Beet Molasses

Beet molasses is conspicuously deficient in magnesium [133] and biotin [134], which can necessitate supplementation with biotin-containing substrates such as blackstrap molasses derived from sugar cane.

From: Biorefineries, 2014

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Mixed Sugar Fermentation by Clostridia and Metabolic Engineering for Butanol Production

T.C. Ezeji, ... N. Qureshi, in Biorefineries, 2014

9.2 Mixed-Sugar Fermentation by Solventogenic Clostridia

In addition to the various carbohydrate substrates mentioned in the introduction of this chapter, butanol can be produced from sugarcane and beet molasses, whey permeate containing lactose, soy molasses, Jerusalem artichoke, corn, and starchy root crops. However, the prices of some of these renewable feedstocks are high and the production of biofuel from them would not be economical, because substrate price is the factor that most significantly affects the economy of biofuel production [12–14]. The use of economically available agricultural residues such as wheat straw, barley straw, corn stover, miscanthus, and switchgrass offers an alternative to using these higher-value substrates. Fermentation of the residues requires pretreatment with dilute sulfuric acid or alkali at high temperature, however, because pretreatment alters the cellulosic biomass structure, making the biomass fibers accessible to the hydrolytic enzymes that release hexose and pentose sugars from the biomass. These fibrous biomass residues contain approximately 30-40% hexoses and 25-30% pentoses. Given that pentose sugars (arabinose and xylose) form a significant part of the biomass, the economics are not favorable if these sugars are not utilized by the culture. This need for pentose utilization is a challenge that has plagued the biomass-to-ethanol process. Hence, the basic objective of this section is to review the pentose-fermentation capabilities of butanol-producing cultures.

Waksman and Kirsh [15] were among the first to report the production of acetone and butanol (AB) from cellulosic or hemicellulosic hydrolyzates, with the process emerging in 1933, followed by xylose fermentation [16,17]. Between 1960 and 1980, few published reports addressed the fermentation of pentoses such as xylose especially as it related to AB. In early 1980s, the fermentation of cellulosic hydrolyzate into butanol gained momentum, and soon several reports were published. In 1982, Soni et al. [18] hydrolyzed bagasse and rice straw to produce AB in batch cultures of Clostridium saccharoperbutylacetonicum. In these studies individual hexose and pentose sugars were not measured, but the authors presumed that the culture utilized both types of sugars for AB production. In a batch system, 17.1 g/L AB was produced with a product yield of 0.34, and the initial and final sugar concentrations in the fermentation medium were 60.0 and 6.0 g/L, respectively. This study was followed by a number of studies that achieved fermentation through the use of either model sugar solutions containing cellulosic sugars or actual cellulosic hydrolyzates [19–21]. Reporting on one such study, Mes-Hartree and Saddler indicated that butanol values of approximately 90% of the theoretical yield were achieved when glucose was used as a substrate [19]. It was also reported that butanol production using five other sugars produced differing yields. Listed in order of decreasing yield, the sugars used were cellobiose, mannose, arabinose, xylose, and galactose. This result indicated that the fermentation of pentose sugars was possible, but to a lesser extent.

In a study, butanol was produced from Pinus radiata (wood) hydrolyzate, which required detoxification prior to fermentation using activated carbon, as well as cation and anion exchange resins. In this study, a maximum butanol concentration of 5.7 g/L was achieved with a theoretical yield of 17%. Although individual sugar concentrations before fermentation were measured, no account of their use for cell growth and fermentation was provided [20]. Further, Marchal et al. used wheat straw, pretreated with alkali, to produce AB using Clostridium acetobutylicum in a single reactor in which hydrolysis and fermentation were combined [21]. For the hydrolysis of cellulosic fibers into individual sugars, a cellulase enzyme from Trichoderma reesei was employed, and the combined hydrolysis and fermentation system produced 17.3 g/L AB. Individual sugars (glucose, cellobiose, xylose, and arabinose) were measured during hydrolysis and fermentation, and Marchal and colleagues demonstrated that all the sugars were used by the culture to produce AB, with the utilization of xylose being slow or incomplete.

In another study on the continuous production of butanol, Groot and Luyben [22] used immobilized cells of Clostridium species DSM 2152 to ferment a mixture of glucose and xylose, and they reported that xylose was utilized by the culture after glucose was completely consumed. Groot and Luyben also noted that, after 700 h of fermentation, the utilization of xylose slowed down. More recently, Qureshi et al. [23, 24] and Ezeji and Blaschek [7] showed that C. acetobutylicum P260 (renamed C. beijerinckii P260) and C. beijerinckii BA101 preferred glucose over xylose. Qureshi et al. [24] produced butanol in an integrated hydrolysis, fermentation, and product recovery system using wheat straw as a substrate. After wheat straw sugars were completely utilized, the bioreactor was continuously fed with a mixture of glucose, xylose, arabinose, galactose, and mannose in a ratio similar to the sugar ratio of wheat straw in order to mimic wheat straw hydrolyzate conditions. After a period of about 200 h, the utilization of xylose was reduced, and xylose accumulated in the fermentation broth. At about 400 h, xylose utilization was considerably reduced, and it completely stopped at 533 h, even though no other sugars were present in the reactor at that time.

Plants in the Soviet Union produced acetone and butanol from molasses and rye starch until 1980 [25]. In a process not published until 2006, these plants used agricultural residues such as hemp waste, corncobs, and sunflower shells to produce this biofuel after hydrolysis [25]. The residues were milled into powder and then hydrolyzed with dilute sulfuric acid for 1.5-3 h, depending upon substrate of choice. Corncobs were hydrolyzed more easily than the others. The so-called pentose hydrolyzates were neutralized with Ca(OH)2 (lime) followed by the addition of these hydrolyzates to rye flour or molasses for fermentation. Fermentation times were observed to increase when pentose hydrolyzates were added to molasses or rye flour. In these fermentations the composition of ABE from the fermentation of pentose hydrolyzates shifted toward ethanol production.

In the early 1990s, a significant number of studies were performed on the production of AB from agricultural hydrolyzates such as corncobs [26]. While testing the various inocula ratios (1.25-10%) in a 4-liter bioreactor, researchers observed that the culture used 38.0-39.1 g/L glucose and 7.1-14.8 g/L xylose to produce 13.5-17.9 g/L total ABE. The reactor inoculated with 10% inoculum used the largest amount of xylose (14.8 g/L) and produced 17.9 g/L total ABE in 38 h of fermentation. The utilization of arabinose ranged from 0 (1.25% inocula) to 0.2 g/L (10% inocula). These studies were performed in batch reactors, however, and the culture in such reactors utilizes a significant amount of xylose as compared to a culture in a continuous reactor.

