Cultivation modes for microbial oil production using oleaginous yeasts – A review

Biochemical Engineering Journal 151 (2019) 107322

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Regular article

Cultivation modes for microbial oil production using oleaginous yeasts – A review

T

Eleni E. Karameroub, , Colin Webba ⁎

a b

School of Chemical Engineering and Analytical Science, The University of Manchester, Oxford Road, Manchester, M13 9PL, UK Department of Mechanical Engineering, University of Bath, Claverton Down, Bath, BA2 2AY, UK

HIGHLIGHTS

of recent advances in oleaginous yeasts cultivation in bioreactors. • Overview appropriate cultivation regime improves substrate utilisation and oil titre. • An fermentation mode is the most beneficial for high biomass and oil yields. • Two-stage modelling is critical contributor to bioprocess design. • Kinetic • Valorisation of by-products can improve the economics of large scale production. ARTICLE INFO

ABSTRACT

Keywords: Microbial lipids Oleaginous yeasts Cultivation methods Two-stage fermentation Kinetic modelling Bioreactors

Microbial oil has been a valuable alternative oil source with applications in the food, cosmetics and biofuels sectors. As concerns for the environment grow, microbially-synthesized oil has emerged as a potential contributor for sustainable production of biodiesel. However, the high costs of its production hinder its large-scale application. Main bottleneck to industrial lipid production is the cost of the fermentation stage. Therefore, it is imperative to design cultivations with little operating requirements and high yields, adjusted to an oleaginous system. This paper provides an overview of the latest advances in oleaginous yeast cultivation in bioreactors. Focus is given to oleaginous yeasts and de novo lipid accumulation due to their high lipid accumulating ability, robustness and versatility across a range of substrates. The advantages and disadvantages of different cultivation modes, feeding patterns and impact on biomass and lipid yield are critically reviewed. The role of biochemical engineering in facilitating understanding of the lipid accumulation kinetics, lipid productivity and its importance as predictive scale up tool is highlighted through a review of existing equations. Perspectives for further bioprocess improvement through kinetic modelling, and valorisation through utilisation of by-products are also discussed.

1. Introduction Microbial oil has recently attracted attention as a potential biodiesel feedstock in a move towards sustainable energy generation with no impact on the food chain [1,2]. Biodiesel production from crops has been partially responsible for the increased prices of edible oils and its conflict with food source utilisation, bringing microbial oil to the fore, as an alternative oil feedstock. Compared to waste oils and non-edible plants, microbially-derived oil is of particular interest thanks to its high quality, suitable composition for biodiesel synthesis and ease of production [3]. With a fatty acid content similar to common vegetable oils [4], microbial lipids are also transesterified to glycerol and fatty acid methyl esters [5,6].

⁎

All microorganisms are capable of producing lipids with functional role to some extent, but only a few of them can accumulate more than 20% of their weight in oil and have been classified as oleaginous [7,8]. Amongst several types of microorganisms, oleaginous yeasts hold a range of advantages, including their ease of cultivation and robustness against bacterial contamination [9,10]. Furthermore, their ability to consume a wide range of inexpensive feedstocks renders them an excellent potential alternative oil source. Although yeast oil has a long history back to the beginning of the 20th century and commercial production was demonstrated for food and pharmaceutical applications, including cocoa butter and single cell protein production [11,12], the lack of understanding behind its

Corresponding author. Present address: Department of Mechanical Engineering, University of Bath, Claverton Down, Bath, BA2 2AY, UK. E-mail address: [email protected] (E.E. Karamerou).

https://doi.org/10.1016/j.bej.2019.107322 Received 6 April 2019; Received in revised form 29 June 2019; Accepted 29 July 2019 Available online 14 August 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.

Biochemical Engineering Journal 151 (2019) 107322

E.E. Karamerou and C. Webb

process development and the cost of sugars restricted its industrialisation. Following a trade-off between microbial oil and vegetable oil, interest on microbial oil re-kindled owing to its potential integration with the biodiesel production process. A major obstacle to large scale microbial oil deployment is the high processing cost, currently not competitive as compared to plant oils [13,14]. In 2008, Ratledge [15] reported that the price of microbial oil would exceed $3,000/t, while in recent estimations the production cost was between $2,300/t and $3,400/t [13,16]. Current research focuses on developing cost-effective approaches to obtain microbial oil. Several types of microorganisms able to consume inexpensive feedstocks have been explored so far [17–20] and waste materials have been investigated as low-cost substrates [19,21,22]. To make the nutrients present in waste materials accessible to the microorganisms without compromising their quality and composition, appropriate pre-treatment methods such as enzymatic hydrolysis of food waste [23] or pre-treatment of lignocellulosic material need to be carried out [24]. Metabolic engineering tools have also been employed to alter cellular metabolic routes, and therefore improve the oil-accumulating ability [25,26]. In a techno-economic study for biodiesel production from yeast oil, the use of glucose accounted for 80% of the raw materials cost, which was about 44% of the overall cost [13]. Sugars of lignocellulosic origin, as presented in a recent review, may represent only 40% of the total production cost of 1 t of yeast lipids [27]. Co-products with pharmaceutical interest can be derived from the yeast fermentations, such as citric acid [28], carotenoids [29], adding value to the fermentation processes. The choice of substrate is a generally flexible parameter, since it can be selected based on local resource availability, but the establishment of robust bioprocesses may clearly impact on the scalability and market presence of microbial biodiesel. Moreover, once it has been proven, a certain strategy can be applied to various strains. However, it is imperative to design efficient fermentation modes with little operational requirements and high oil productivities. A significant amount of research is focusing on optimising process parameters and advancing fermentation strategies in conventional equipment with the aim of improving the conversion of raw materials. Recently, research on large scale production of microbial lipids has been reported [30] and integration of experimental and computational tools is underway [31]. In principle, the bioprocess should ensure that any provided substrate is utilised efficiently and the productivity is high enough to create the minimum waste. According to this, oil contents above 40% should be targeted and these are translated into less oil-free biomass, consequently less waste to be treated at the end of the fermentation [15]. The

lipogenesis conditions should be also taken into consideration for an effective fermentation design. A combination of excess carbon and a limiting nutrient (such as nitrogen, sulphur or phosphorus), is generally a prerequisite for lipid synthesis, the so-called ‘de novo’ lipid accumulation [8,12,32–34]. When carbon and nitrogen are available in the medium, cell proliferation is the prevalent process. Upon the consumption of nitrogen the carbon excess is converted into storage lipids [35]. This is likely to cause growth inhibition in a system operating in batch mode where a large initial amount of carbon source is supplied. Therefore, fed-batch and continuous cultivations are advantageous over batch modes in terms of avoiding inhibition and leading to higher productivities. Nevertheless, a specific amount of nitrogen must be provided to support cell density and make the oil yield meaningful. Hence, fermentations consisting of two stages, where at first biomass production is the target and after which lipid accumulation takes part provide a potential solution. The yield is also influenced by the channelling the excess of carbon source to other metabolites such as acids, polysaccharides and pigments. The feeding rate affects the carbon flux to metabolite production as well as oxygenation conditions [36,37]. This paper provides an overview of the literature concerning existing operational modes and experimental configurations that have been successfully applied for the cultivation of oleaginous yeasts, using bioreactors. A comparison of the advantages and weaknesses of these different modes is made and the scale-up potential with the use of kinetic modelling is critically discussed to highlight promising operational aspects of lipid production with the aim of suggesting an efficient cultivation mode. 2. Cultivation modes 2.1. The batch mode: a good screening tool for process development In principle, batch fermentation is a closed system, where all the reaction components (cells, substrate) are supplied at the beginning and the reaction takes place without external intervention [38]. It is a basic and the most studied method for oil production and is performed either in flasks with uncontrolled culture conditions (e.g. pH, aeration) or bioreactors, where these culture parameters can be better controlled. Batch fermentation in flasks is commonly used for media optimisation works or when yeast strain capabilities are being explored [39–42], while in bioreactors to identify optimal operating conditions for lipid production, such as pH [43], aeration rates [44], agitation speed [45] and initial media composition [46]. Results obtained from batch cultivations can be seen in Table 1. For example, Kitcha et al. [42],

Table 1 Summary of yields obtained via batch fermentation of oleaginous yeasts on different substrates at shake-flask and bioreactor scale. Strain

Method

C/N ratio

Scale

DCW (g/L)

Oil content (%)

Yarrowia lipolytica Lipomyces starkeyi Trichosporon dermatis Trichosporon coremiiforme Rhodosporidium toruloides Metschnikowia pulcherrima Yarrowia lipolytica Candida freyschussii Trichosporonoides spathulata Yarrowia lipolytica Rhodotorula graminis Yarrowia lipolytica Rhodotorula glutinis Candida viswanathii Lipomyces starkeyi Rhodosporidium diobovatum Metschnikowia pulcherrima Rhodosporidium toruloides

Crude glycerol Crude glycerol Soyabean oil, corncob acid hydrolysate Corncob acid hydrolysate Crude glycerol Xylose Crude glycerol Pure glycerol Crude glycerol Pure glycerol Corn stover hydrolysate Glucose Pure glycerol Glucose Hemicellulose hydrolysate Crude glycerol

45.9 84.89 N.R. 108.7 60 333 100 52 N.R. 69.69 N.R. 76.85 698 100 50 N.R.

