6. Improving quality and quantity of alginate by means of fermentation strategies
Several studies were carried out at the beginning of 80's, which described alginate production by A. vinelandii either in batch [59,60] or continuous cultures [61-63]; however to our knowledge, none of these processes has yet been adopted for the industrial production of microbial alginates. Using bioengineering tools, several new experimental strategies were reported, only recently. These make it possible to obtain higher yields of alginates, with certain characteristics which are suitable for particular applications and are thus more competitive for the microbial polymer market. In this section we will describe and discuss the most recent advances regarding the influence of fermentation parameters, which determine the production and composition of alginate, as well as the few reports about the scale-up of the process and novel fermentation strategies for the production of alginate.
6.1 Influence of dissolved oxygen tension (DOT) and mixing
Aeration and mixing are critical parameters for the optimal production of polysaccharides. Reports have been published about the influence of these parameters on the concentration and chemical characteristics of the alginate, synthesized by A. vinelandii [47,64,65]. It is important to point out that dissolved oxygen tension (DOT) can be controlled, either by manipulating the agitation rate of the culture, or by varying the proportions of nitrogen or oxygen present in the gas inflowing through mass flow controllers [47,66]. The advantage of this latter method is that it is possible to independently evaluate the effects of hydrodynamics and oxygen transfer conditions. In the previous reports (cited in previous reviews [67,68]) the dissolved oxygen tension (DOT) was controlled by varying the agitation rate of the bioreactor and, for this reason, it was not possible to discriminate between the influence of the oxygen in the bulk liquid and the agitation speed, on alginate production.
Data obtained under non-nitrogen fixing [47,66,69] and nitrogen-fixing conditions , indicate that alginate production, as well as the molecular mass of the polymer, are strongly influenced by dissolved oxygen tension (DOT) and the stirring speed of the culture. Peña et al.  have found that under high DOT (5 % of air saturation), the bacteria produced more alginate (4.5 g/L) than that obtained at low (0.5 %) oxygen tension (1.0 g/L) in cultures conducted at 300 rpm. On the other hand, the higher the stirring speed (from 300 to 700 rpm), the higher the specific growth rate and alginate production rate in cultures where the DOT was constant at 3 %. However, low agitation speed (300 rpm) lead the culture to produce a polymer of high molecular mass (680 kDa), whereas a low molecular mass (352 kDa) alginate was obtained from cultures conducted at high (700 rpm) stirring speed. At 700 rpm, the mean molecular mass (MMM) increased to a plateau between 1 and 3 % DOT and then decreased to a minimum of 0.11 × 106 g/g mol at 7 %. Microscopic observations revealed the presence of cellular aggregates when the culture was conducted at 300 rpm. Oxygen gradients occurring within big aggregates (more than 1000 μm) may be responsible for this phenomenon . It is important to point out that with a high agitation rate, the MMM of the alginate dropped towards the end of the culture in all conditions evaluated, which was probably due to AlgL activity .
Sabra et al.  reported that in phosphate-limited continuous culture, the specific rates of oxygen consumption and alginate formation of A. vinelandii increased as a function of the DOT of the culture, obtaining a specific alginate production rate of 0.2 g/g.h at a dilution rate of 0.22 h-1 at 5 % of DOT . Furthermore, in the same study, the authors reported that both the molecular mass and the L-guluronic acid content increased with the DOT, reaching a maximal molecular mass of 800 kDa and a guluronic acid content of 50 % in the cultures conducted at 10 % of air saturation. Sabra et al.  proposed that under nitrogen-fixing conditions, the bacterium builds an alginate capsule, with the composition varying in accordance with the external DOT and this may also help to protect the nitrogenase system against oxygen damage.