Studies on the use of hexose and pentose sugars for butanol production suggested that butanol-producing cultures have limited capability to utilize pentose sugars, with fermentation of xylose being most limited [19,21,24]. If these cultures are employed to produce butanol on a continuous basis, they could stop fermenting xylose, thus resulting in the loss of significant amounts of cellulosic sugars. To utilize all the cellulosic sugars in continuous, long-term fermentations, cultures were developed using metabolic engineering techniques to enhance capability of butanol production from mixed sugars. The details of these studies, along with elimination of acetone production, are described in Section 9.3 of this chapter.

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Biodiesel from microbial oil

L.F. Bautista, ... V. Garre, in Advances in Biodiesel Production, 2012

8.3.1 Carbon sources rich in simple carbohydrates

Raw materials that are basically rich in sucrose, sugar cane, either in the form of cane juice or cane molasses and beet molasses, have been extensively used for microbial growth. Molasses is a by-product of the sugar industry readily available at low cost, which consists of water, approximately 50% (w/w) total sugars (sucrose, glucose, fructose), suspended colloids, heavy metals, vitamins and nitrogenous compounds, and so on (Najafpour and Shan, 2003). Another by-product used for microbial growth that is rich in a monosaccharide, lactose, is cheese whey (Guimarães et al., 2010).

Few studies have analysed the accumulation of lipids suitable for transformation into biodiesel in oleaginous microorganisms grown in molasses media (Table 8.1). One remarkable study analysed these parameters in 11 yeasts growing in molasses media (Karatay and Dönmez 2010). Several of these strains showed high levels of biomass and lipids, with promising results for Candida lipolytica, Candida tropicalis and Rhodotorula mucilaginosa. Although their lipids were transformed into FAMEs, the process was not optimized and would require a detailed study.

Although this type of carbon source is probably the best among the low cost raw materials, the presence of heavy metals in high concentrations may cause a critical problem during fermentation because they can inhibit the growth of microorganisms, influence the pH of the substrate and are involved in the inactivation of the enzymes associated with biosynthesis of products. Different pretreatments of molasses may reduce this problem (Roukas, 1998; Kotzamanidis et al., 2002), but will add costs to biodiesel production. The yeast Trichosporon fermentans was grown using cane molasses pretreated with sulphuric acid and produced 36.4 g L− 1 of biomass with a lipid content of 38.2%. The lipid content obtained with waste molasses was much lower than that with nitrogen-limited culture medium owing to a low carbon/ nitrogen ratio (C/N) molar ratio (about 12.5). Thereby, addition of various sugars to the pretreated waste molasses increased the C/N molar ratio and consequently enhanced the lipid accumulation of T. fermentans up to 53.6% (Table 8.1). Similar to vegetable oils, the lipid mainly contained palmitic acid, stearic acid, oleic acid and linoleic acid and the unsaturated fatty acids amounted to about 64% of the total fatty acids. This microbial oil was transesterified to biodiesel by base catalysis after removal of free fatty acids and a high methyl ester yield of 92% was obtained (Zhu et al., 2008).

Trends in biofuels production tend toward the isolation or generation of microorganisms able to produce biofuels themselves. The lipases present in many microorganisms can catalyse the transesterification process of lipids in the presence of methanol. A recent survey of 81 yeasts isolated from raw milk identified a Cryptococcus curvatus strain that produced FAMEs when lysates of the cell were incubated with methanol. The biomass of this strain grown in beet molasses and cheese whey media showed a transesterification rate of its triglycerides higher than 97% (Takakuwa and Saito, 2010). Although FAME levels were low (around 70 mg L− 1), the strain grew well in both carbon sources and further improvements could be made to increase FAME levels.

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Integration of Ethanol Fermentation with Second Generation Biofuels Technologies

Caroline Häggström, ... David B. Hodge, in Biorefineries, 2014

8.5 Case Study: Aerobic Cultivation of S. cerevisiae TMB-3400-FT30-3 on Dilute Acid-Pretreated Softwood Hydrolyzate

The following example of fed-batch aerobic yeast cultivation using dilute acid-pretreated softwood hydrolyzate demonstrates one fermentation integration approach applied for cellulosic biofuels processes. This case study investigates the requirements for a culture media to be used for on-site aerobic cultivation of the Saccharomyces cerevisiae TMB-3400-FT30-3, a xylose-utilizing yeast adapted to dilute acid pretreatment hydrolyzate inhibitors [129]. Specifically, researchers investigated the ways in which cell mass yield was impacted by relative percentages of concentrated beet molasses, undetoxified softwood dilute acid hydrolyzates, and total nitrogen in the form of ammonium sulfate. The hydrolyzate was produced at the pilot plant in Örnsköldsvik, Sweden, managed by SEKAB E-Technology, using the dilute sulfuric acid pretreatment of Norway spruce (Picea abies) at 200 °C, pH 2, and 7 minutes residence time. Table 8.3 shows the composition of the hydrolyzate in terms of sugars and inhibitors, with inhibitor quantification performed as described previously [130]. As seen in Table 8.3, in addition to significant levels of pretreatment-derived inhibitors, the broth contained a large amount of pentose sugars, which constituted 34% of the total sugars. The hydrolyzate was not detoxified, and the pH was adjusted to 5.5 with 10 M NaOH. The neutralized hydrolyzate was then filter-sterilized before use. Concentrated commercial sugar beet molasses was obtained from Danisco (Örtofta, Sweden) and contained 448 g/L sucrose (Table 8.3) as determined by HPLC.

