text:
Effects of slurry concentration and rotational speeds of drum drier to the characteristics of pregelatinized breadfruit (Artocarpus altilis) flours

Breadfruit is one alternative carbohydrate source in Indonesia. However, breadfruit is seasonal and climacteric crops; it should be processed immediately to produce flour. Native breadfruit flour has many limitations since it is difficult to be solubilized and swollen in cold water. The flour is modified physically by pregelatinization to improve its properties. Pragelatinized breadfruit flour can be used as instant food, porridge, and baby food. This research is aimed to investigate the effect of slurry concentration (20 and 30%) of native breadfruit flour and rotational speeds (4, 6 and 8 rpm) of double drum drier to the chemical composition and physico-chemical properties of the pregelatinized flours. The results showed that pregelatinization did not influence to the chemical composition. Pregelatinization of the flour improved the water solubility and swelling power on room (30°C) and hot (70°C) solutions, reduced the freeze-thaw stability, caused partial gelatinization which is monitored by losing the birefringence of starch granule, and darkened the flour. The pregelatinization also reduced the gelatinization temperature and had low final viscosity. Low concentration of slurry and low rotation speed of drum increased the gelatinized part of the flour, which is influenced to the cold and hot water solubility.

Introduction
Breadfruit (Artocarpus altilis) or sukun (local name) belongs to the Moraceae a family of about 50 general and over 1000 species. The species originated in Oceania, and is grown across the Pacific region. It is now found in many countries in tropical zones across the world, including in Africa, Australia, South America, South and Southeast Asia [1]. It has long been an important staple crop and a primary component of traditional agroforestry systems in Oceania [2].
The plant Artocarpus altilis, seedless variety, is a fruit-producing plant. In Indonesia, breadfruit is harvested twice a year. The fruit can be eaten in all stage of maturity, since it is a good source for carbohydrate, vitamin and minerals [2]. Breadfruit is a large, round, starchy fruit borne by a tree. It is classified as climacteric fruit, which has fast respiration process [3]; therefore it has short shelf-life. In order to anticipate abundant breadfruit during the peak harvest session and to prolong its shelf life, the fruits should be processed immediately.
Breadfruit is consumed after cooking by boiling, steaming, roasting, baking and frying. Breadfruit has been processed into many forms for utilization in the food industry. It can been processed into starches [4] into flour [5,6] and other modified products [7]. Native breadfruit flour has some limitation in application as food products. It has low solubility and swellability in room temperature. Studies on the modification of breadfruit starch involving heat-moisture-treatment and annealing, as forms of physical modification collectively referred to as hydrothermal treatment which entail modification of temperature and moisture content have been carried out and reported by Adebowale et al. [8]; and Ojifolokun and Adeniran [7]reported the study explored the potential of substituting breadfruit into cassava to obtain gari analoque.
Pregelatinization is one of the physical methods used to modify starch by using drum dryer, spray dryer or extruder [9]. This modification method affects physicochemical and functional properties of  [10]. Due to starch granule disruption, pregelatinized flour can absorb water and increase viscosity immediately even with cold water [11,12]. For powder production from fruit or root and tuber, drum drying is considered a special method for numerous materials with many benefits such as good characteristics of product and economic efficiency of the process. Pregelatinized starch is primarily used as a thickener in many instant products, such as baby food, soups and desserts, due to its ability to form pastes and dissolve in cold water. The use of pregelatinized starch is preferred in sensible foods because it does not require heating to form a paste [13]. The four variables which govern the operation of drum are steam pressure, rotational speed, and film thickness and feed material characteristics. The steam pressure or heating medium temperature will regulate the drums temperature. The rotational speed of the drum determines the all performance of dryer [14]. This research is aimed to investigate the effect of slurry concentration of native breadfruit flour and rotational speeds of double drum drier to the chemical composition and physico-chemical properties of the pregelatinized flours.

Materials
The breadfruit used in this study was collected from local farmer in Jawa Barat. They were on the optimum green maturity, and taken in polypropylene plastic bags to the laboratory to use and process immediately.

Processing of Breadfruit into Flour
Fresh fruits were peeled and washed with clean water, and then sliced, blanched with hot steam for one minute, and dried at 60 o C for 6-8 h. Dried chips were milled into flour, and the flour was screened by 60-mesh standard sieve.