Shake flask Shake flask Shake flask Shake flask Shake flask Shake flask 5-L bioreactor 2-L bioreactor 5-L bioreactor 3.7-L bioreactor 1-L bioreactor 1.5-L bioreactor 2-L bioreactor 7-L bioreactor 1.3-L bioreactor 7-L bioreactor

6.5 16.2 26.7 20.4 14.85 4.5 25 14.6 11.3 4.68 48 4.46 5.3 17 9.6 13.6

31 54.3 43.4 37.8 41.76 22.8 20 32 44.3 22.3 34 45.51 33 32.94 26.1 50.7

2 8.8 11.6 7.7 6.20 1.1 5 4.7 5.01 1.04 16.32 2.03 1.68 5.6 2.51 6.90

[51] [53] [54] [40] [48] [55] [56] [57] [43] [58] [59] [60] [44] [61] [62] [63]

Crude glycerol Glucose

N.R. 211

30-L bioreactor 3.5-L bioreactor

11 36.2

22 65.1

2.42 23.56

[64] [52]

N.R. Not reported. 2

Lipids (g/L)

Ref.

Biochemical Engineering Journal 151 (2019) 107322

E.E. Karamerou and C. Webb

examined in batch mode the extent of glycerol assimilation by 23 newly isolated yeast strains. Amongst them, two, Kodamaea ohmeri and Trichosporonoides spathulata, accumulated between 30 and 43% w/w oil content and total biomass, 10 g/L. Batch cultivation in flasks was used to investigate glycerol tolerance, using the yeast Candida freyschussii, prior to conducting bioreactor fermentations based on the outcomes from the flask cultivations [47]. The effect of crude glycerol impurities, altogether or separately, on growth and lipid accumulation of Rhodosporidium toruloides was studied in batch mode using flasks, in order to investigate the feasibility of supplying the crude material as a carbon source [48]. The same research group used batch mode to investigate how growth and lipid synthesis were affected by components present in saccharified liquid from food waste and it was found that the yeast accumulated 52.67% w/w lipids at 50 g/L sugar concentration and C/N ratio of 73 g/g [49]. Due to the small amount of nitrogen provided in a batch mode, the yields in cellular mass can be low. The initial high C/N ratio is not maintained, while the C/N cannot be controlled after the initiation of the culture and is variable. The oleaginous yeasts will continue growing until the exhaustion of nitrogen, so the residual C/N continuously increases until carbon gets consumed. A high initial C/N ratio requires large amounts of carbon added at the beginning and the high substrate concentration can sometimes lead to inhibition of growth, stressful conditions and discontinuity in cell and oil yields [50]. For example, use of ammonium sulphate and yeast extract at 0.5 g/L each, limited the final cell concentration of Yarrowia lipolytica strains to 4.2–8.2 g/L, in flasks using glycerol at 30 g/L [51]. The total nitrogen concentration must have been around 0.16 g/L assuming a 10% nitrogen content in yeast extract. On the contrary, the growth of Rhodosporidium toruloides was not inhibited by high glucose concentration (150 g/L) in the presence of NaCl and the strain reached cell concentration of 36.2 g/L with 65.1% w/w oil content, while the lipid yield on glucose was close to maximum achieved experimental levels (0.21 g/g) [52]. Despite the simplicity of batch cultivation, other modes with regulated C/N ratio levels can provide better substrate utilisation with higher yields.

(Table 2). Fed-batch cultivation of C. freyschussii on glycerol resulted in a 6-fold increase in the oil concentration, while the oil productivity was twice as high as that of batch fermentation [57]. In practise, the addition of medium dilutes the cell concentration and the fermentation can be characterised as either fixed volume (when broth is removed and the same volume of feed solution is added), or as variable volume, such as in the case of a constant rate feeding. The dilution effect can be lower, when the cellular density is high enough, although it is better to increase the concentration of the feed solution and to minimise the added volume to the bioreactor. 2.2.1. Fed-batch cultivations with pulsed medium addition The feeding patterns that are employed vary per case and are usually determined by ‘feedback control’ [66]. Most commonly, additional carbon source is added, when its levels in the broth are quite low [67]. For example, every time the residual glucose was 5 g/L, a concentrated glucose solution was added in pulses to achieve a target range of 5–25 g/L [55]. Similarly, Thiru et al. [68] fed concentrated glycerol solution to Cryptococcus curvatus in a 6-L bioreactor every time the residual glycerol was less than 3 g/L (in total 7 times in a period of 134 h). Tsakona et al. [69] used the same criterion to supply concentrated flour-rich hydrolysate every time the residual glucose concentration fell below 20 g/L during a 200-h cultivation of L. starkeyi, which achieved remarkably high cell density (Table 2). In another approach [46], increase in dissolved oxygen was used as an indication of reduced metabolic activity of C. curvatus, linked to the lack of carbon source, in order to supply additional glycerol. This resulted in a very high cellular concentration of 118 g/L with 25% w/w oil content, while the biomass yields in batch mode were below 10 g/L. However the high biomass yield can be attributed to a dense inoculum size. Additionally to its role in providing carbon, the feed of crude glycerol was used to control the pH of wastewater medium at 5 g/L for the cultivation of Trichosporon oleaginosus yielding 65.63 g/L of biomass and 54.53% w/w oil content. In other cases, the time interval between additions of feed is fixed. In fed-batch fermentation of R. glutinis, appropriate amounts of glycerol (30 g/L) and yeast extract were added in pulse every 24 h starting during the late exponential phase and almost doubled the batch cell and lipid concentrations to 9.4 g/L and 2.58 g/L [44]. In a fermentation of R. toruloides, crude glycerol was supplied after 72 h of batch phase, every 24 h in pulses to achieve final concentrations of 70–90 g/L [45]. Cell concentration of 23.1 g/L and lipid concentration of 9.4 g/L were obtained compared to 12.1 g/L and 6.1 g/L respectively, obtained in batch cultures.

2.2. Increasing the cell density: Fed-batch cultivation Fed-batch cultivation (Fig. 1) has long been considered as an effective mode for promoting higher cell densities [65]. Clearly, initial substrate inhibition can be avoided and a large amount of carbon is eventually converted to cells and lipids by the end of the fermentation

Fig. 1. (A) Schematic representation of fed-batch cultivation. S0 is the concentration of substrate in the feed medium, Xi, Si, Li are the concentrations of cells, substrate and lipids in the broth respectively and Fin is the flowrate of the feed to the bioreactor and (B) Summary of major advantages found in fed-batch cultivation mode. 3

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Table 2 Overview of different feeding patterns used during fed-batch cultivation leading to high cell densities and lipid contents. Strain

Method

Scalea

DCW (g/ L)

Oil content (%)

Ref.

C. viswanathii R. toruloides

Batch for 48 h and 4 pulses of glucose every 24 h (no nitrogen feed). Intermittent feed of glucose (pulses) to target residual concentration 50 g/L, every time the glucose concentration dropped between 0-5 g/L. Continuous feed of glucose to target 5 g/L in the broth. Batch for 16 h. Three pulses of glycerol at increasing amount to target C/N ratios of 20, 30, 45 respectively. Addition of pure glycerol 87% concentrated every time the dissolved oxygen was low. Batch phase for 72 h, then crude glycerol, salts and nitrogen were added at regular intervals. Batch for 68 h, then 3 pulses of glucose:xylose mixture and nutrients were given. Batch for 30 h, 6 glucose pulses every 20 h. Batch phase at 40 g/L glucose, then addition of glucose to achieve 20 g/L for lipid accumulation. Batch phase with 60 g/L glucose, then 5 pulses of glucose to maintain always ≤20 g/L of residual glucose in the medium. Batch at around 50 h and exponential supply of crude glycerol at variable feeding rate.

7-L bioreactor 15-L bioreactor

21 89

50 52.2

[61] [70]

15-L bioreactor 15-L bioreactor

127.5 43.82

61.8 49.99

[70] [72]

2-L bioreactor 2-L bioreactor 3-L bioreactor 30-L bioreactor Shake flasks 15-L bioreactor

118 31.2 42.1 104.1 11.4 106.5

25 44.2 36.7 82.7 63.6 67.5

[46] [73] [65] [74] [75] [76]

50-L airlift bioreactor 2-L bioreactor 2-L bioreactor 2-L bioreactor 30-L bioreactor 3.5-L bioreactor

39.2

43.3

[71]

10.05 30.5 65 50.4 37.2

60.70 30 32 45 64.5

[77] [57] [57] [68] [52]

3-L bioreactor

109.8

57.8

[69]

T. oleaginosus C. curvatus C. curvatus L. starkeyi C. curvatus Cryptococcus sp. R. toruloides R. glutinis R. glutinis C. freyschussii C.curvatus R. toruloides L. starkeyi a

Batch for 12 h with 95 g/L glycerol, followed by 4 pulses of glycerol and nitrogen source at C/N 85. Batch for 30 h and two pulses of pure glycerol when residual glycerol was getting consumed. Batch for 30 h and four pulses of glycerol and nitrogen source Pulses of crude glycerol every time its residual concentration fell below 3 g/L. Batch mode until glucose dropped below 10 g/L, followed by pulses of glucose 60% w/v every time glucose was consumed until 272 h. Batch until glucose dropped below 20 g/L (˜100 h), followed by pulses of concentrated glucose, every time the residual glucose dropped below 20 g/L over the course of 200 h.