Table 8.3. Compositions of Undiluted Feedstocks Used for Yeast Cultivation

Sugars and inhibitors Softwood dilute acid hydrolyzate concentration (g/L) Concentrated beet molasses
Sucrose – 448
Glucose 10.1 –
Mannose 22.1 –
Galactose 1.5 –
Xylose 11.7 –
Arabinose 5.8 –
Total sugars 51.2 448
Acetic Acid 2.3 –
HMF 0.8 –
Phenolics 2.8 –
Formic Acid 0.7 –
Levulinic Acid 0 –

8.5.1 Media Requirements for Aerobic Growth

An effective cultivation medium must contain all the components required for growth with significant sources of C and N and generally minor requirements for trace minerals and often vitamins [131,132]. Sugar cane and sugar beet molasses and corn syrups are generally used as carbon sources for aerobic yeast production, depending on local economic factors. Beet molasses is available as a low-cost carbon source in Europe, and, in addition to its sugar content, it contains many growth-promoting nutrients, such as proteins, amino acids, vitamins, and nitrogen, which may vary with producer and the time of year. Beet molasses is conspicuously deficient in magnesium [133] and biotin [134], which can necessitate supplementation with biotin-containing substrates such as blackstrap molasses derived from sugar cane. For the commercial production of yeast biomass, the beet molasses-derived media is generally supplemented with P, N, Mg, and S in the form of (NH4)2SO4, (NH4)3PO4, NH4Cl, H3PO4, MgSO4, and potentially the vitamins biotin and thiamine [133].

In this case study, the cultivation was begun with beet molasses as the carbon source, followed by feeding with softwood hydrolyzate or hydrolyzate-beet molasses mixtures, because the slow addition of hydrolyzate during the fed-batch process offsets its inhibitory effects [135,136]. The batch phase was run for a minimum of 16 hours, and when sugars and ethanol were depleted, as verified by the dissolved oxygen profile and HPLC validation, the feeding of hydrolyzate commenced. The feed consisted of hydrolyzate and/or molasses, salts, supplemental vitamin solution, and distilled water. In order to maintain respirative metabolism during the fed-batch phase, the feeding rate was adjusted after every OD measurement based on maintaining a maximum sugar uptake rate, qs (g sugar/g biomass/h), which was assumed to be 0.360 g sugar (as monomer) /g cell mass / h. The feeding rate, F (L/h), was calculated according to:

(8.1)F=Vt⋅X⋅qsSFeed

Where:

V(t) = Volume of the reactor (mL)

[SFeed] = Total sugar concentration of the feed (46.1 g/L)

[X] = Biomass concentration (g/L)

(8.2)DCWg/L=0.8184⋅OD600−0.6318

For this case study, nitrogen was also added in the form of (NH4)2SO4, based on the assumption that yeast organisms supplied with 0.114 grams of elemental nitrogen per gram DCW are not nitrogen-limited, as demonstrated by the findings of Albers et al. [137], and the nitrogen was loaded into the reactor based on the assumed biomass yields of 0.2 g DCW/g sugar in the batch phase and 0.5 g DCW/g sugar in the fed-batch phase. The levels of nitrogen were set to 50% of the base case to identify nitrogen limitation. The resulting media balances yielded for the batch media were as follows: (NH4)2SO4, 1.1 g/L at 100% N; KH2PO4, 0.21 g/L; and MgSO4, 3.3 g/L; fed-batch media: (NH4)2SO4, 12.0 g/L at 100% N; KH2PO4, 2.4 g/L; and MgSO4, 3.3 g/L; final overall mixture: (NH4)2SO4, 8.6 g/L at 100% N; KH2PO4, 1.7 g/L; and MgSO4, 3.3 g/L. Supplemental biotin (50 μg/L, filter-sterilized) was used in all media.

Figure 8.2 shows examples of cultivation kinetics during the fed-batch phase without the addition of supplemental vitamins, and it indicates a 100-fold increase in the dry cell weight, corresponding to between six and seven doublings, reaching cell densities of 10–15 g/L. This yield can be compared to commercial aerobic yeast production, which requires 10 to 12 days and reaches cell densities as high as 80 g/L [133].

Figure 8.2. Example of kinetics from select cultivation experiments.

It was not possible to grow the yeast solely on hydrolyzate, possibly due to the high inhibitor concentration, the lack of supplemental nutrients in the beet molasses, or a combination of both. A supplementation of only 6.7 g/L of concentrated beet molasses (corresponding to 10% of the sugars supplied) was found to cause no significant reduction of the biomass yield from its near maximum value of 0.45 g biomass/g sugar (Figure 8.3a). Below this value, a statistically significant reduction in yield was observed, however. Reducing the nitrogen, supplied as ammonium sulfate, from 0.12 to 0.06 g N/g biomass also resulted in a statistically significant decrease in the yield. One potential reason for the decrease in the yield is the decreased ATP availability during aerobic growth on xylose due to the use of the pentose phosphate pathway. An important result from the mass balance is that, at a maximum theoretical biomass yield of approximately 0.50 g/g, the final yeast titer asymptotically approaches the maximum yield on pure hydrolyzate. For a process that can be performed within reasonable reactor and operating constraints, the fed-batch operating region is limited to a Vfeed/V0 ratio from 1 to 3. This ratio is the variable chosen in this work to control the overall fraction of molasses sugars in the reactor, and it has the potential to yield biomass concentrations in the range of 14–17 g/L. It should be considered that commercial yeast production can yield cell densities as high as 80 g/L [114], requiring significantly more concentrated hydrolyzates. The authors of this study suggested that the lower yields resulted from two factors. The first of these may be the loss of ATP related to to cell maintenance due to the increased energy demands associated with the stresses placed on the cell during growth on hydrolyzates. The second factor leading to decreased yields is that the sugar source is not differentiated in the yield so that the yeast may produce a lower aerobic biomass yield on xylose, as reported in the literature [70]. Figure 8.3b shows the levels of correlation between the amount of residual pentose sugars and the biomass yield. As previously observed, the complete utilization of pentose sugars by recombinant Saccharomyces cerevisiae strains is often difficult to achieve due to the relatively high Km value for xylose utilization via either of the transport pathways [138].

Figure 8.3. Example of cell mass production using softwood hydrolyzate supplemented with concentrated sugar beet molasses, showing (a) biomass yields as a function of molasses supplementation to the reactor and (b) biomass yields as a function of residual pentose sugars in the reactor. Open symbols represent the final yield. Solid symbols represent the transient yield during the fermentation.