Production of Pregelatinized Flour
The slurry (A1-20% and A2-30%) was prepared by mixing the flour with distilled water, and then dried in double drum drier with different rotational speeds (R1-4 rpm, R2-6 rpm, and R3-8 rpm). The drier has two stainless steel drums with 12 in outer diameter and 8 in cylinder lenght, and 80±5ºC surface drier plate temperature. Then the dried flour was milled and screened by 60-mesh standard sieve. Samples were kept in polyethylene bag, sealed and stored at room temperature until used.

Flour Characteristics
The flour was analyzed its proximate components (moisture, ash, crude fiber, crude protein, crude fat, and carbohydrate by difference contents) according to AOAC methods [15], starch content (Luff Schorl method [15], the whiteness of flour by Colortec, shape and size of starch granules by using polarized light microscope.
The viscoamylographical properties of each flour was studied by using Brabender Viscoamylographer. Flour solubility and swelling power at 30 o C and 70 o C, paste clarity, and freezethaw stability were analyzed by using modified Perez et al. [16]. Viscosity of 5% flour paste was analyzed by using Brookfield viscosimeter.

Results and Discussion
Breadfruit should be harvested at the stage of maturity that is required for the method of preparation for consumption. In this research, the breadfruit is harvested as "green mature". At this stage the fruit has completed its development and has reached the dimensions of a mature fruit but has not begun to ripen and is still very firm. The flesh is very starchy and not sweet to the taste.
Native breadfruit flour (A0R0) and six pregelatinized flour were analyzed for their chemical composition (Table 1). It has shown that all flour contained relative low minor components such as fat (1.004-3.671%) and protein (3.694-5.028%), but high in carbohydrate and fiber. The starch is the main carbohydrate in breadfruit flour. The starch content in native flour was determined as 31.6%. Similar results also reported by Wooton and Tumaalii [5], besides the starch the flour also contained lots of total sugar with fructose is predominant sugar found in less mature fruits.
The protein and fat contents showed decreasing values compared to native flour. The major cause of this significant decrease in the crude protein content may be the pre-gelatinization process, which allows elution and  [17]. In food application, this composition is a key to achieve desirable product quality as well as nutrition value and even the lipid content in flours was low, it could affect to the flour properties dramatically such as swelling and pasting properties [18]. Pregelatinized flour is flour that has undergone a cooking process until complete or partial gelatinization and a simultaneous drying process. Since the process is physical process, it did not affect to the chemical composition, as shown in Table 1 for different slurry concentration and rotational speeds of drum dryer. The main consequence of this treatment is the destruction of the granular structure, resulting in complete granular fragmentation, and the absence of birefringence properties as shown in Figure 1.  Table 2 showed that pregelatinization slightly increased the solubility and swelling power at 30 o C and 70 o C, paste viscosity and clarity. Murayama et al. [17] reported that the solubility of the native flours showed slight changes between 50 and 70℃, and The native breadfruit flour has high tendency to retrogradation as shown in high freeze-thaw stability and pasting properties (Figure 2 and Table 3 for A0R0). Similar pattern for breadfruit starch also reported by Rincon and Padilla [4]. Pregelatinization effectively affected to the cold swelling flour with low (30 o C) initial gelatinization temperature. Higher slurry concentration and higher rotational speed caused the increasing of maximum viscosity and reduced the tendency to retrogradation.

Conclusion
Breadfruit flour contained high amount of carbohydrate mainly starch, and low minor components such as protein and fat, but the pregelatinization process did not influence to the chemical composition. Pregelatinization of the flour improved the water solubility and swelling power on room and hot solutions, reduced the freeze-thaw stability, caused partial gelatinization which is monitored by losing the birefringence of starch granule, and darkened the flour. The pregelatinization also reduced the initial gelatinization temperature and had low final viscosity. Low concentration of slurry and low rotation speed of drum increased the gelatinized part of the flour, which is influenced to the cold and hot water solubility.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Dissection of two quantitative trait loci with pleiotropic effects on plant height and spike length linked in coupling phase on the short arm of chromosome 2D of common wheat (Triticum aestivum L.)