All bioreactors reported in Table 2 are stirred tank unless specified.

were only slightly higher than in the cases of pulsed and constant feedings. In contrast to Zhao et al. [70], who utilised commercial glucose as carbon source, Yen et al. [71] compared feeding modes using crude glycerol. The complexity of this substrate can be responsible for not obtaining clearly conclusive results from the various feeding modes. A recent study utilised a variable glucose feeding rate directed by the glucose consumption rate to maintain a certain C/N ratio throughout the fermentation of T. oleaginosus and obtained a high cell density of 132 g/L with 54% w/w oil content [78]. On the other hand Rhodosporidium azoricum under the same feeding regime obtained lower cell density of 79 g/L, which is also a high value. In this fermentation mode, a key variable is the cellular uptake rate, which is strain dependent. Experimental data are required for its determination and the feeding rate should be adjusted according to the cellular requirements to direct the carbon flux to the desired product.

Fed-batch fermentation with glycerol supply doubled the lipid titre and cellular concentration of C. freyschussii, but when glycerol and nitrogen were supplied together, the lipid and cell concentrations were about four times higher (Table 2) [57]. This indicates that more nitrogen was necessary for building biomass, affecting the final lipid yield but also the overall glycerol consumption. The experimental design should be such to supply adequate amount of nutrients for growth at the right times. Scale-up of a fed-batch process from a 6-L to a 30-L bioreactor, resulted in comparable yields at both scales (69.2 g/L, 48% w/w and 50.4 g/L, 45% w/w biomass and oil content at 6 L and 26 L respectively). The composition of the feed medium and the time of feeding can have an effect on the lipid productivity, although results can be strain dependent. Nevertheless, in pulsed feeding modes, especially if the feed depends on manual operation there might be periods of time when the cells have depleted the nutrients and might experience starvation until the next feed.

2.3. Continuous cultivations

2.2.2. Fed-batch cultivations with continuous medium supply Regulation of the substrate feeding mode has great impact on the productivities of oleaginous yeasts [36,66,70]. An appropriate substrate feeding mode makes the conversion of the carbon source to lipids more efficient (Fig. 2). Nevertheless, the number of studies investigating the effect of different substrate feeding rates in a fed-batch cultivation are to-date limited. The effect of pulsed and continuous glucose feedings on the growth and lipid productivity of R. toruloides was examined by Zhao et al. [70]. In their work, continuous glucose feeding, which maintained low residual glucose (5 g/L) improved, by 43% and 5%, the cell concentration and lipid content respectively, compared to glucose pulses (Table 2). Maintaining residual glucose concentration at 5 g/L was better than the case of 30 g/L residual glucose, as it did not inhibit growth [70]. After finding that carbon and nitrogen pulses increased the biomass production of C. freyschussii to 62.5 g/L with a 32% w/w oil content, Raimondi et al. [57] fed the same medium continuously and the cell and oil concentration were 1.3 times higher in the latter case (82 g/L and 28 g/L respectively). Yen et al. [71] examined the effect of pulsed, constant and exponential feeding of crude glycerol during fed-batch cultivation of R. glutinis. Although the exponential feeding gave the highest growth rate and least residual glycerol, the biomass, lipid content and productivities

In continuous operation, both carbon and nitrogen sources are supplied and a constant C/N ratio can be maintained during steady state [50]. A schematic representation of continuous cultivation is given in Fig. 3A. The cell concentration, the growth and the substrate uptake rate are constant and either growth or lipid accumulation can be targeted by modifying the residual nitrogen concentration. Continuous fermentation is better than batch mode in terms of lipid productivity and economic feasibility as cells can be harvested and processed throughout the process [79]. Continuous supply of nutrients avoids inhibition caused by a large amount of substrate and cells are always provided with fresh medium, as in the fed-batch mode with continuous feed supply. The main factor affecting the productivities in a continuous system is the dilution rate, D. Extensive studies on the effects of dilution rate on yeast growth and lipid production have been carried out by Papanikolaou et al. [80]. They studied dilution rates from 0.02 to 0.13 h−1 and detected higher glycerol levels in the broth at higher dilution rates, while lipid synthesis was favoured at low dilution rates (Fig. 3B). Similar studies have shown that high dilution rates favour cellular growth (lipid-free biomass synthesis) but not oil accumulation [65,81–83]. Table 3 summarises the cell densities and lipid titres obtained during continuous cultivations under different dilution rates. In 4

Biochemical Engineering Journal 151 (2019) 107322

E.E. Karamerou and C. Webb

Fig. 2. Feeding strategy versus lipid yield: Application of an appropriate feeding strategy can lead to increased lipid production.

simulated lipid and cellular yields were mainly steady over the range of D from 0.01 to 0.22 h−1, while at a C/N of 50, the yields were reduced when D was greater than 0.06 h−1 [84]. Shen et al. [81] used mass balances for steady state to describe the relationship of dilution rate with lipid production rate and yield as well as substrate consumption. This model allowed optimum dilution rates to be identified for the best lipid production rate. The values between 0.05 and 0.09 h−1 fitted well the experimental data. Rakicka et al. [36], applied a different feeding concept to an engineered strain of Y. lipolytica to improve utilisation of larger glycerol amounts. They maintained a constant dilution rate of 0.01 h−1 (Table 3) but increased gradually the glycerol concentration in the stock solution from 100 g/L to 250 g/L. This technique fed more glycerol gradually and resulted in higher oil yield and productivity (59.8 g/L of cells with 24.2 g/L lipids) than a fed-batch cultivation with 250 g/L stock glycerol solution (49.1 g/L of cells and 22.6 g/L of oil).

continuous fermentation of glucose using R. toruloides, under carbon limitation, dilution rates from 0.02-0.19 h−1 did not clearly affect the biomass or lipid production, while under nitrogen limitation, a drop in both cell mass and lipid yield was observed with increasing dilution rate [81]. At low dilution rates, the cells have more time (˜24 h) to consume the available nitrogen, first to grow and then convert carbon to lipids [62]. A similar study using C. curvatus on acetate showed that the lipid content decreased with increasing dilution rate at nitrogenrich and nitrogen-limited conditions [82]. Despite the drop in the specific lipid formation rate the acetate consumption rate increased with increasing D, because of the increase in cell mass yield. Beligon et al. [84], constructed a predictive model based on Monod kinetic expressions including carbon, nitrogen and oxygen terms in the growth rate and lipids equations in order to simulate lipid production from acetic acid using continuous fermentation. At a low C/N ratio of 10, the

Fig. 3. (A) Schematic representation of continuous cultivation. S0 is the concentration of substrate in the feed medium, Xi, Si, Li are the concentrations of cells, substrate and lipids in the broth and bleed collection bottle respectively, Fin is the flowrate of the feed to the bioreactor and Fout is the flowrate of the bleed from the bioreactor. (B) The effect of dilution rate on the biomass yield on glycerol (YX/S), lipid-free biomass on glycerol (YXf/S) and lipids on glycerol (YL/S), adapted from [75].

5

Biochemical Engineering Journal 151 (2019) 107322

E.E. Karamerou and C. Webb

continuous fermentation targets high cell concentration through sequential batch cycles (Fig. 4). In this approach, after an initial batch growth phase, a portion of the broth is removed and the remaining cells are used as a concentrated inoculum (˜10%) for the next batch cycle in the same bioreactor vessel, to provide a very large inoculum and reduce downtime (for cleaning etc.). The lag phase is reduced thanks to the concentrated cell suspension and the removal of the majority of potentially toxic metabolic products from the previous cycle [89]. These cultivations result in high cell densities and lipid contents compared to batch mode [90,91]. An overview of Draw-and-Fill cultivations of oleaginous yeasts is given in Table 4. Repeated batch of C. curvatus in shake flasks at different C/N ratios, improved the oil content of the first batch cycle from 24% w/w to 44% w/w at the end of the fourth cycle for the highest C/N ratio applied [92]. However, they take longer (˜238 h) and the prolonged time can result in a decrease in the cellular activity, as has been observed with R. toruloides Y4 on glucose [70]. For example, the final biomass increase of C.curvatus grown on Volatile Fatty Acids (VFAs) between the third and fourth cycle was 0.73 g/L, while it was only 0.09 g/L between the fourth and fifth cycle [93]. Research in the same group showed recently that draw-and-fill cultivation of C. curvatus at high concentrations of acetic acid resulted in secretion of lipids [91]. In particular, the maximum intracellular lipid production occurred at the end of the second cycle, after which extracellular lipids appeared, due to damage in the cell membrane. Decrease in the specific growth rate was also observed during 5 cycles of repeated batch of R. glutinis on palm oil mill effluent [94]. Although the biomass and lipid yield (10.9 g/L of dry weight and 67.27% w/w) were slightly higher than the yields in batch (biomass 9.15 g/L and 60.62% w/w lipid content), the specific growth rate decreased from 0.024 h−1 to 0.009-0.013 h−1 during the last cycles. C. curvatus was subjected to three cycles on 8 g/L VFAs coming from continuous macroalgae anaerobic fermentation, as a carbon source [95]. All cycles had similar final cell concentrations and the lipid titre increased by 17% between the first and second cycle. A drop in the oil yield from nearly 50% w/w to 30% w/w was noticed at the beginning of each cycle. Sample analysis showed that VFA uptake was reduced at the beginning of each cycle, with consequent drop in the previously achieved oil content. Possibly, slight lipid degradation occurred due to the lower uptake rate while production of lipid-free biomass was the key process at the start of a new cycle or the culture needed time to adjust to the fresh medium. It could be concluded that the main reason for the similar biomass yields can be the dilution effect of the existing biomass when the new medium is added. The removal of broth takes away a big portion of active cells. Moreover, it should be taken into account that at large scale, the culture will require time to reach steady environment after the addition of fresh medium, due to mixing deficiency to match the environment of the remaining cell population, increasing the time the culture will reach a homogeneous environment. Nevertheless, this cultivation mode allows