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Nutritional Requirements in Fermentation Processes

Willem H. Kampen, in Fermentation and Biochemical Engineering Handbook (Third Edition), 2014

5.0 The Source of Trace and Essential Elements

Minerals supply the necessary elements to cells during their cultivation. Typical biological functions of the main elements were listed in Table 4.1. Table 4.8 shows the trace element composition in samples of Puerto Rican blackstrap and Dutch beet molasses in the year 1986. Phosphorous occurs principally in the form of sugar-phosphates, such as the nucleotides which compose DNA, RNA, and ATP. Phosphorus is assimilated in its inorganic form where the phosphate ion is esterified. The P-atom does not change in valence and remains as part of a phosphate group. Upon the death of the cell, it is again liberated as inorganic phosphorus through hydrolysis. Sulfur is present to the greatest extent in the amino acids cysteine and methionine. It is also commonly supplied as H2SO4 for pH adjustment, and as ammonium sulfate and potassium bisulfate. Many of the other elements are found complexed with enzymes: e.g., Mg2+ with phosphohydrolase and phosphotransferase, K+ with pyruvate phosphokinase (and Mg2+), and Na+ with plasma membrane ATP-ase (and K+ and Mg2+) [1].

Table 4.8. Average Composition of (trace) Elements in Puerto Rican Blackstrap Molasses in 1986 and One European Beet Molasses Sample of the Same Year [5]

Element Blackstrap Beet Molasses
B 0.041 0.0003
Ca 0.86 0.42
Co 0.000054 0.00006
Cu 0.0028 0.0005
Ρ 0.071 0.012
Fe 0.0158 0.0115
Mg 1.14 —
Mn 0.0057 0.0018
Ni 0.000123 —
Na 0.058 0.083
Pb 0.75 0.78
K 2.68 3.39
Sr — 0.004
Zn 0.011 0.0034

Requirements for trace elements may include iron (Fe2+ and Fe3+), zinc (Zn2+), manganese (Mn2+), molybdenum (Mo2+), cobalt (Co2+), copper (Cu2+), and calcium (Ca2+). The functions of each vary from serving in coenzyme functions to catalyze many reactions, vitamin synthesis, and cell wall transport. The requirements are generally in very low levels and can sometimes even be supplied from quantities occurring in water or from leachates from equipment. Trace elements may contribute to both primary or secondary metabolite production. Manganese can influence enzyme production. Iron and zinc have been found to influence antibiotic production. Primary metabolite production is usually not very sensitive to trace element concentration, however, this is a different matter for secondary metabolite production. Bacillus licheniformis produces the secondary metabolite bacitracin [7]. A manganese concentration of 0.07×10−5 M is required, but at a concentration of 4.0×10−5 M manganese becomes an inhibitor. Streptomyces griseus produces streptomycin as a secondary metabolite. For maximum growth it requires a concentration of 1.0×10−5 M of iron and 0.3×10−5 zinc, while a zinc concentration of 20×10−5 M becomes an inhibitor. Aspergillus niger produces citric acid as a primary metabolite. Concentrations of 2.0×10−5 M of zinc, 6.0×10−5 M of iron and/or 0.02×10−5 M of manganese act as inhibitors [8]. It produces only citric acid from glucose or sucrose during an iron deficiency and/or proper Cu/Fe ratio in the fermentation media. Raw materials such as molasses may have to be treated to remove iron. Manganese enhances longevity in cultures of Bacillus sp., iron in Escherichia sp., while zinc suppresses longevity of Torulopsis sp [8].

Cells are 80% or more water and in quantitative terms this is the major essential nutrient. Water is the solvent within the cell and it has some unusual properties, like a high dielectric constant, high specific heat and high heat of vaporization. It furthermore ionizes into acid and base, and has a propensity for hydrogen bonding. In most fermentations, microorganisms inhabit hypotonic environments in which the concentration of water is higher than it is within the cell. The cell walls are freely permeable to water, but not to many solutes. Water tends to enter the cell to equalize the internal and external water concentrations. Many eukaryotes and nearly all prokaryotes have a rigid wall enclosing the cell, which mechanically prevents it from swelling too much and undergoing osmotic lysis. The product of osmotic pressure and the volume containing one gram-molecule of solute is a constant. Thus, osmotic pressure is directly proportional to the concentration and

P=(0.0821)(T)/V

for a substance, where, Ρ is measured in atmospheres, V in liters, and T in degrees Kelvin. A solution of 180 g/L of glucose (MW=180) at 30°C, contains 1 gram-molecule per liter. Thus, V=1 liter and K=303 K. Hence, the osmotic pressure Ρ=24.9 atm. (366 psi).

All the required metallic elements can be supplied as nutrients in the form of the cations of inorganic salts. K, Mg, Ca and Fe are normally required in relatively large amounts and should normally always be included as salts in culture media. Table 4.9 shows which salts are soluble and which are insoluble in water [9], as well as commonly used inorganic and trace elements and concentration ranges.

Table 4.9. Solubility of the Common Salts [5] and Commonly used Inorganic and Trace Elements and Concentration Ranges

Salt Soluble Insoluble
Nitrates All
Sulfides Na, K, Ca, Ba All others
Chlorides All others Ag, Hg, Pb
Carbonates Na, K All others
Sulfates All others Pb, Ca, Ba, Sr
Phosphates Na, K All others
Silicates Na, K All others
Acetates All
Oxalates All others Ca (depends upon concentration)
Source Quantity (g/L)
KH2PO4 1.0–5.0
MgSO4·7H2O 0.1–3.0
KC1 0.5–12.0
CaCO3 5.0–17.2
FeSO4·4H2O 0.01–0.1
ZnSO4·8H2O 0.1–1.0
MnSO4·H2O 0.01–0.1
CuSO4·5H2O 0.003–0.01
Na2MoO4·2H2O 0.01–0.1

(from Stanbury & Whitaker, 1984).

Oxygen is always provided in water. Some organisms require molecular oxygen as terminal oxidizing agents to fulfill their energetic needs through aerobic respiration. These organisms are obligately aerobic. For obligate anaerobes molecular O2 is a toxic substance. Some organisms are facultative anaerobes and can grow with or without molecular O2. Lactic acid bacteria have an exclusive fermentative energy-yielding metabolism, but are not sensitive to the presence of oxygen [4]. Saccharomyces cerevisiae produces ethanol anaerobically and cell mass aerobically, and it can shift from a respiratory to a fermentative mode of metabolism.

