Growth and lipid accumulation by L. starkeyi in SBP hydrolysates
When lignocellulosic hydrolysates are used as substrate for microbial productions, it is necessary to find a compromise between the need to start from a high sugar concentration and the necessity to minimize the toxic effect of inhibitors. Indeed, during pre-treatments not only sugars are released but also inhibitors like weak organic acids. In our case, we noticed the presence of acetic and lactic acid (Figure 1A), which can be mainly ascribable to the initial contamination of the SBP with acid-producing bacteria, as reported by Kühnel and colleagues [26].
To test the toxic effects of SBP hydrolysate and of acetic and lactic acid on the growth of L. starkeyi we carried out different spot assays. Cells were spotted on solid SBP media and on solid minimal media added with acetic or lactic acid in the corresponding concentrations as present in SBP hydrolysates. SBP hydrolysates originated from 1 to 7 % of initial total solid (TS) (Figure 1B). The higher was the concentration of SBP hydrolysate, the higher was the inhibition of L. starkeyi’s growth, which was possible until 3 % SBP (Figure 1B.2). Acetic acid showed to cause a strong inhibition on cell growth starting from the concentration of 1 g/L (Figure 1B.3). On the contrary, no inhibition was observed for lactic acid at all the tested concentrations (Figure 1B.4). When both weak organic acids were combined (Figure 1B.5), the growth inhibition was similar to the one observed in the presence of the corresponding SBP hydrolysate.
With the aim to better determine the proper concentration of SBP to be used, L. starkeyi was grown in SBP hydrolysates from 1 to 7 % TS. Poor or no growth was observed at low (1 and 2 % TS) and high (6 and 7 % TS) concentrations of SBP hydrolysates (Figure 2A and Table S1 in Additional file). At low TS percentages, sugars content is probably not enough to sustain growth, whereas at high TS percentages acetic acid is reaching an inhibitory concentration (Figure 1). 3, 4 and 5 % TS supported yeast growth without apparent impairment due to inhibitors, allowing the cultures to reach a comparable final OD and µ after 72 hours of growth (Figure 2A and Table S1 in Additional file). Because of the heterogeneous nature of this raw substrate, the use of diverse SBP stocks can generate hydrolysates with different amounts of sugars and inhibitors, despite the precaution taken for standardising as much as possible the procedure. To avoid possible growth delay or inhibition due to this heterogeneity, and consequent discrepancies, we selected 3 % SBP hydrolysate as a suitable medium for reproducible L. starkeyi cultivation. Moreover, on solid media 3 % SBP hydrolysate is the higher concentration where we observed growth (Figure 1B.2).
The capability of L. starkeyi to metabolize glucose, arabinose, lactic acid and acetic acid present in SBP hydrolysates was determined by analysing the concentration of these compounds at different time points. As previously observed [24, 27, 28], L. starkeyi is able to co-consume glucose and acetic acid, but arabinose and lactic acid were not assimilated throughout the cultivation time (Figure 2B).
Under these conditions, cells accumulated about 19.2 % of their dry weight as intracellular oils, leading to a production of 0.5 g/L of lipids after 144 hours (Table 1). These values are low if compared with the results obtained by cultivating the same strain on other residual substrates [29-31]: very likely this depends on the low C/N ratio of the media, which is not suitable to efficiently redirect the yeast metabolism towards lipid biogenesis.
Growth and lipid accumulation in SBP hydrolysate blended with molasses
To unbalance the C/N ratio, we evaluated the effect of the addition of different concentrations of molasses to 3% SBP hydrolysate on L. starkeyi growth and lipid accumulation. As shown in Figure 3A, the final OD reached by cultures directly correlated with the concentration of molasses up to 4% molasses. At 4% and 6% molasses cells grow to a comparable final OD and this situation could be caused by the presence in 6% molasses of moderately-toxic amount of acetic acid (Figure 3E, square symbol) and/or by the high sucrose concentration that might cause osmotic stress (Figure 3C, square symbol). In respect to main metabolites, it can be observed that cells co-consumed glucose and acetic acid from about 24 hours of cultivation and started to also co-consume sucrose after about 48 hours (Figure 3B, C, E), as also reported in literature [24]. As observed in SBP hydrolysate alone, arabinose and lactic acid were not metabolised (Figure 3D, F).