Table 3 The impact of the dilution rate on the cell concentration and lipid titre under continuous cultivation mode. Yeast

Carbon source

D (h−1)

DCW (g/ L)

L (g/L)

Ref.

Y. lipolytica

Crude glycerol Acetic acid

R. toruloides

Glucose

L. starkeyi

Sugarcane bagasse hydrolysate Glycerol

8.1 4.9 3.8 5.05 1.6 0.77 8.67 3.19 1.63 13.3 10.0 59.8

3.5 1.1 0.3 3.35 0.63 0.12 5.36 1.17 0.21 7.1 4.33 24.2

[80]

C. curvatus

0.03 0.08 0.13 0.01 0.06 0.11 0.02 0.10 0.20 0.03 0.06 0.01

Y. lipolytica

[82] [81] [65] [36]

However, large amounts of citric acid (50.2 g/L) were secreted from the beginning of the cultivation. The feeding rate of carbon source is quite important for the carbon flux and cellular metabolism to product synthesis and energy/maintenance [80,85]. The long-term operation of a continuous culture and the constant biomass levels can be preferable compared to a batch cultivation and allow for continuous cell harvesting. Nevertheless, a long-term fermentation can result in blockages due to media sedimentation or biofilm formation if the cell density is high, while it imposes the risk of contamination and compromise on the yield due to cellular changes. Moreover, in a process where the product is intracellular, it would be better to maintain steady number of cells and not bleed, to increase the lipid yield (continuous fermentation with cell recycling). Cryptococcus albidus reached high cell density of 24.8 g/ L with 35.5% w/w oil content, when cultivated under continuous fermentation with cell recycling through a hollow fibre membrane at dilution rate 0.35 h−1 and bleed ratio 0.05 h−1 [86]. After 40 h batch growth, the continuous phase started at only cell recycling and stepwise increase of D, while after 80 h, partial bleed started at stepwise increasing rate until 192 h. Higher bleed rates affected negatively the lipid titre and yield, which indicates that there should be a balance between the dilution rate and bleed rate for optimal cell density and lipid accumulation. The lipid productivity was between 0.41 and 0.69 g/L/h for all bleed ratios tested [86]. Cell recycling is a common method for extracellular products (e.g. succinic acid, citric acid), where it is necessary to maintain a high cell concentration with continuous secretion of the product [87–89]. 2.4. Repeated batch (Draw-and-Fill) In the repeated batch (Draw-and-Fill) cultivation mode, a semi-

Fig. 4. Schematic representation of repeated batch cultivation. 6

Biochemical Engineering Journal 151 (2019) 107322

E.E. Karamerou and C. Webb

Table 4 Common draw-and-fill cultivation strategies and their respective cell and lipid production values. Strain

Carbon source

Cultivation method

DCW (g/L)

Lipids (g/L)

Ref.

C. curvatus

VFAs

4.53

1.79

[96]

C. curvatus

VFAs

Five 15-h batch cycles in shake-flasks with full medium replacement, total duration of draw-andfill ˜70 h. Four batch cycles in shake flasks with full medium replacement, total cultivation lasted for 35 h.

N.R.

[91]

R. toruloides

Glucose

101.66

15.34 (intracellular) 5.43 (extracellular) 61.4

[70]

R. glutinis

Palm oil mill effluent VFAs

7.5

2.25

[94]

2.4

1.35

[95]

C. curvatus

Three 90-120 h cycles in a 15-L bioreactor with 90% of the broth removed, total fermentation lasted for 360 h. Seven 48 h cycles in a 2-L bioreactor with 50% of the medium removed, total fermentation duration was 336 h. Three cycles with 90% of the medium removed in a 1-L bioreactor, total duration 168 h.

N.R. Not reported.

for regular cell harvesting throughout the process, which then translates the produced biomass and oil into a cumulative quantity.

2.5.2. Two stage cultivations with feed supply The two-stage concept can be adapted to fed-batch or continuous fermentations, where the two stages are modified easily by altering the nutrient supply and/or rate. Previous studies have shown that addition of just the carbon source during the final feedings of a fed-batch cultivation is able to boost the lipid yield [70]. Fontanille et al. [102] employed VFAs as carbon source during the second stage and glycerol or glucose during the first stage (Table 5). In order to maintain a constant C/N ratio throughout the cultivation, ammonium sulphate was supplied at a basal level. The principle behind this approach was that the bioconversion of VFAs was easier when enough biomass had been obtained [102] and led to 41.02 g/L of biomass with 34.59% w/w oil content. Many other strategies followed the approach of pulse-feeding carbon source (glucose or glycerol) only when its concentration had fallen below a critical value to maintain it above a certain level without nitrogen supply [68,70,74]. A few comparison studies were performed between one-stage and two-stage cultivations. T. spathulata was cultivated on crude glycerol media [43]. After 60 h of batch cultivation, crude glycerol and ammonium sulphate were pulse-fed every 12 h (one-stage), to enhance biomass production (17.3 g/L), while glycerol-only pulses (two-stage) gave higher lipid content (56.4%w/w) and slightly lower cell concentration (13.8 g/L) than the single stage. A combination of these two approaches could be very advantageous by feeding nutrient-rich medium to extend the cell production phase and supply some more carbon source for lipid synthesis later. The importance of the extended growth phase was demonstrated in fermentation of R. glutinis on pure glycerol [44]. One stage fed-batch cultivation with three glycerol and yeast extract pulses every 24 h was extended by two more glycerol and yeast extract containing pulses, followed by one glycerol only pulse. This resulted in 44% improvement in cellular density (16.8 g/L) and 20% in oil content (34.62% w/w) compared to the one stage experiment. Likewise, in a fermentation of C. curvatus on crude glycerol the transition from carbon and nitrogen-containing pulses to glycerol only feed, improved the cell concentration from 31.2 g/L to 32.9 g/L and the lipid content from 44.2% w/w to 52% w/w (Liang 2010). Similarly, a two-stage approach was applied in fermentations of R. glutinis on corncob hydrolysate [103]. The two-stage fermentation, with pulses of detoxified hydrolysate along with nitrogen, followed by carbon source alone, in this case undetoxified hydrolysate, resulted in an increase of 21% in the oil content and despite the slightly lower biomass concentration, higher lipid titre was obtained compared to that of onestage cultivation with carbon and nitrogen supply (Table 5). The twostage concept was applied in fed-batch fermentation of sucrose by R. glutinis. After an initial batch phase, nitrogen-limited exponential feeding took place to drive cell concentration to 70 g/L, followed by sucrose-only feed, which led to 106 g/L of cells with 63% w/w lipid content [104]. The two stages can also be differentiated by separate feed rates to supply the desired nutrients at a rate suitable for each stage’s product, biomass or lipids. Karamerou et al. [85] supplied after a 24-h batch phase of R. glutinis, glycerol and nitrogen continuously at a

