibiotics, a thermodynamically controlled process may be an alternative to kinetically controlled coupling.

Growth inhibition of Monodus subterraneus by free fatty acids

Monodus subterraneus is a microalga, which is known for its high eicosapentaenoic acid (EPA; 20:5omega3) content. To produce EPA commercially, high volumetric productivities of microalgae are required. These high productivities can be reached in flat panel photobioreactors with small optical paths that have to be operated at high cell densities (>10 g/L). However, at these cell densities a reduction of productivity is observed. This growth inhibition is probably caused by growth inhibitors released by the microalgae, which have been suggested to be fatty acids. Our aim was to investigate if free fatty acids produced by M. subterraneus inhibited growth of this species. Therefore a bioassay was developed and saturated, unsaturated and poly-unsaturated fatty acids occurring in Monodus were tested on their growth inhibiting properties. Growth of M. subterraneus was completely inhibited at a saturated concentration (96 microM) of palmitoleic acid (16:1omega7). But, the saturated fatty acid palmitic acid (16:0) and the mono-saturated oleic acid (18:1omega9) were much stronger inhibitors. Growth was inhibited for 50% already at concentrations of 0.4 microM 16:0 and 3 microM 18:1omega9, respectively. These fatty acids probably cause the growth inhibition in high cell density cultures of M. subterraneus.

Thermodynamically controlled synthesis of cefamandole

The direct enzymatic synthesis of the antibiotic cefamandole is presented as an alternative for the current processes based on either chemical synthesis or kinetic enzymatic synthesis. The influence of pH and temperature on the apparent equilibrium constant was measured; from these data, the maximum cefamandole concentration was predicted. In spite of a beneficial apparent equilibrium constant at pH 3.75, less product was formed as compared to the concentration at the optimum pH (4.25). This effect is caused by the low solubility of one of the substrates at pH 3.75.The maximum product concentration (at the optimum pH of 4.25) was found to be 22 mM which is a significant concentration. The thermodynamically controlled synthesis may prove to be an alternative for the currently used processes if it can be combined with an effective in-situ cefamandole removal step.

Marine biotechnology in education: a competitive approach

This paper describes the development of a practical, which is taught to third year biotechnology students. We wanted to motivate the students by making them responsible for a research project. Competition was added as a stimulus for interaction between the students. A virtual company called CaroTech employed the students for 2 weeks. They worked in groups of two persons and each group was responsible for a 0.8 l flat panel photobioreactor. They had to produce as much beta-carotene as possible using the marine alga strain Dunaliella salina in this photobioreactor. On the first day, students developed a strategy to obtain optimal algal growth rate. They putted this plan into practice the second day and while cultivating the organism, they developed a second strategy how and when to stress the alga to initiate beta-carotene production. At the end of the ninth day, the total amount of beta-carotene was measured. To stimulate competition, the group that produced the most beta-carotene obtained half a point bonus on the final practical mark. On the tenth day, each group presented their results and an evaluation of their chosen strategies to the CaroTech board. Most groups were successful in growing algae. In the second phase some groups failed to stress the alga. The best group produced more than two times beta-carotene than the runner-up. The students were motivated by being responsible for their own results and the competitive approach. All students liked the practical and indicated that they learned a lot by following this practical.

Process Design for Enzymatic Adipyl-7-ADCA Hydrolysis

Adipyl‐7‐ADCA is a new source for 7‐aminodeacetoxycephalosporanic acid (7‐ADCA), one of the substrates for antibiotics synthesis. In this paper, a novel process for enzymatic 7‐ADCA production is presented. The process consists of a reactor, a crystallization step, a membrane separation step, and various recycle loops. The reactor can either be operated batch‐wise or continuously; with both types of processing high yields can be obtained. For batch reactors chemical degradation of 7‐ADCA can be neglected. For continuous reactors, chemical stability of 7‐ADCA is a factor to be taken into account. However, it was shown that the reaction conditions and reactor configuration could be chosen in such a way that also for continuous operation chemical degradation is not important. Downstream processing consisted of crystallization of 7‐ADCA at low pH, followed by a nanofiltration step with which, at low pH, adipic acid could be separated from adipyl‐7‐ADCA and 7‐ADCA. The separation mechanism of the nanofilter is based on size exclusion combined with charge effects. Application of this filtration step opens possibilities for recycling components to various stages of the process. Adipic acid can be recycled to the fermentation stage of the process while both adipyl‐7‐ADCA and 7‐ADCA can be returned to the hydrolysis reactor. In this way, losses of substrates and product can be minimized.

In situ product removal during enzymatic cephalexin synthesis by complexation

In this paper, ‘complexation’ indicates the formation of clathrate type inclusion compounds of cephalexin with naphthalene derivatives. These inclusion compounds readily crystallise in solution, resulting in specific co-crystals of complexing agent and cephalexin with a set ratio between both components. Complexation is used for in situ product removal during enzymatic kinetic cephalexin synthesis to prevent undesired hydrolysis. In order to achieve this, beneficial reaction conditions have to be matched with conditions that are beneficial for complexation. In the work described here, a pH of 7.5 and a temperature of 293 K meet these requirements best. The results were compared to predictions obtained with a model originally developed for cephalexin synthesis and which is now extended with complexation. For 1,5-dihydroxy-naphthalene, the course of the reaction was predicted accurately. For 2-naphthol, this was not the case; synthesis was enhanced and hydrolysis reduced compared to the model predictions for immobilised enzyme. On the other hand, the course of reactions could be predicted accurately by the model for liquid enzyme. Apparently, the reduced reaction rate (30% residual activity) is such that mass transfer can keep up with it and diffusion limitation was lifted resulting in higher cephalexin concentrations. The effect of in situ complexation on productivity is discussed. It was found that complexation has a beneficial effect on overall cephalexin productivity and in most cases, hydrolysis is suppressed. The effects were most pronounced for liquid enzyme in combination with complexation with 1,5-dihydroxy-naphthalene for which, also experimentally, the highest cephalexin concentrations were measured.

How to use Nile Red, a selective fluorescent stain for microalgal neutral lipids.

The use of Nile Red for rapid monitoring of the neutral lipid content in microalgae has gained interest over the last decade, since neutral lipids are feedstock for renewable transportation fuel. In this review, we discuss the main considerations needed to make an NR protocol reliable for staining neutral lipids in microalgae. Cell wall permeability must be enhanced by using stain carriers: DMSO (5% v/v to 25% v/v), glycerol (0.1 to 0.125mg/mL), or EDTA (3.0 to 3.8mg/mL). Temperatures between 30 and 40°C facilitate the diffusion of NR through the cell wall without incurring excess quenching. Good NR-lipid interaction requires using a low NR/cell ratio; the NR concentration must be between 0.25μg/mL and 2.0μg/mL, and the cell concentration >5×10(4)cells/mL. In order to have the maximum and stable NR fluorescence, it is necessary to scan the excitation/emission wavelengths for up to a 40-min of incubation time. We outline a five-step method to customize the Nile Red protocol to a specific strain: 1) Evaluate the strains suitability by checking for the presence of neutral lipid, 2) Select of the best excitation/emission wavelength, 3) Optimization of incubation time, stain carrier, dye concentration, and temperature, 4) Prepare single-strain algal cultures with different lipid contents to calibrate NR fluorescence with neutral-lipid content, and 5) Correlate NR fluorescence intensity to neutral lipid content for the same strain. Once the protocol is customized, the NR method allows for rapid and reliable monitoring of neutral lipid content of a microalgae strain.