Trujillo-Roldán et al.  have made clear that alginate polymerization occurs by making chains with very uniform molecular mass distributions, which have low dispersion throughout the culture, regardless of the strain used (wild type or AlgL mutant) and of culture time (Figure 4). In addition, the MMM of these families is strongly affected by DOT, increasing to a plateau (between 1 and 3 % of DOT) and then decreasing at higher DOT values (Figure 4). These results indicate that the polymerase is highly affected by DOT. It is possible that transcription of alg8, encoding the polymerase, is affected by DOT as is the case for algA, algC and algD transcription in P. aeruginosa . Trujillo-Roldán et al.  reported that the alginate-lyase is not essential for the production of alginate; however, when the enzyme is present (as in the wild type), its role is restricted to a post-polymerization step, with its activity reaching a maximum in the pre-stationary phase of growth. The action of alginate-lyase is evidenced by a drop in the MMM of the alginate families.
6.2 Influence of medium components
It has been reported  that the components of the culture medium play an important role in determining the alginate production in the case of A. vinelandii. Recent literature has focused on the study of phosphate and nitrogen and how these affect the concentration and quality of the alginate produced . According to these authors, alginate production was not affected by phosphate and nitrogen concentration. In addition, they reported that the depolymerization of the alginate may be related to the concomitant occurrence of two or more limitations (low levels of oxygen, nitrogen or phosphate) or to the energetic state of the cells.
Peña et al.  have reported how (3N-morpholino)-propane-sulfonic acid (MOPS), a component used in the medium in order to keep the pH constant during cultivation of A. vinelandii influences the quality of the alginate in terms of its chemical composition and also the rheological behavior of alginate-reconstituted solutions. This compound had an important affect on the acetyl content of the alginate and in turn on the physico-chemical properties of this polymer. A higher acetylation of alginate was obtained when 13.6 mM of MOPS was supplemented to the medium. This value was twice as high as that obtained when no MOPS was used. The higher acetylation resulted in greater viscosity in the alginate solutions, but it exhibited less pronounced pseudoplastic behavior. These changes in the functional properties of the polymer can have great value in terms of specific applications of alginate in food and pharmaceutical fields.
The inoculation process represents an important aspect during alginate production, even though it is generally considered to be irrelevant. A typical inoculation consists of a pre-culture consisting of between 1 and 20 % (v/v) of the working volume of the production fermenter, where the exhausted culture medium is added to the new medium in the fermenter, together with bacterial cells. By washing the cells prior to inoculation, Trujillo-Roldán, et al.  have shown that it is possible to obtain alginates of a higher molecular mass (1200 kDa) than those obtained in cultures conventionally inoculated (350 kDa). These results suggest that components in the exhausted inoculum broth affect alginate characteristics, and should therefore be considered in strategies designed for alginate production.
6.3 Use of CO2 to prevent alginate degradation
A. vinelandii is a bacterium exhibiting a high respiration rate and thus also a high CO2 generation rate. A study about the influence of carbon dioxide on the production and quality of alginate in batch cultures, conducted in a 1 L bioreactor under constant dissolved oxygen tension of 3 %, was carried out by Seáñez et al. . Bacterial growth and alginate production were affected by the CO2 addition. In terms of growth rate and alginate production, inhibitory (0–8 %) and stimulatory effects (13 %) were observed, and a total growth inhibition was obtained when using 25 % CO2 in the inlet gas stream. Studies about the de-polymerization of alginate using broth extract from cultures developed with and without CO2 showed that high CO2 concentrations inhibit either the synthesis or the activity of AlgL 
6.4 Novel fermentation strategies
Saude & Junter  have reported the production of alginate by batch cultures of A. vinelandii, immobilized in a system constituted by a gel layer and a microporous membrane structure. The immobilization of A. vinelandii cells favored the production of alginate with a high molecular mass (MM) and low polydispersity, as compared to conventional free-cell cultures grown in shake flasks. Cheze-Lange et al.  reported the advantages of continuous production of bacterial alginate by A. vinelandii, coupled to a system of membranes of varying nominal pore sizes. According to these authors, the yields of alginate with respect to sucrose were significantly higher than in the batch process; however, the molecular mass of the polymer and the polydispersity were very similar to those of the alginate obtained from the batch experiments.