Sodium and chloride ions are respectively the principal extracellular cations and anions in animals and plants. Potassium is the principal intracellular cation. Candida intermedia, (SCP) single cell protein, grows better on normal alkanes with sources of N, O, and P, if small amounts of ZnSO4·7H2O are added [7]. Takeda, et al., reported that yeast can be grown by continuously feeding a medium consisting of hydrocarbon fractions boiling at 200° to 360°C, small amounts of inorganic nitrogen, inorganic salts and organic nitrogen to which the fermentation waste liquor previously used or CSL and ethanol are added [7]. This suggests that SCP production on hydrocarbons may be an outlet for stillage from ethanol-from-beet molasses plants or whey. Table 4.10 shows the approximate composition of such concentrated stillage from a European producer [5].

Table 4.10. Composition of a Concentrated French Stillage (Ethanol-from-beet molasses)

Moisture 31.4%
True protein 4.7%
Betaine 10.6%
L-pyroglutamic acid 7.2%
Lactic acid 4.1%
Acetic acid 1.3%
Butyric acid 1.3%
Other organic acids 1.8%
Glycero l 5.4%
Raffinose 0.3%
Glucose+fructose 0.9%
Melibiose 1.4%
Inositol 0.7%
k+ 8.1%
Na+ 1.7%
Ca2+ 0.4%
Mg2+ 0.03%
Fe3+ 0.02%
P2O5 0.34%

The combination of minerals is also important in regulating the electrolytic and osmotic properties of the cell interior. In most cases, the complex industrial carbon and/or nitrogen sources supply sufficient minerals for proper fermentation.

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Sugar Strategies for Biomass Biochemical Conversion

Hongzhang Chen, Lan Wang, in Technologies for Biochemical Conversion of Biomass, 2017

6.6.1 Sources and History of Sugar Platform Materials

From the perspective of fermentable carbon cost, the development of sugar platform materials has passed through three stages: sucrose materials, starchy materials, and lignocellulosic biomass. Of which, the industrial fermentation technologies of sucrose materials and starchy materials are quite mature, while the utilization of lignocellulosic biomass is still in the initial stage (Bai, Anderson, & Moo-Young, 2008).

Commonly used raw materials of sugar in the fermentation industry include sugarcane, sugar beet, molasses, and sugar grass. Sugarcane is rich in sugar and water, being an important source of raw materials for sugar in the fermentation industry. Compared with sugarcane, sugar grass juice provides sugar containing quite a bit of reducing sugar. Molasses is a viscous, brown semifluid, which is the by-product of the sugar refining industry. Molasses can be divided into sugarcane molasses, sugar beet molasses, grape molasses, and maize molasses, and all contain lots of fermentable sugar, which is regarded as an important source of sugar platform compounds.

With the development of technologies, besides the sugars that can be directly used, humans begin to obtain sugars that can be used by microorganisms directly or indirectly from noncarbohydrates; one of these sugar sources is starch. Starchy materials commonly used in the fermentation industry include grains, potatoes, wild plants, and by-products of agrotechny. Rice, maize, and wheat provide starchy materials and high annual yields. Generally speaking, starch mainly exists in the seeds and tubers of plants. For example, the starch content of rice is about 62%–86%, wheat 57%–75%, and potatoes more than 90%. At present, such grains are the main raw materials used in the fermentation industry.

However, one of the disadvantages of using grains as the main resource for the production of sugar is the issue of the reasonable distribution of resources in view of the fact that grains are also food for humans. Even if nongrain starchy materials are used, problems around occupying arable land still exist. Owing to the aggravation of the energy crisis and environmental pollution, more and more researchers are paying attention to the high-value utilization of renewable resources, especially lignocellulosic biomass (Hahn-Hägerdal, Galbe, & Gorwa-Grauslund, 2006). Lignocellulosic biomass is regarded as the traditional waste of agroforestry, and thus utilization of lignocellulose can not only reduce the waste of resources, it can also bring out extraneous income, realizing the “multi win” of resource, environment, and economy.

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Vegetable-based feedstocks for biofuels production

S. Pinzi, M.P. Dorado, in Handbook of Biofuels Production, 2011

4.4.1 Sucrose-containing feedstocks

The main feedstock for ethanol production is sugar from cane and beet. Sugar is converted into bioethanol by ethanologenic fermentation. The most employed microorganism is Saccharomyces cerevisiae due to its capability to hydrolyse cane sucrose into glucose and fructose, two easily assimilable hexoses (Sanchez and Cardona, 2008). Yeasts such as Schizosaccharomyces pombe present the additional advantage of tolerating high osmotic pressures (high amounts of salts) and high solids content (Bullock, 2002). Among bacteria, Zymomonas mobilis provides higher ethanol yield, up to 97% of theoretical maximum (Claassen et al., 1999). The disadvantage of its use during fermentation is the formation of a polysaccharide (which increases the viscosity of fermentation broth) and sorbitol, which decreases the efficiency of the conversion of sucrose into ethanol (Lee and Huang, 2000).

Sugar cane and sugar beet

Feedstock for bioethanol production is essentially composed of sugar cane (Fig. 4.7) or molasses (by-product of sugar mills) and sugar beet (Fig. 4.8) (UNCTAD, 2006). Two-third of the world sugar production is from sugar cane and one-third is from sugar beet. Sugar cane is grown in tropical and subtropical countries, while sugar beet is only grown in temperate climate countries.

4.7. Sugar cane.

(Photo courtesy of Hannes Grobe)

4.8. Sugar beet.

(Photo courtesy of Biofuels Center of North Carolina [http://www.biofuelscenter.org])

While Brazil is the world’s largest producer, in European countries, Spain is the largest producer of bioethanol, and beet molasses are the most utilised sucrose-containing feedstock (Cardona and Sánchez, 2007). Sugar beet crops are grown in most of the European Union (EU) member states, providing 90% of the total EU demand of sugar. The advantages of sugar beet are a lower cycle of crop production, higher yield and high tolerance of a wide range of climatic variations, low water and fertiliser requirement. Compared to sugar cane, sugar beet requires 35–40% less water and fertiliser (Balat et al., 2008).

Sorghum bicolor

Sorghum bicolor crop, also known as sweet sorghum (Fig. 4.9), is heat tolerant and is one of the most drought-resistant crops as it has the capability to remain dormant during the driest periods, so it can grow in marginal land (Yuan et al., 2008). Sweet sorghum is one of the most promising candidates for bioethanol production in developing countries because it produces grains with high starch content, stalks with high sucrose content and leaves and bagasse with high lignocellulosic content (Smith and Buxton, 1993). It has been found that the production of ethanol from the hemicellulose and cellulose in bagasse is more favourable than burning it to make power in North China, although ethanol produced from the juice is very sensitive to the price of sugar (Gnansounou et al., 2005).