2.5. The need for advanced cultivation modes In microbial oil production works, the aim is to increase the lipid yield on the carbon source and the lipid productivity to improve process economics. Metabolic engineering and/or process optimisation can manipulate the carbon flux to lipid synthesis [25,97]. Once the nitrogen source is exhausted, lipid production takes place. Each cultivation phase has different nutrient requirements; rich medium is required for the growth phase while only the carbon source is needed for the oil accumulation phase [98]. Hence, fermentations consisting of two stages, where at first biomass is produced and then oil accumulation takes place provide a potential solution [99]. A number of cultivation modes (batch, fed-batch and continuous) have been developed based on this two-stage regime. 2.5.1. Two stage batch cultivations It is worth reviewing the case of two-stage cultivation by combining two batch cycles, with cells from the first batch used as concentrated inoculum for a second batch [98–101]. In this case, nitrogen-rich media promote growth in the first stage and nitrogen-limited media induce lipid synthesis in the second stage. For instance, R. toruloides AS 2.1389 was cultivated in shake flasks, using glucose-containing media with low C/N ratio (C/N = 15) for 4 days, then the cells were re-suspended in acetic acid-based media under a higher C/N ratio of 200 [90]. In this way, a preferred carbon source such as glucose can be used for biomass production and a potentially inhibitory carbon source, such as acetic acid can be used as substrate when a certain level of biomass has been achieved and growth is not the primary goal. The final biomass was 6.75 g/L with 50.1% w/w lipid content, higher than the 4.21 g/L and 38.6% w/w achieved during repeated batch fermentation. However, in that study, a lag-phase was noticed after the inoculation of the cells into the acetic acid media, which limited somehow the final productivity. In a very similar approach, L. starkeyi was first cultivated on glucose-based media in a 15-L vessel and then all cells were re-suspended in a 7-L bioreactor with only glucose as the sole nutrient [101]. This method resulted in an increase of 83.5 g/L and 64.7 g/L in cellular mass and lipid concentration, reaching final values of 104.6 g/L of cells with 64.9% w/w lipid content, respectively. However, all these two-stage batch methodologies require two vessels at least to be carried out. Such a case is not easily applicable at large scale because it requires unloading of the previous vessel and loading of the second if two different vessels are used, where in such a case many bioreactors are required. If the same bioreactor is used throughout the process, it can be considered as a modification of the repeated batch approach, cleaning in place of the first vessel and sterilization are needed in order to be filled with the new media, while the cells need to be maintained at low temperature until re-inoculation. These operations would require down-time where no cultivation is performed, adding extra costs to the overall process. 7

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52.7 25.8

[109]

56.4 52 34.62 34.59 47.2 53 40.8 57.3 65.5 56.4 13.8 32.9 16.8 41.02 70.8 30.63 23 20.54 97.4 11.3

[43] [73] [44] [102] [103] [85] [85] [105] [108] [86]

50.1 64.9 54.2 69.5 6.75 104.6 16.8 22.93

[90] [101] [99] [106]

rate twice as high as the glycerol uptake rate of the growth stage and then glycerol feed only at twice the glycerol uptake rate of the lipid stage and this increased the lipid titre by 22% (Table 5). Two-stage continuous cultivation of C. albidus resulted in high oil content of 56.4% w/w, lipid titre 11.3 g/L and 0.57 g/L/h lipid productivity, all higher than the values obtained at the end of the growth stage (28.3% w/w, 2.8 g/L and 0.14 g/L/h respectively) [86]. Setting higher dissolved oxygen (DO) concentration for the growth phase and lower DO with additional glycerol supply for the lipid stage, improved lipid production of T. oleaginosus and it could reduce energy consumption by providing only the necessary oxygen for the yeast [105]. The two-stage biomass of 20.54 g/L was higher than that of constant DO experiments (20.54 g/L), as well as the oil content (57.3% w/w). Other researchers used temperature shift from 30 °C to 25 °C during the lipid phase to induce lipid accumulation in shake flasks using Rhodosporidium fluviale [106]. To optimise both lipid and carotenoids production from R. toruloides, a dual pH strategy was applied alongside the two-stage cultivation [107]. After a batch period of 20 h, glucose and nutrients were supplied to enhance growth and lipid production at pH = 4, followed by a glucoseonly stage at pH 5 for further lipid but also carotenoids production. This approach resulted in higher yields of biomass than constant pH experiments, indicating that flexible process design adjusted to the requirements of lipogenesis, can further optimise bioproduction and process parameter variations such as pH and aeration rate can be easily applied at various scales.

Two-stage Batch Cultivations R. toruloides Cultivation for 120 h in glucose media (C/N = 15) and resuspension to C/N = 200 acetic acid-based media in flasks. L. starkeyi Growth in glucose rich media in a 15-L bioreactor and resuspension in a 7-L bioreactor in glucose-only media. C. curvatus Growth in N-acetylglucosamine seed media and resuspension to N-glucosamine solution in shake flasks under non-sterile conditions. R. fluviale Growth in glycerol-based media and 30 °C for 72 h, followed by resuspension to glycerol-only solution at 25 °C until 144 h in shake flasks. Two-stage Fed-batch Cultivations T. spathulata Batch growth for 60 h in glycerol and ammonium sulphate, followed by glycerol-only pulses every 12 h in a 5-L bioreactor. C. curvatus Batch growth for 72 h, followed by glycerol and ammonium chloride feeds and later glycerol-only pulses in a 2-L bioreactor. R. glutinis Batch growth for 24 h, then glycerol and yeast extract pulses every 24 h, followed by a glycerol-containing pulse in a 2-L bioreactor. Y. lipolytica Growth in glycerol media for about 60 h, then VFAs were supplied in a 7-L bioreactor. R. glutinis Until 80 h detoxified corncob hydrolysate was fed along with nitrogen, while between 80 and 199 h undetoxified hydrolysate was fed alone in a 5-L bioreactor. R. glutinis Batch growth for 24 h, then constant feed of glycerol and yeast extract at 2 mL/L/h until 96 h and from 96 to 144 h glycerol only feed at 2.85 mL/L/h in 2-L bioreactor. R. glutinis Batch growth for 24 h, then every 24 h until 96 h of glycerol and yeast extract media were added and at 96 h and 120 h glycerol solution was supplied in a 2-L bioreactor. T. oleaginosus Batch growth with 50-60% DO until 30 h, then addition of glycerol and switch to 20-30% (DO) until 70 h. L. starkeyi Growth in xylose media in a 10-L bioreactor and resuspension in a 7-L bioreactor in non-sterile xylose solution. C. albidus After batch growth, continuous phase started at a growth-promoting 1-L bioreactor with C/N = 8 mol/mol while the bleed was pumped to a second lipid-promoting 1-L bioreactor with C/N = 61 mol/mol, both at D = 0.05 h−1. Y. lipolytica Growth phase at 60% DO and shift to 30% during the lipid accumulation phase at a 5-L bioreactor.

Principle of operation Strain

Table 5 Overview of two-stage batch, fed-batch and continuous cultivations using oleaginous yeasts fermentations at bioreactor scale on various carbon sources.

Cell concentration (g/L)

Oil content (%)

Ref.

E.E. Karamerou and C. Webb

3. The role of biochemical engineering in microbial oil process scale-up Modelling is a useful engineering tool for process design, as it can be used to define kinetic expressions, predict experimental conditions and eventually it can provide guidelines for scale-up. This necessary engineering input can bring forwards insights and a view on the process, based on stoichiometry and mass balances. Although there is a plethora of experimental works on microbial oil production from different yeasts and substrates, the computational approaches are limited. Over recent years, there has been a significant modelling effort on microbial oil kinetics using yeasts [110,111] but there is a lot of scope for further research, since oleaginous systems are not straightforward to generalise. Initially, Ykema [112] proposed a model to predict the impact of C/N ratio on lipid yield and productivity under continuous mode of operation. Their approach utilised experimental data from continuous fermentation of Apiotrichum curvatum on glucose and was based on a generalised stoichiometric equation of the biochemical oil synthesis reaction. Later works focused on the kinetics of the process rather than the cost estimation and based the mass balances on the stoichiometric cell and lipids production for specific mode of operation. Economou et al. [111] developed a batch model for the growth of the oleaginous fungus M. isabellina on glucose. This approach assumed a well-mixed system, double substrate growth rate dependence on carbon and nitrogen with a combination of Andrews and Monod expressions (Eq. (1)). Lipid accumulation does not start before nitrogen becomes extinct (Eq. (2)). The model could predict well batch fermentations with different C/N ratios and even though it could predict lipid degradation, that was not necessary to be included in their results.

µSN = µSNmax

S KS + S +

S2 K i1

N KN + N

(1)

Where μSN (h−1) is the specific growth rate with respect to the carbon and nitrogen sources, μSNmax is the maximum specific growth rate with respect to the carbon and nitrogen sources (h−1), S is the concentration of sugars (g/L), N the nitrogen source concentration (g/L), KS and KN are the saturation constants for sugars and nitrogen respectively (g/L), Ki1 is the inhibition constant from high sugar concentration (g/L). 8

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qL = qLmax

S KLS + S +

S2 Ki 2

k1 k1 + N

parameters for continuous cultivation and to predict the growth of the oleaginous yeast on acetate.

(2) −1

Where qL is the specific lipid formation rate (h ), qLmax is the maximum specific lipid formation rate (h−1), KLS is the saturation constant for lipids (g/L) and k1 is a constant number ensuring that lipid accumulation takes place only when N comes close to zero. Even though Economou et al. [105] initially included lipid degradation in their equations, they did not observe experimentally such phenomenon taking place and removed this term from the model. A different approach to batch cultivation was taken by Meeuwse et al. [110], who modelled the cultivation of M. isabellina using glucose as carbon source. Their batch model described the different phases of fermentation (exponential growth, lipid accumulation and lipid degradation) with switch from one phase to another by introduction of fixed time expressions. They also took into account carbohydrate production, CO2 production and O2 consumption. During the growth phase, lipids are produced in proportion to the biomass growth (Eq. (3)), during the lipid accumulating phase lipid formation rate is slowing down (Eq. (4)) and lipid degradation is attributed only to maintenance (Eq. (5)).