Lipid accumulation in intact cells of L. starkeyi grown in SBP hydrolysate added with molasses was monitored over time by Fourier Transform Infrared (FTIR) micropectroscopy. FTIR is an efficient tool for rapidly following up microbial lipid accumulation at different stages of growth [32-39], since it can analyze intact cells identifying specific molecular groups by their absorption bands. Starting from the spectra obtained sampling the cultivations over time, Figure 4 illustrates the temporal evolution of the CHx stretching band area, between 3050 and 2800 cm−1, and of the ester carbonyl band area, between 1760 and 1730 cm−1, after normalization for the total protein content, given by the amide I band area (Figure S1 in Additional file). The analysis showed that the addition of molasses at concentrations higher than 1 % significantly improved the final intracellular lipid production (Figure 4). In the presence of 6 % molasses, intracellular lipids were the 47.2 % of cellular dry weight and lipid production reached 9.3 g/L after 144 hours (Table 1).
From these data it seems that SBP can be considered more as a nitrogen supplementation than as a carbon source. Indeed, we determined nitrogen content in 3 % SBP, which is 27.0 mg/L, 17.5 mg/L from primary amino acids and 9.5 mg/L from ammonia. Ammonium sulphate is one of the compounds commonly used to fulfil the need of nitrogen in yeast media formulations [40, 41]. We therefore formulated a medium where ammonium sulphate at different concentrations was blended with molasses to compare L. starkeyi growth and lipid accumulation in the presence of SBP or ammonium sulphate as nitrogen source. As in sugar beet pulp the nitrogen is present mainly in its organic form, we supplemented the medium with a higher ammonium sulphate concentration [13, 14], since it represents a less bioavailable inorganic nitrogen source. Despite the higher availability of nitrogen, we observed that growth was significantly reduced in the presence of all the (NH4)2SO4 concentrations (Figure 5A) if compared with the growth observed using only residual biomasses (Figure 3A). As a consequence, we observed lower values of lipid production, even after 144 h of cultivation (Table 1). SBP hydrolysate therefore resulted to be superior to ammonium sulphate in supporting growth and lipogenesis in L. starkeyi when blended with molasses.
Growth and lipid accumulation in molasses pulse-fed batch cultures
FTIR data on lipid accumulation in SBP hydrolysate added with molasses showed that lipid accumulation was faster at low concentrations of molasses (1 % and 2 %), or even in its absence, during about the first 48 h of growth (Figure 4). Because of the existence of a correlation between lipid accumulation and cell density that was independent from the concentration of molasses (Figure S2 in Additional file), the slightly delayed accumulation of lipid observed in the presence of 6 % molasses might be a consequence of the slowed growth shown in Figure 3A and discussed above.
The use of pulsed feeding fermentations has the advantage to avoid the possible stress imposed by high concentrations of substrates and to temporally separate cell growth and lipid accumulation, which has been described as a successful method to obtain high lipid production [42, 43]. Considering this, we cultivated cells in SBP hydrolysate with pulse feeding of molasses at different time intervals (0, 24 h and 48 h) and low concentrations (1 %), followed by a pulse feeding at 72 h with a higher molasses concentration (3 %). When a pulse feeding of molasses was applied, we observed a faster growth during the first hours compared with the batch culture (Figure 6A and Table S1 in Additional file). At the end of the fermentation, cells reached a similar OD (Figure 6A; Table 1) and consumed almost all sucrose, glucose and acetic acid (Figure S3 in Additional file).