Asami et al.  studied the behavior of alginate synthesis by A. vinelandii in batch experiments conducted in bubble column and shake flasks. They found that the productivity and the fraction of GG-blocks of the alginate obtained in the bubble column were higher than those obtained in the shake flasks. In the bubble column, the production of GG-blocks in the late exponential growth phase was higher than that obtained in the stationary phase. However, the authors did not explain the reasons why the fraction of GG blocks changed under varying conditions, for example because of shear stresses and oxygen tension.
Priego et al.  used exponentially fed-batch cultures with the aim of determining the effect of specific growth rate on alginate production and on its molecular characteristics. In this study, particular care was taken in terms of the experimental conditions in order to study only the effect of μ, whilst discriminating the effect of other culture variables. The conclusion reached from this study was that the specific growth rate of A. vinelandii negatively affects the molecular mass of the alginate and to some extent, the alginate/biomass and alginate/sucrose ratio. This effect was particularly pronounced at very low specific growth rates (0.03 h-1) where the Yp/x, Yp/s and MMM increased by up to 2.3, 10 and 14 times higher, respectively, than those obtained at a specific growth rate of 0.21 h-1 (such as that found in conventional batch cultures). These findings are highly relevant for the reliable production of high molecular mass alginates.
6.5 Scaling-up of alginate production
The transfer of results obtained in plate to shake-flasks and in turn to stirred tank fermentors is troublesome and in general, poorly understood. There are very few reports covering aspects referring to the scale-up of the process for alginate production. Trujillo-Roldán et al.  reported scale-down studies, where conditions occurring in large scale fermentors were simulated in laboratory fermentors. In this study, A. vinelandii was cultured under DOT oscillating conditions, in fermentors. Exposure to oscillating DOT with wave periods of 1200 and 2400 s only slightly affected the growth of A. vinelandii and alginate production. In contrast, small changes to the average amplitude of the wave drastically affected alginate mean molecular mass and its distribution. These data suggest that poor DOT control in alginate fermentation, caused for example by high viscosity and/or insufficient mixing, could lead to the loss of polymer quality in terms of its molecular weight.
The mean molecular mass of alginates produced by A. vinelandii in shake flasks can reach values of up to 1900 kDa and viscosities of up to 520 cps, for broths containing about 5 g L-1 of alginate . However, when the process has been translated to laboratory fermentors (1 L), in which pH and DOT were kept constant, the molecular mass and viscosity of the broths were considerably lower, obtaining alginates with a molecular mass of less than 0.68 × 106 Da and viscosities lower than 100 cps for an alginate concentration of around 5.0 g L-1 [47,66,73] (Figure 5).
Using the specific power consumption (P/V) as criterion, the study carried out by Reyes et al.  revealed that in order to scale-up from flasks to fermentor, the initial power drawn did not permit the behavior of shake flask cultures to be reproduced (particularly broth viscosity-concentration profiles and mean molecular mass). Drastic differences in the power drawn evolution may be occurring during the cultures developed in shake flasks and the stirred-bubbled fermentor. Decreasing initial P/V in the fermentor or during cultivation, permitted the molecular characteristics of the alginate obtained in shake-flasks to be matched  (Figure 5).
More recently, Peña et al.  have rigorously studied both the evolution of the specific power consumption and the oxygen transfer rate, occurring in shake flasks in cultures of A. vinelandii, with the purpose of better understanding the behavior of alginate production in shake flasks and in order to develop strategies for the scaling-up of the process. These studies revealed that power consumption increased exponentially during the course of fermentation (up to 1.4 kW m-3) due to an increase in the viscosity of the culture broth. At the end of fermentation, when the viscosity and alginate concentration reached a maximum, a slight drop in the power consumption was observed. It is important to point out that the analysis of molecular mass distributions of the alginates suggests that in the shake flask cultures, DOT conditions may be more homogeneous than those present in a stirred fermentor, where control of DOT and pH is lacking .