4.9. Sorghum bicolor.

(Photo courtesy of Daniel Georg Döhne)
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Biofuel-driven biorefineries for the co-production of transportation fuels and added-value products

R. Van Ree, ... B. Van Der Drift, in Handbook of Biofuels Production, 2011

21.2.1 Bioethanol

Current bioethanol production technologies are based on the conversion of carbohydrates derived from sugar cane, sugar beet, maize or cereals (i.e. wheat, barley) into ethanol. In addition, bioethanol can be derived from a number of other agricultural commodities such as cassava, or from residues or waste streams from other agro-industrial processes, including cane or beet molasses and starchy residues.

A number of by-products or co-products are produced during the conversion of biomass to ethanol. Most prominent by-product from ethanol production from corn, wheat or barley is so-called DDGS, which is a protein-rich fibrous residue that is primarily sold as animal feed. DDGS is formed by combining insoluble residues from the fermentation step with soluble residual streams from the distillation step, and drying the combined product. The market price of DDGS devaluated in the last 20 years due to increased production volumes saturating the feed market. Other high added-value products need to be found for DGGS to maintain its co-product status.

A common by-product of sugarcane derived ethanol is bagasse, which is the fibrous residue of the sugar cane stem after extraction of soluble sugars. Bagasse is commonly used to generate electric power and heat at the sugar mill facility to supply the energy needed for the bioconversion process.

Upgrading of process residues like DDGS and bagasse to higher added-value bio-based products (i.e. chemicals, materials) – turning the processes into biofuel-driven biorefineries – maximises the sustainable valorisation of the raw biomass materials, increasing the market competitiveness of the bioethanol produced. DDGS is high in protein content (over 30%) which, if isolated, can be used potentially for the production of chemical precursors (Brehmer, 2008). Bagasse can also have many other applications such as the production of fibre boards or the production of high added-value specialty chemicals, i.e. xylitol from xylose-rich effluents from acid hydrolysis of sugarcane (Baudel, 2005).

Another by-product of bioethanol production is CO2, which in certain cases is marketed as gas for industrial use.

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Anaerobic biodigestion for enhanced bioenergy generation in ethanol biorefineries

L.T. Fuess, M.L. Garcia, in Bioenergy Systems for the Future, 2017

5.3.1 Energetic potential (EP) for vinasses from various feedstocks

In short, EP constitutes the energy potentially recovered as biogas from the organic content found in vinasse. In this study, the energetic analysis was initially applied to vinasses from various feedstocks: sugarcane (juice—SGJ—and blends of juice and molasses—SJM), beet molasses (BML), corn (CRN), cassava (CSV), and lignocellulosic materials (LCL). Table 5.2 compiles the raw data used in the estimates, including the compositional characteristics of vinasse (COD) and typical performance data on COD removal and methane production from AD systems reported elsewhere. EP calculations were based on Eq. (5.1), so that an average vinasse production rate (VPR) of 13 m3 m− 3EtOH was considered (BNDES and CGEE, 2008):

Table 5.2. Reference data on vinasse characterization and typical performance of AD systems applied to vinasses from various feedstocks

Crop Organic content (COD) AD performance
(g L1) OLR ERCOD MYa
(kgCOD m− 3 d− 1) (%) (N m3 kg− 1CODremoved)
Sugarcane (juice) 30.4 18.3 76.0 0.329b
Reference Wilkie et al. (2000) Costa et al. (1986)
Sugarcane (juice + molasses) 45.8 26.5 71.7 0.307
Reference Costa et al. (1986), Prada et al. (1998) Souza et al. (1992)
Beet (molasses) 55.5 6.0 88.2 0.330
Reference Vlissidis and Zouboulis (1993) Vlissidis and Zouboulis (1993)
Corn 60.2 7.5 92.2 0.284
Reference Wilkie (2008) Agler et al. (2008)
Cassava 62.2 11.3 85.1 0.258
Reference Luo et al. (2009), (2010), and (2011) Luo et al. (2009)
Lignocellulosic materials 61.3 10.0 84.4 0.316c
Reference Wilkie et al. (2000) Good et al. (1982)

Parameters: COD, chemical oxygen demand; OLR, organic loading rate; ERCOD, COD removal efficiency; MY, methane yield.

a
Values corrected to standard temperature and pressure conditions (STP) if not indicated in the reference study
b
Temperature not indicated
c
Refer to eucalyptus wood hydrolysate.
(5.1)EP=VPR⋅COD⋅ERCOD⋅MY⋅LHVCH4

In Eq. (5.1), the terms EP, VPR, COD, ERCOD, MY, and LHVCH4 are, respectively, the energetic potential of vinasse (MJ m− 3EtOH), the average generation rate of vinasse (13 m3 m− 3EtOH), the chemical oxygen demand of vinasse (kgCOD m− 3), the COD removal efficiency (dimensionless), the methane yield (N m3 kg− 1CODremoved), and the lower heating value of methane (50 MJ kg− 1 or 35.72 MJ N m− 3, Heywood, 1988). Performance data compiled in Table 5.2 refer to the application of thermophilic temperature conditions (50–55°C) to the reactors, which may be considered a feasible alternative for vinasses, based on two main advantages: (i) Vinasse is released from distillation columns at approximately 85–95°C (Dias et al., 2012; Moraes et al., 2015), naturally reaching the adequate temperature for the treatment without requiring energy inputs, and (ii) thermophilic systems may handle higher OLRs compared with mesophilic ones, which enables the use of more compact reactors and consequently improves the economics of full-scale anaerobic processes (Wilkie et al., 2000).

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Sugar- and Starch-Based Biorefineries

Rainer Höfer, in Industrial Biorefineries & White Biotechnology, 2015

6.3 Higher Carboxylic Acids

6.3.1 Citric Acid

Citric acid (2-hydroxypropane-1,2,3-tricarboxylic acid) is a weak organic tricarboxylic acid with three different values of pKa (3.1, 4.7, and 6.4). It can exist either in an anhydrous (water-free) form or as a monohydrate. At room temperature, citric acid is a white hygroscopic crystalline powder (Figure 4A.31).

Figure 4A.31. Citric acid.