FTIR measurements showed that the pulse-feeding cultivation allowed a higher intracellular lipid accumulation compared to the batch culture throughout all the fermentation (Figure 6B and C). The ratio between the lipid accumulation measured by FTIR and cell density, as a matter of fact, was higher in fed-batch compared with batch cultures (Figure S2 in Additional file), confirming that molasses pulse feeding is effective in increasing the intracellular lipid content.
The difference in lipid accumulation among batch and fed-batch cultures was not evident when lipid content was measured as the ratio of their weight to dried cell biomass (lipid content %, Table 1), but we cannot exclude that lipid extraction was not completely efficient. In fact, compared to lipid extraction technologies, spectroscopic analysis have the advantage to evaluate lipid accumulation in intact cells avoiding possible lipid loss [32]. Moreover, the addition of molasses increased not only lipid production but also lipid yield (g/g), and this improvement was more pronounced in pulse-fed compared to batch cultivations (Table 1).
Molasses fed-batch fermentations were also applied using ammonium sulphate as an external nitrogen source. Under these conditions, the growth of L. starkeyi was better than in batch cultures and was not affected by the concentration of ammonium sulphate (Figure 5B, Table 1). After 144 h of growth, cell dry weight, lipid production and lipid content were not affected by the different concentrations of ammonium sulphate. These performances were higher than in batch cultures, but lower than in molasses pulsed-fed cultivations with SBP hydrolysate (Table 1).
Table 1. Biomass and lipid production by L. starkeyi.
Cultivation mode
|
Molasses
|
SBP
|
(NH4)2SO4
|
CDW (g/L)
|
Lipid production (g/L)
|
Lipid content (%)
|
Lipid yield (gl/gs)
|
Batch
|
-
|
3 %
|
-
|
2.5 ± 0.4
|
0.5 ± 0.3
|
19.2 ± 4.2
|
0.080 ± 0.039
|
6 %
|
3 %
|
-
|
19.7 ± 1.2
|
9.3 ± 0.3
|
47.2 ± 2.6
|
0.167 ± 0.006
|
6 %
|
-
|
0.5 g/L
|
5.0 ± 1.3
|
2.0 ± 1.0
|
30.2 ± 3.8
|
ND
|
6 %
|
-
|
1.0 g/L
|
4.2 ± 0.4
|
1.9 ± 0.7
|
36.4 ± 5.1
|
ND
|
6 %
|
-
|
2.0 g/L
|
2.2 ± 0.6
|
0.9 ± 0.1
|
29.6 ± 2.7
|
ND
|
Pulsed fed-batch
|
6 %
|
3 %
|
-
|
20.5 ± 2.2
|
9.7 ± 0.7
|
49.2 ± 0.9
|
0.178 ± 0.012
|
6 %
|
-
|
0.5 g/L
|
15.6 ± 0.1
|
6.1 ± 0.3
|
38.3 ± 2.6
|
ND
|
6 %
|
-
|
1.0 g/L
|
16.2 ± 0.1
|
6.7 ± 0.4
|
38.9 ± 3.8
|
ND
|
6 %
|
-
|
2.0 g/L
|
16.1 ± 0.5
|
6.9 ± 0.6
|
41.0 ± 4.4
|
ND
|
Cells were grown in 6 % molasses with the addition of 3 % SBP hydrolysate or (NH4)2SO4 in batch and pulsed fed-batch cultures. CDW, lipid production, lipid content and lipid yield were calculated after 144 h of growth. Lipid yield (gl/gs) was calculated considering the amount of glucose, sucrose and acetic acid consumed by cells. ND: not determined.
Composition of biodiesel deriving from oils produced by L. starkeyi on sugar beet residues
The lipids produced by L. starkeyi in SBP hydrolysate without and with molasses in batch and fed-bath cultures were transmethylated by alkaline catalysis and the resulting fatty acid methyl esters (FAMEs) were determined by gas chromatography. The main fatty acids produced by L. starkeyi in SBP hydrolysate with or without molasses were oleic (18:1) and palmitic (16:0) acids (Table 2). If compared with the composition of fatty acids of cells grown in SBP hydrolysate alone, the addition of molasses increased the percentage of palmitic acid (16:0) and reduced the percentage of linoleic acid (18:3), overall increasing and reducing the content of monounsaturated and polyunsaturated fatty acids, respectively (Table 2).