L (t ) =

fL0 1

fL0

X (t )

qL =

fL0 1

fL0

+ qLmax (1

rX = µmax

k d (t t12 ) )

Xmax

rL = qL

t23)

S O X 1 KS + S KO + O

1+e 1+e

1 I

100 I 1 1

100

µ (N ) I1 I1

1

1+e 1+e

1 I

100 I 2 2 100

L % I2 I2

Where rL is the specific lipid production rate, qL is the lipids formation rate (h−1), I1 and I2 are the inflection points for shift to lipids production and shift to non-lipids production respectively, μ(N) is the specific growth rate Monod expression for nitrogen and L% is the lipid content. Robles-Rodriguez et al. [114] developed a model for describing the metabolism, lipid and citric acid production by Y. lipolytica on glucose under fed-batch cultivation. Their model structure was very similar to that of Economou et al. [111] with growth rate dependence on carbon and nitrogen, growth and glucose inhibition on lipid and citric acid production described by an Andrews relationship. The difference in their work was that lipid production was competing with citric acid synthesis. Furthermore, citric acid production is inhibited by the increasing amounts of the acid. They also developed a model based on intracellular quota (‘variable yield coefficients’), which maintained the glucose inhibition term but based the growth rate on intracellular nitrogen quota (Eq. (9)). The latter model explained better the kinetics on nitrogen and was used to optimise the feed rates in fed-batch mode and decrease citric acid formation. Nevertheless, this is not common for kinetic modelling of yeast fermentations and most researchers use unstructured model systems.

(4)

Where kd is the lipid production rate degradation rate constant (h ), t12 is the transition time from growth phase to lipid synthesis phase (h) and Xmax is the maximum cell concentration (mol of carbon/m3).

mL Xmax (t

(7)

(8)

−1

L (t ) = L (t23)

S N O X K S + S KN + N K O + O

Where rx is the biomass production rate (g/L/h), μmax is the maximum specific growth rate (h−1), S, N, O are the concentrations of carbon and nitrogen sources and oxygen and KS, KN, KO the respective saturation constants (g/L).

(3)

e

(6)

Where qL is the specific lipid formation rate (g/g/h), CL and CXf is the lipid and lipid-free biomass titres (g/L) and D is the dilution rate (h−1).

Where L and X are the lipids and biomass concentrations respectively (mol of carbon/m3), fL0 is the minimum fraction of biomass that is lipids (mol of carbon in lipids/mol of carbon in biomass), t is time (h).

L (t ) =

CL D CXf

(5)

Where mL is maintenance coefficient on lipids and t23 is the transition time (h) from lipid synthesis to lipid degradation. They also described experimental data from Economou et al. [111] well enough with small expected deviations in initial profiles for a starting concentration of sugars of 12 g/L. Their other modelling work [113] for continuous fermentation investigated three different limitations: carbon, nitrogen or dual limitation. It simulated well experimental data for biomass, lipid concentration and substrates (carbon, nitrogen) but it was not enough for CO2 production and O2 consumption since those values were not included in the model parameter optimisation [113]. Other works that dealt with modelling of continuous cultivation predicted the dependence of the lipid accumulating rate on the dilution rate. Shen et al [81] used mass balances based on continuous cultivation of R. toruloides on glucose to describe the specific lipid formation rate profile (Eq. (6)). The model was in good agreement with the experimental data and showed that for low dilution rates the specific lipid formation rate increases, it reaches a peak at 0.06 h−1 and decreases rapidly with further increased dilution rate. However, the interaction of the other process variables (biomass, glucose and nitrogen consumption) was not shown in that work. Another mathematical description of continuous fermentation was developed by Beligon et al. [84] using the yeast C. curvatus. The authors developed a model, based on a stoichiometric reaction for biomass, lipids production and maintenance, which incorporates oxygen in the growth rate, making it essentially a triple-substrate dependent model (Eqs. (7) and (8)). Differently from others, they described lipid formation as a sigmoid expression, continuing a function of the residual nitrogen. Furthermore, the point where lipid synthesis stops was determined by a sigmoid expression that contains the lipid content (Eq. (8)). After developing the model for fed-batch culture, they used it predictively to optimise the

µ = µmax 1

Q0 qN

S K S1 + S +

S2 KI 1

(9)

Where Q0 is the minimum nitrogen quota at which cell growth can take place (g of nitrogen/g total biomass), qN is the internal nitrogen quota (g of nitrogen/g total biomass), KS1 is the saturation constant for the carbon source (g/L) and KI1 is the inhibition constant (g/L). Aside from de novo lipid accumulation, ex novo lipid synthesis to produce edible oils and special fatty acids is of great interest to the food sector. Ex novo lipid accumulation is a primary anabolic activity independent to nitrogen limitation conditions, that occurs when yeasts are cultivated on hydrophilic substrates such as fats [115,116]. The fatty substrate needs to be hydrolysed by microbial enzymes, lipases, into free fatty acids (FFA) and glycerides in order to enter the cell [117]. During cultivation on fats, intracellular lipid degradation in support of lipid-free biomass is a common phenomenon, even though extracellular carbon is not extinct and may be a result of unbalanced uptake rates or reserve lipids saturation [118]. Though mathematical description of ex novo systems is limited, Papanikolaou et al. [119] constructed a three-differential equations batch model, describing biomass and lipid production of Y. lipolytica from fatty substrates, as well as lipid-free cells production from lipid degradation at low extracellular carbon uptake rates or concentrations (Eq. (10)). The specific growth and lipid accumulation rates are described by linear 9

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Table 6 Comparison of estimated model parameters generated by computational optimisation of oleaginous yeasts and fungi systems. Cultivation mode

Carbon source

μmax (h−1)

qLmax (h−1)

YL/S (g/g)

YX/N (g/g)

KS (g/L)

Ref.

Batch Fed-batch Batch Continuous Batch Batch Batch Batch

Sugars Glucose Glucose Glucose Industrial fats Glucose Glucose/Stearin Olive oil

0.566 0.415 0.21 0.2 0.23 0.15 0.32 0.215

0.786 0.03 0.09 n.a. 0.16 n.a. 0.003 0.0022

0.242 0.276 0.56 (Cmol/Nmol) 0.41 (Cmol/Nmol) 0.63 n.a. 0.52 0.750

18.21 23.23 5.5 (Cmol/Nmol) n.a. n.a. 40.1 60.4 n.a.

1.256 1.517 n.a. n.a. n.a. n.a. n.a. 0.011

[111] [114] [110] [112] [119] [120] [122] [123]

n.a. Non applicable.

relationships from their depending variables: fatty substrate or lipids (Eqs. (11)–(13)). The model described well the kinetic behaviour of the system, including cell mass synthesis from lipid degradation.

dSF = qL Xf dt

µ Xf (L) Xf

µ Xf (S ) = µ Xf (SF ) µ Xf (L) = µ Xf (L)

qL = qLmax

max

1 YXf / L

fatty acids concentration in the substrate (g/L), rE is the specific hydrolysis rate of the substrate (h−1), E is the lipase activity (g/L), α is a constant, μXfSFFAi is the specific growth rate on the substrate fat (h−1), Xf is the lipid-free biomass concentration (g/L), μXfLi is the specific growth rate on lipids (h−1), b is a number with units h−1 for converting the concentration to rate in Eq. (16) and kd is the inactivation constant of lipase (h−1).

(10)

SF SF 0

(11)

µ Xf SFFAi = µ Xf SFFAimax

L max

(12)

Lmax

SF SF 0

qL = qLmax

(13)

Where SF is the fatty substrate concentration (g/L), t is the time (h), qL is the specific lipid accumulating rate (h−1), qLmax is the maximum specific lipid accumulating rate (h−1), Xf is the concentration of lipidfree cells (g/L), μXf(L) is the specific growth rate on lipids (h−1), μXf(S) is the specific growth rate on fatty substrate (h−1) and YXf/L is the lipidfree biomass yield on lipids (g/g). SF0 and SF are the initial fatty substrate concentration and the fatty substrate concentration respectively (g/L), L and Lmax are the lipids and maximum lipids concentrations respectively (g/L). The kinetic behaviour of Y. lipolytica cultivated in the presence of glucose and stearin was simulated by a model constructed for biomass production from nitrogen and glucose and citric acid synthesis, complemented by a differential equation for fatty substrate conversion to biomass and lipids. The specific growth rate is therefore a function of residual and initial nitrogen in the media (Eq. (14)). Interestingly, de novo lipid oil accumulation takes place when both a sugar and a fatty substrate are present, while growth is primarily attributed to the consumption of fat. This model was applied to different scenarios of glucose and fat mixtures and matched well the biochemical phenomena taking place in the system [120].