It is a primary metabolic product formed in the Krebs cycle used by all aerobic organisms to generate energy and as such found in small quantities in virtually all plants and animals.

Although industrial-scale citric acid production originally was based on the physical extraction from citrus fruit or lemon juice, resp., fermentation is the major route to commercial citric acid since Pfizer began industrial-level production using this technique. All substrates containing glucose, including sucrose, beet molasses, corn syrup, and dextrose from enzyme-treated starch, are well-suited for the manufacture of citric acid. Processes employed are surface fermentation in shallow pans or submerged fermentation in bubble column fermenters using Aspergillus niger mold and submerged fermentation by yeast (Candida guilliermondii, C. lipolytica), with A. niger still being the main industrial fermenting organism. After the mold is filtered out from the fermentation broth, citric acid is recovered by solvent extraction or more commonly by calcium citrate precipitation, followed by treatment with sulfuric acid to convert the calcium citrate to calcium sulfate and citric acid and then removing the calcium sulfate by filtration. The final processing involves crystallization, centrifuging, fluidized bed drying, and classification. The product is sold as an anhydrous or monohydrate acid.67 About 70% of total citric acid production is used as an acidulent, flavor enhancer and preservative in food and carbonated beverages, about 15% as detergent builder cobuilder or cleaner in liquid household detergents, detergent powders, hard-surface cleaners, and fabric softeners. Other applications include the pharmaceutical industry as antioxidant or in the form of iron citrate as a source of iron for the body; the use as chelating agent and pH adjuster in hair conditioners and shampoos or in metal finishing and cleaning; as a setting retarder in gypsum plaster; the use as starting material for a number of citrate esters, such as acetyl tributyl citrate, tributyl citrate, and tri-2-ethylhexyl citrate used as plasticizers for a variety of vinyl resins and films, or in some cellulose acetate and cellulose nitrate gums and resins.68

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Process Modeling and Techno-Economic Evaluation of an Industrial Bacterial NanoCellulose Fermentation Process

Fernando Dourado, ... Miguel Gama, in Bacterial Nanocellulose, 2016

Process Simulation: Bacterial NanoCellulose Production

For this study, a survey of the literature was first conducted, to gather information on the type of strains, CM, and fermentation conditions. Table 12.1 summarizes the collected data. For the simulation of the biotechnological BNC production, the starting point and criteria of choice were the selection of one publication demonstrating the use of a commercial acetic acid bacterium strain that exhibited good comparative yields, while using, totally or partially, low cost substrates. The use of a commercial strain allows to obviating regulatory issues when considering the implementation of an industrial (commercial) bioprocess; regarding the fermentation method, static culture was selected to better approximate this study to the production of nata de coco; as for the CM, low cost substrates, mostly from the food industry have been widely explored in several biotechnological processes, to lower production costs, and were chosen for this study. Among the several existing possibilities (Table 12.2), the paper from Keshk et al. [31] was selected to retrieve the relevant data. Briefly, BNC was produced from beet molasses using Komagataeibacter xylinus ATCC 10245; from the fermentation experiments with beet molasses, BNC yield was calculated to be of 7 g L−1 (dry mass), following a 7-day static culture fermentation process. The composition of the CM, as extracted from the manuscript [31] is shown in Table 12.2.

Table 12.2. Composition and estimated purchase prices of the main raw materials for BNC production

Stock (g/L) Density (kg/L) Culture Media (g/L) Price (USD$/Tona)
Beet molasses 570 1.2 20 200.00
Yeast extract 1.5 5 2,000.00
Peptone 20 7,945.00
Disodium phosphate 2.7 500.00
Citric acid 0.8 1.2 500.00
Water 1 2.50
Downstream washing
NaOH 10 2000
a
An average price of the stock components was calculated on the basis of the values from different suppliers and literature.

The conceptual project and economic analysis of BNC fermentation was done using Super-Pro Designer software, version 9.0, for Windows operating system. The plant was arbitrarily projected to process 60,000 L month−1 of CM. Assuming hydrated BNC contains 99% water, this production volume would yield 42 tons month−1, that is, 504 ton year−1 of BNC. Fig. 12.1 displays a flowsheet of the BNC fermentation process as retrieved from the software. Table. 12.3 describes the main equipment (type and number of used equipments, their size, and cost).

Figure 12.1. Super-Pro process flowsheet of the fermentative production of bacterial cellulose.

Table 12.3. Equipment specifications and costs (2015 prices)

Name Type Units Size (Capacity) Purchase Cost (US$ unit)
V-101 Blending tank 3 4,111.11 L 248,000
SBR-100L Seed bioreactor 1 137.60 L 521,000
SBR-1000L Seed bioreactor 1 1,389.97 L 1,280,000
Cleanroom Generic box 2 12,340.29 L 500,000a
PZ-101 Pasteurizer 1 20,012.85 L  h−1 81,000
FL-101 Filler 1 414.96 units  h−1 25,000
BX-101 Packer 1 41.50 unit h−1 20,000
GR-101 Grinder 1 10,122,21 kg  h−1 122,000a
FSP-101 Flow splitter 1 11,051.61 kg  h−1 1,000a
PZ-102 Pasteurizer 1 2,775.00 L h−1 31,000
WSH-101 Washer (bulk flow) 2 10,122.21 kg  h−1 20,000a
Unlisted equipment 966,000
Total 4,831,000
a
Estimate costs obtained from local companies. Remaining price estimates were provided by Super-Pro simulation software.

Inoculum propagation is widely reported to be achieved by successive propagation of biomass and CM at a ratio of 1:10 (biomass:CM), as used in this work. For the sake of simplicity, propagations below 100 L were omitted in the design, as these can easily be done at laboratory scale (the inoculum from ATCC 10245 could then be transferred to the 100 L seed fermenter). As such, two seed fermenters with 100 (SBR-100 L) and 1000 L (SBR-1000 L, Fig. 12.2) capacities were considered for biomass growth (Fig. 12.2, “Inoculum Propagation” stage). Also, a simplified version of the culture CM preparation and pasteurization was chosen. For academic purposes, a single entry containing the mixture of the CM components was fed to a storage tank (V-101) before pasteurization (PZ-101). The pasteurized CM is then sequentially fed to each of the seed fermenters. Each seed fermenter operates for 3 days. The bacteria and additional pasteurized CM (up to a total volume of 10,000 L) are then combined and transported to a “cleanroom” for the fermentation under static conditions. This generic unit represents a controlled environment room with minimum level of pollutants, operating at 30 °C for 7 days, to simulate static culture conditions. Two clean rooms were considered for this process (Fig. 12.2 and Table. 12.1. for a description of the equipments) as explained: the static culture step (“Cleanroom-10,000,” Fig. 12.2a) is the dominant scheduling bottleneck of the entire process, that is, its occupation time per batch exceeds any other equipment in the process, thus leading to large plant cycle times (roughly 10 days, Fig. 12.2a). By including an additional cleanroom operating in staggered mode (“STG01 Cleanro,” Fig. 12.2b), lower production cycle times can be obtained (roughly 5 days). This allows to increase from 41 to 81 batches year−1.