If on one hand microbial lipids are emerging as important platforms for the sustainable production of biodiesel, on the other hand immobilized lipases are considered as alternative catalyst of transesterification reactions that are consistent with the development of green processes [44-46]. For this reason, we evaluated the FAMEs profile deriving from lipids produced by L. starkeyi in SBP hydrolysate fed with molasses also using the immobilized lipase Novozym 435. Due to the inhibitory effect of methanol on lipase activity [46, 47], a small amount of methanol (2.5 %) was added to the reaction every 24 h for four times (Figure S4 in Additional file). Compared to the alkaline catalysis, the enzymatic reaction was less efficient and more time consuming (144 h to reach about 65% of FAMEs; Figure S4 in Additional file). Considering the transesterification, the use of lipase reduced the percentage of linoleic acid (18:2) and increased that of stearic acid (18:0) (Table 2).
Table 2. Fatty acid profile of L. starkeyi.
Cultivation mode
|
Molasses
|
SBP
|
(NH4)2SO4
|
Catalyst
|
C14:0
|
C16:0
|
C16:1
|
C18:0
|
C18:1
|
C18:2
|
C18:3
|
M
|
P
|
S
|
CN*
|
Batch
|
-
|
3 %
|
-
|
NaOH
|
0.34 ± 0.08
|
31.7 ± 3.7
|
6.5 ± 0.2
|
2.4 ± 0.3
|
43.8 ± 4.4
|
8 ± 3.4
|
0.43 ± 0.1
|
53.4 ± 0.1
|
8.4 ± 3.6
|
38.2 ± 3.5
|
61.2
|
6 %
|
3 %
|
-
|
NaOH
|
0.32 ± 0.04
|
33.2 ± 1
|
4.9 ± 0.6
|
3.5 ± 0.6
|
51.9 ± 0.5
|
2.8 ± 0.6
|
< 0.1
|
57.7 ± 0.3
|
3 ± 0.4
|
39.3 ± 0.1
|
62.9
|
Pulsed fed-batch
|
6 %
|
3 %
|
-
|
NaOH
|
0.38 ± 0.05
|
32.7 ± 1.9
|
4.4 ± 0.5
|
3.8 ± 0.5
|
52.5 ± 3
|
3.3 ± 0.9
|
< 0.1
|
57.9 ± 3.4
|
3.3 ± 0.8
|
38.7 ± 2.5
|
62.9
|
6 %
|
3 %
|
-
|
Novozym 435
|
0.45 ± 0
|
34.2 ± 0.5
|
4.8 ± 0.2
|
5 ± 0.3
|
51.6 ± 1
|
1.5 ± 0.1
|
< 0.1
|
56.4 ± 1.2
|
1.8 ± 0.2
|
41.9 ± 0.9
|
63.6
|
6 %
|
-
|
0.5 g/L
|
NaOH
|
0.26 ± 0.03
|
33.9 ± 0.5
|
2.8 ± 0.1
|
5.2 ± 0.3
|
50 ± 1.1
|
6.3 ± 0.6
|
< 0.1
|
53.4 ± 1.1
|
6.5 ± 0.6
|
40.1 ± 0.8
|
62.7
|
Fatty acids produced by L. starkeyi on 3 % SBP hydrolysate with or without the addition of 6 % molasses in batch and pulsed fed-batch cultures. M: monounsaturated fatty acids, P: polyunsaturated fatty acids, S: saturated fatty acids. * CN was calculated using the equation: CN = 61.1 + 0.088(% 14:0) + 0.133(% 16:0) + 0.152(% 18:0) – 0.101(% 16:1) – 0.039(% 18:1) – 0.243(% 18:2) – 0.395(% 18:3) [48].