µ = µmax

N N0

(14)

dE = dt

rE E

i=1

i=1

µ Xf Li Xf + bXf

kd E

K1j SFFAj

SFFAi K SFFA2i + SFFAi

(17)

(18)

Apart from the fermentation improvement, by means of efficient cultivation mode with full substrate utilisation and high productivities, process valorisation can be done by combined bioprocessing, utilisation of by-products and wastes.

n

µ Xf SFFAi Xf +

j = 1, j i

4. Other ways to improve profitability of microbial oils

(15)

n

n 1

Where KSFFAi is the saturation constant on free fatty acids in the substrate (g/L), K1j is the coefficient of substitutable substrates interaction for the fatty substrate (g/g), qL and qLmax are the specific and maximum specific lipid formation rate respectively and KSFA2i is the saturation constant of lipids on the fatty substrate (g/L). The above-mentioned studies are all good approaches to modelling oil production based mainly on adaptations of Monod kinetics. These models describe adequately two different systems, resulting generally in a good agreement between the estimated parameters for the maximum specific growth rate and the yield of lipids on carbon source across different works (Table 6). There is a variation between estimated values for the maximum specific lipid synthesis rate and the values for the saturation constants, but this can be attributed not only to small differences in the equations developed and the parameter optimisation, but also to the system described and the diverse effects of the carbon source on the cellular growth. Overall, higher lipid yields on carbon source (0.750 g/g) are observed in the case of fatty substrates (Table 6). However, the models have been mainly based on the use of glucose as carbon source and the assumptions applied are not widely applicable to other systems due to the specificity of oil accumulation and the strain dependent responses to different factors. Integration of the oxygen mass balance in the kinetic equations as shown by Beligon et al. [84] would definitely help in providing a better description and fitting of experimental data such as the growth rate and the lipid formation rate. Since oleaginous yeasts are aerobic microorganisms, oxygen dependence should be part of the growth rate equations. Though most models manage to describe well several systems, a universal model that can have better value for scale up while being able to predict the operating conditions found at scale is therefore needed.

A different and more complex approach to ex novo lipid accumulation was recently developed by Vasiliadou et al. [121] who advanced existing kinetic models on lipid synthesis from fats by introducing equations for lipase mass balance (Eq. (15)). The model also includes the deactivation of lipase, linking it to both biomass production and the substrate fat hydrolysis rate (Eq. (16)). Similarly to other approaches, the specific growth rate is related to the substrate fat (Eq. (17)) while the lipid production rate is described by Monod kinetics (Eq. 18).

dSF = dt

SFFAi K SFFAi + SFFAi +

(16)

Where SF is the fatty substrate concentration (g/L), SFFAi is each free 10

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4.1. Combined bioprocessing: Synergistic co-cultivation of oleaginous microorganisms

be greater at larger scale [52,82,127,128]. Application of selective culture conditions, such pH, inoculum size and/or combinations of them favours only the growth of a certain oleaginous yeast strain, rendering it dominant population. Moustogianni et al. [129] applied a two-stage concept with the growth phase of Thamnidium elegans aseptically, followed by lipid accumulation under non-aseptic conditions and addition of optimised thyme essential oil concentration, as antimicrobial agent, during fed-batch cultivation at bioreactor, resulting in 30% w/w lipid content. Microbial contamination of cultivation of a cold-adapted Y. lipolytica on non-sterile cheese whey medium was prevented by the combination of low temperature (15 °C), low pH 5.5 and inoculum size 3% v/v of the yeast [130]. Waste-derived media might contain toxic compounds that some yeasts have the ability to tolerate. Open cultivation of R. glutinis on high concentration of molasses minimised the extend of bacterial contamination and did not prevent the yeast reaching its highest oil content, 64.8% w/w [131]. Non-sterile cultivation of R. glutinis on starch wastewater was also demonstrated at a pilot-scale 300-L bioreactor using the draw-and-fill operation mode [132]. The uncontrolled pH, the non-sterilised media and the little nutrient enrichment prevented culture contamination. Optimised culture conditions and possible adjustment of culture parameters to specific strain requirements or selection of suitable substrate and its concentration may prevent bacterial contamination. While in most cases low pH cultivation is adopted, other strategies are of interest to potentially reduce utilities and operating costs associated to the cultivation stage.

Recently, co-cultures of yeast and microalgae were applied at laboratory scale in order to benefit from the potentially synergistic characteristics of the two systems. The yeast growth results in CO2 release, which the microalgae use and produce O2 for the yeast. Moreover, in co-cultures of yeast and microalgae, microalgae might release organic carbon or secondary metabolites that can be taken up by the yeast and support lipid accumulation and/or growth [124]. There are many factors to be taken into account, such as the pH of the culture, which can be acidified by the yeast growth, the different optimal temperatures for yeast and algal growth, the matching of the growth rates (microalgae grow slower than yeast) and there might be an imbalance on the production and utilisation of gases. By engineering these parameters properly, suitable bioreactor design can be achieved, such as including membranes permeable to gases or nutrients. One cultivation approach consisted of co-culture of the yeast Rhodotorula glutinis and the microalga Chlorella vulgaris in a double system bubble column photobioreactor [125]. For research purposes, this process is interesting but imposes several technical challenges making its large-scale implementation difficult. The oleaginous yeast C. curvatus was cultivated using repeated-batch mode on VFAs from mixed continuous culture grown on sludge and marine biomass [95]. Roltz et al. [126] proposed a proof of concept combined strategy of producing ethanol from sweet sorghum sugars, followed by microbial oil production from the residual sugars and nitrogen from the ethanol step. The biomass concentration was 21 g/L with 28% w/w oil content. In this way, zero cost of raw materials is achieved and waste creation is moved to the end of the overall process, better than having to process waste twice if the fermentations were separate.

4.3. By-products from oil production Overflow metabolites that are produced alongside microbial lipids consume part of the carbon source for their formation (Fig. 5). Some are produced by default and some because of the operating conditions, so these pathways could be modified either by modification of the operating conditions or metabolic engineering in order to avoid their formation. One of them, CO2, is a standard respiration product. The next, is the fat-free biomass, which is not actually a by-product but is composed of carbohydrates and proteins. Some yeasts can synthesise citric acid (Y. lipolytica) from the TCA cycle [28,85,133]. Various yeasts produce pigments, which are in general valuable materials and their formation conditions are similar to those for oil accumulation (e.g. nitrogen limitation) [71,94,134]. There is also the possibility for cells to produce volatile organic acids under low dissolved oxygen conditions, a fact that can be seen through pH decrease of the culture [135,136]. In some cases, the by-products are of value and if they cannot be avoided, they can further valorise the process through co-production of value added products. Since oil recovery requires cell disruption, in all cases defatted biomass is a waste that needs disposal, however it can serve as a nutrient source (e.g. fermentation feedstock or animal feed).

4.2. Robust cultivation conditions At large scale, pH control increases the production cost. Use of microorganisms, especially yeasts that can tolerate pH changes is desirable [10]. Moreover, alkali addition would inevitably result in accumulation inside the broth and complicate the final separation processes. The drop in pH can make the broth unfriendly to external microorganisms, as was shown by a study, where the oleaginous yeast M. pulcherrima was cultivated in an open tank and the low pH along with the antimicrobial compounds the yeast produced, ensured a reasonable level of monoculture [9]. If yeasts that do not need pH control are utilised, these strains would be robust enough to require little external supply, resulting in a simple fermentation process easier to scaleup. A few works have explored the possibility of cultivating oleaginous yeasts at non-aseptic conditions to lower the energy demands of the fermentation stage, related to the sterilisation of the media, which will

Fig. 5. Potential by-products of oleaginous yeasts fermentation. 11

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Table 7 Comparison of different cultivation modes for the production of microbial oil. Cultivation mode

Advantages

Disadvantages

Batch

Short duration. Simple to operate. First step in process development. Good tool for screening experiments. Moderate substrate additions avoid cell growth inhibition. Feed adjusted to strain-specific metabolism. Waste substrates can be fed moderately. Constant carbon and nitrogen levels. Constant production yields at steady state. It can be coupled with continuous processing.

High initial carbon source concentration may cause cell growth inhibition. Uncontrolled C/N ratio throughout the fermentation. Low nitrogen content may limit cell growth.

Fed-batch Continuous

Repeated batch (Draw-andFill)

Two-stage batch Two-stage fed-batch

Lag phase is minimised or excluded. High inoculum achieves faster cell growth. Enables the application of limitation and combinations of different substrates. Enables regular harvesting. Combination of media can be performed. Good for research purposes. Achieves high biomass and lipid yields Extends growth phase and higher biomass makes lipid titre higher for a given content. Enables the combination of carbon sources.

No standard regime, optimisation of feed according to each strain. Pulsed additions of medium at large scale require longer time to reach homogenisation in the broth. The presence of nitrogen, even at low amounts may compromise lipid accumulation. Optimisation required for optimal N levels. Long operating period; risk of undesired mutations and blockages. Potential degeneration of strain’s oil producing capacity over the time-course. Potential loss of cellular activity over the time-course.