Figure 12.2. Equipment utilization chart of the simulated BC fermentation process.

(A) Multiple batches operation with one cleanroom; (B) multiple batches operation with two cleanrooms, one of which is in “staggered mode” (“STG01 Cleanro”).

Upon fermentation, the resulting BNC sheets are collected, cut into cubes (GR-101), and washed with NaOH and water (WSH-101). The cubes are then packed (in plastic bags and cardboard boxes; FL-101 and BX-101) and stored (“Downstream Processing” stage).

Based on the inserted data (regarding unit operations, reaction kinetics, raw materials, etc.), Super-Pro Designer can estimate the equipment size and cost (Table 12.3) by using built-in cost correlations from data derived from a number of vendors and literature sources (data from some of the equipments were obtained from direct contact with several companies as these were not available in Super-Pro). The total cost of the equipment is of US$ 4.83 million.

Table 12.4 summarizes the resulting estimation of the capital investment costs, operating costs, and profitability analysis of the BNC fermentation process simulation. Some of the financial data were calculated based on information outlined by Peters and Timmerhaus [46]. The total capital investment (TCI) for an industrial facility capable of producing 504 tons year−1 of BNC was estimated to be of near US$ 13 million; near 71% of which correspond to direct costs (DC) from equipment and installation, piping, instrumentation, insulation, electrical facilities, building costs, yard improvements and auxiliary facilities, and land. Indirect costs (29% of the TCI) consist of engineering and construction costs. Contingency charges (US$ 966,199.99) are extra costs added into a project budget to accommodate for variations in the cost estimates. These allow to compensating for unpredictable expenses, minor process changes, price fluctuations, and estimating errors.

Table 12.4. Cost structure (in US$) and profitability analysis of the BC fermentation process

Estimation of Capital Investment Cost
Direct costs (DC) 9,178,799
 Purchased equipment (PE) 4,831,000
 Installation, including insulation and painting 828,171
 Instrumentation and controls 552,114
 Piping 690,142
 Electrical 483,000
 Buildings, process, and auxiliary 690,142
 Service facilities and yard improvements 276,057
 Land 828,171
Indirect costs (IC) 2,070,428
 Engineering and supervision 579,719
 Construction expense and contractor’s fee 552,114
 Contingency 966,199
Fixed capital investment (FCI) 11,276,834
Working capital (WC) 1,691,525
Total capital investment (TCI) 12,968,359
Estimation of the Annual Product Manufacturing Cost
Manufacturing costs (MC) 5,390,511
 Direct production cost (variable costs) 2,184,022
 Raw materials 169,280
 Operating labor (OL) 1,269,130
 Direct supervisory and clerical labor (DS & CL) 222,097
 Utilities (electricity, steam, chilled water) 80,000
 Maintenance and repairs (2–10% FCI) 338,305a
 Operating supplies (0.5–1% FCI) 84,576a
 Laboratory charges (10–20% OL) 126,913a
 Fixed charges 2,108,768
 Depreciation (13% FCI machin. & equip. + 2–3% FCI buildings) 1,465,988a
 Local taxes 281,920a
 Insurances 281,920a
 Plant overhead costs (50–70% OL, DS, & CL, and M & R) 1,097,720
General expenses (GE) 2,023,417
 Administrative costs (2–6% of TPC) 440,000a
 Distribution and selling costs (2–20% of TPC) 385,000a
 Research and development costs (5% of TPC) 550,000a
 Financing (interest) (0–10% TCI) 1,584,551
Total product cost (TPC): MC + GE 7,413,928
Profitabilitya (504,000 kg year−1, at a selling price of US$ 25 per kg−1)
  Total income: selling price  × quantity of product 12,600,000
  Gross income: total Income − total product cost 5,079,790
  Taxes: 30–40% gross income 1,777,926
  Net Profit: gross income − taxes 3,301,863
  Rate of return: net profita100/TCI 10%
  Payout period: FCI/(net profit + depreciation) 4  Years
a
Calculated as described in Ref. [46].

The annual manufacturing (product or production) costs (TPC), totaling US$ 7.4 million include elements that contribute directly to the cost of production (such as direct operating costs, fixed charges, and plant overhead costs), and general expenses. Operating labor (with 58%) is the most representative of the direct production costs (which total US$ 2.18 million). Fixed charges, totaling US$ 2.1 million, relate to the physical plant in itself, thus unaffected by the productivity levels. These include depreciation, local taxes, insurances, and rent. Depreciation (70% of the fixed charges) is a time-dependent operating cost representing a fixed capital loss mostly due to equipment and facilities wear out and obsolescence. Plant overhead costs (little over US$ 1 million) includes charges for services not attributable to the cost of the product, such as medical service, safety and protection, storage facilities, plant superintendence, cafeteria, janitorial services, administrative and accounting services, etc. The general expenses (US$ 2 million) cover the management costs and to develop new processes (Research & Development). Administrative costs include salaries for administrators, accounting, legal support, and computer support, as well as office supply and equipment, administrative buildings, etc. The sum of the manufacturing costs, MC [73% of the total product manufacturing costs (TPC)] and general expenses, GE (27% of the TPC) make up the TPC (US$ 7.4 million).

Based on the above capital expenses, profitability analysis was done considering a market price for BNC at US$ 25 kg−1 (corresponding to packed BNC cubes as the final selling product). The resulting net profit amounts to US$ 3.3 million  year−1. The return on investment (of 10%) is the ratio of profit to investment and measures how effectively a company converts the invested capital into profit (i.e., it represents the return per dollar invested). Finally, the payback period, representing the length of time necessary to recover the capital investment, was calculated to be of 4 years.

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