Requires two-vessels: much downtime for cleaning/sterilisation. Occupation of several vessels: costly process. Prolonged operation. Needs to be optimised for specific strains.

under fed-batch fermentation, such as 0.54 g/L/h on glucose [76], 0.44 g/L/h on sugarcane juice [30], while lipid productivity as high as 1.6 g/L/h was found under two-stage cultivation of L. starkeyi on glucose [101]. By regulating the feed supply and composition, appropriate C/N ratio is maintained allowing for higher cell densities to be produced, alongside better carbon to lipids conversion. If the productivity is high enough, there is higher possibility for microbial lipids to be competitive to conventional oils. The two-stage strategy, where cell production is extended by rich medium supply prior to lipid synthesis, is the most appropriate mode for targeting high lipid titres. This strategy might extend the fermentation time but not significantly as that found in repeated batch fermentation. Two-stage fermentation offers flexibility over process design and can be based on the modification of parameters such as feed composition, feed rate, dissolved oxygen, pH and others. The increasing interest in developing two-stage fermentation modes [104] shows their potential to significantly enhance microbial oil profitability. Furthermore, description of the system’s kinetics enables a better understanding on the fluxes and the relationships between key variables while predicting different conditions at scale. In addition, some oleaginous yeasts are having the ability to modify the composition of both waste fats and oils in order to create targeted edible oils with pre-determined composition such as cocoa butter [143,144]. When cultivated on fatty-based feedstocks, yeasts tend to incorporate unsaturated fatty acids for growth purposes whereas saturated fatty acids are accumulated intracellularly [118]. This is of great importance, as selective removal of fatty acids from a low-cost matrix can be done economically and energetically-friendly while paving the way towards sustainable microbial oil scale up [145]. Supplementation of sugar-based media with oils usually results in enhanced lipid production and increased lipid-free biomass as the sugar is mainly used for lipid biosynthesis and the fat for supporting the growth needs. The application of optimal composition ratio of sugar/fat enables a controlled end-product intracellular lipid profile. Two-stage cultivation with growth of yeast on glucose first and then lipid accumulation on a mixture of glucose and stearin was demonstrated [146], suggesting that a wide range of combinations can be used. Lipid accumulation on only fat or supply of fat together with sugars at certain stages confirms also the operational advantage of two-stage cultivation strategies on utilising efficiently the nutrients provided while targeting high lipid yields.

Researchers have explored the possibilities of feeding de-oiled biomass back to another culture of oleaginous yeasts and this has brought up interesting results [68,137]. 5. Discussion Though large-scale microbial lipid production for fatty acids production has been explored in the past, its commercial consolidation as a biodiesel feedstock is hindered by the high processing costs. Driven by the potential perspectives of microbial lipids as alternative biodiesel feedstocks, a great amount of research has been focused on seeking cost-effective improvements, such as strain identification, use of waste raw materials, optimal bioprocessing conditions and cost-effective downstream processing operations [6,13,138–140]. The fermentation stage plays a major role in the overall costs. To make lipid production economically viable, the selection of the cultivation mode is key in order to support high lipid production titres while providing the necessary nutrients at the right growth phase. In addition, key aspects to be considered during the selection of the cultivation mode are the control level required (i.e. sterile vs. non-sterile conditions or pH control strategy), the energy input and utilities needed (aeration, heating) and the downtime operational periods (low vs. high) required at large scale [140]. The advantages and disadvantages related to each cultivation mode are summarised in Table 7. Batch fermentation is a simple method to operate and a common tool for screening studies, but the low C/N ratios and yield limitations associated do not render this cultivation mode as the most preferential approach (Table 7). Additional media supply, under fed-batch and continuous cultivation mode, can result in improved yields by simply providing more nutrients and extra carbon source. Amongst the reviewed modes, repeated batch and pulsed fedbatch are the most widely applied strategies due to their higher yields, better substrate utilisation rates and easier up-scaling. However, in order to get optimal fermentation efficiency, bioprocess design should be adjusted to the strain metabolism and performance, as emphasized across the present review. The phase for media supply, and the frequency of feeding should not be only based on the detection of low nutrient levels but also on combined understanding between the uptake rate and the strain tolerance to the supplied nutrients [138,141,142]. High productivities have been achieved by oleaginous yeasts cultivated

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6. Conclusions

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The latest trends and challenges on the bioprocess design and cultivation strategies developed for targeting improved lipid production by oleaginous yeasts were overviewed in the present review. This review has emphasized how the selection of the cultivation mode impacts on the cell generation, lipid yield and process scalability. Amongst all the different cultivation strategies developed during the last decade, twostage fermentation stands out as the optimal approach for targeting lipid production due to its easier up-scaling, higher cell and lipid yields, while offering the flexibility of operating under diverse configurations and being applied to a wide range of oleaginous yeast strains. Kinetic modelling of oleaginous systems under different operating modes and carbon sources plays a pivotal role in providing a deeper understanding not only on the trade-off between cell and lipid production, but also on the active biochemical pathways to facilitate the bioprocess design. To this end, several state-of-the-art kinetic models developed in the last decade were comprehensively reviewed in the present review, with an emphasis on the growth and lipid accumulation rate equations as key variables. High productivities above 1 g/L/h will improve process economics by reducing the bioreactor size and consequently associated energy costs. The higher productivity is expected to reduce significantly the operating costs and the capital expenditure of lipid production plants. Exponential feeding responsive to the growth rate or variable feeding rate according to the carbon source consumption rate need to be further developed to provide higher cell and lipid yields with increased productivity. All these bioprocess improvements with integration of biochemical engineering tools has resulted in higher productivity and enhanced substrate conversion values that are essential to address the cost implications associated and to foster the commercialisation of microbial lipids. References [1] B. Bharathiraja, S. Sridharan, V. Sowmya, D. Yuvaraj, R. Praveenkumar, Microbial oil – a plausible alternate resource for food and fuel application, Bioresour. Technol. 233 (2017) 423–432, https://doi.org/10.1016/j.biortech.2017.03.006. [2] Q. Li, W. Du, D. Liu, Perspectives of microbial oils for biodiesel production, Appl. Microbiol. Biotechnol. 80 (2008) 749–756, https://doi.org/10.1007/s00253-0081625-9. [3] D.E. Leiva-Candia, S. Tsakona, N. Kopsahelis, I.L. García, S. Papanikolaou, M.P. Dorado, A.A. Koutinas, Biorefining of by-product streams from sunflowerbased biodiesel production plants for integrated synthesis of microbial oil and value-added co-products, Bioresour. Technol. 190 (2015) 57–65, https://doi.org/ 10.1016/j.biortech.2015.03.114. [4] S. Bellou, I.E. Triantaphyllidou, D. Aggeli, A.M. Elazzazy, M.N. Baeshen, G. Aggelis, Microbial oils as food additives: recent approaches for improving microbial oil production and its polyunsaturated fatty acid content, Curr. Opin. Biotechnol. 37 (2015) 24–35, https://doi.org/10.1016/j.copbio.2015.09.005. [5] P. Thliveros, E. Uçkun Kiran, C. Webb, Microbial biodiesel production by direct methanolysis of oleaginous biomass, Bioresour. Technol. 157 (2014) 181–187, https://doi.org/10.1016/j.biortech.2014.01.111. [6] A. Patel, N. Arora, K. Sartaj, V. Pruthi, P.A. Pruthi, Sustainable biodiesel production from oleaginous yeasts utilizing hydrolysates of various non-edible lignocellulosic biomasses, Renewable Sustainable Energy Rev. 62 (2016) 836–855, https://doi.org/10.1016/j.rser.2016.05.014. [7] J.P. Wynn, C. Ratledge, Oils from Microorganisms, in: Bailey’s Ind. Oil Fat Prod. John Wiley & Sons, Inc., 2005, https://doi.org/10.1002/047167849X.bio006. [8] M. Athenaki, C. Gardeli, P. Diamantopoulou, S.S. Tchakouteu, D. Sarris, A. Philippoussis, S. Papanikolaou, Lipids from yeasts and fungi: physiology, production and analytical considerations, J. Appl. Microbiol. 124 (2017) 336–367, https://doi.org/10.1111/jam.13633. [9] F. Santamauro, F. Whiffin, R. Scott, C. Chuck, Low-cost lipid production by an oleaginous yeast cultured in non-sterile conditions using model waste resources, Biotechnol. Biofuels 7 (2014) 34, https://doi.org/10.1186/1754-6834-7-34 http://www.biotechnologyforbiofuels.com/content/7/1/34. [10] I.R. Sitepu, L.A. Garay, R. Sestric, D. Levin, D.E. Block, J.B. German, K.L. BoundyMills, Oleaginous yeasts for biodiesel: current and future trends in biology and production, Biotechnol. Adv. 32 (2014) 1336–1360, https://doi.org/10.1016/j. biotechadv.2014.08.003. [11] C. Ratledge, Microbial Lipids, in: Biotechnol. Set, Wiley-VCH Verlag GmbH, 2001, pp. 133–197, https://doi.org/10.1002/9783527620999.ch4g. [12] S. Papanikolaou, G. Aggelis, Lipids of oleaginous yeasts. Part II: technology and potential applications, Eur. J. Lipid Sci. Technol. 113 (2011) 1052–1073, https:// doi.org/10.1002/ejlt.201100015.

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