Key message Two QTL with pleiotropic effects on plant height and spike length linked in coupling phase on chromosome 2DS were dissected, and diagnostic marker for each QTL was developed. Abstract Plant height (PHT) is a crucial trait related to plant architecture and yield potential, and dissection of its underlying genetic basis would help to improve the efficiency of designed breeding in wheat. Here, two quantitative trait loci (QTL) linked in coupling phase on the short arm of chromosome 2D with pleiotropic effects on PHT and spike length, QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2, were separated and characterized. QPht/Sl.cau-2D.1 is a novel QTL located between SNP makers BS00022234_51 and BobWhite_rep_c63957_1472. QPht/Sl.cau-2D.2 is mapped between two SSR markers, SSR-2062 and Xgwm484, which are located on the same genomic interval as Rht8. Moreover, the diagnostic marker tightly linked with each QTL was developed for the haplotype analysis using diverse panels of wheat accessions. The frequency of the height-reduced allele of QPht/Sl.cau-2D.1 is much lower than that of QPht/Sl.cau-2D.2, suggesting that this novel QTL may be an attractive target for genetic improvement. Consistent with a previous study of Rht8, a significant difference in cell length was observed between the NILs of QPht/Sl.cau-2D.2. By contrast, there was no difference in cell length between NILs of QPht/Sl.cau-2D.1, indicating that the underlying molecular mechanism for these two QTL may be different. Collectively, these data provide a new example of QTL dissection, and the developed diagnostic markers will be useful in marker-assisted pyramiding of QPht/Sl.cau-2D.1 and/or QPht/Sl.cau-2D.2 with the other genes in wheat breeding. Electronic supplementary material The online version of this article (10.1007/s00122-019-03318-z) contains supplementary material, which is available to authorized users.

Introduction
Wheat (Triticum aestivum L.) is an important food crop worldwide, providing calories and proteins consumed by humankind (Fischer et al. 2014;Shiferaw et al. 2013). Plant height (PHT) is a crucial trait related to plant architecture and yield potential (Cadalen et al. 1998;Peng et al. 1999; Communicated by Yunbi Xu.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s0012 2-019-03318 -z) contains supplementary material, which is available to authorized users. Sakamoto and Matsuoka 2004). Consequently, the use of dwarfing genes to reduce PHT and improve yield has been one of the main strategies in breeding modern high-yielding hexaploid bread wheat varieties. For example, during the green revolution, the introduction of semi-dwarf varieties into wheat (T. aestivum L.) appropriately reduced PHT and contributed significantly to a worldwide increase in potential grain yield (Peng et al. 1999). PHT is known to be typically under polygenic control (Bellucci et al. 2015;Tang et al. 2007). Thus, identification of QTL/gene controlling PHT would help to improve the efficiency of designed breeding in wheat.
To date, 24 genes influencing PHT have been identified and designated reduced height genes in wheat (McIntosh et al. 2017). Of these 24 major genes, Rht-B1b and Rht-D1b, which are located on chromosomes 4B and 4D, respectively (Pearce et al. 2011), were cloned and most widely used in wheat breeding (Borner et al. 2002;Cadalen et al. 1998). They encoded DELLA proteins, which are transcriptional regulators that reduce response to gibberellin. Thus, Rht-B1b and Rht-D1b belonged to the group of dwarfing genes that is insensitive to gibberellic acid (Pearce et al. 2011). The characterization of these two genes has enhanced our knowledge about PHT determination in wheat, and functional markers developed for them have been used in wheat breeding (Akman and Bruckner 2012;Borrell et al. 1991;Tang et al. 2009).
The other extensively used gene in wheat breeding is Rht8, which is derived from the Japanese variety Akakomugi and was introduced into Southern European wheat breeding in the 1930s by the Italian breeder Strampelli, together with the photoperiod-insensitive, early flowering Ppd-D1a allele (Lorenzetti 2000). Rht8 was mapped on the short arm of chromosome 2D, and a closely linked SSR marker named Xgwm261 was detected ). The 192-bp allele of Xgwm261 corresponds to a height-reducing phenotype of Rht8, attributing to a 7-8 cm reduction in PHT without pleiotropic effects on other agronomic traits ). Since then, the 'diagnostic' 192-bp allele was used to survey the presence of Rht8 in wheat cultivars (Ahmad and Sorrells 2002;Asplund et al. 2012;Bai et al. 2004;Chebotar et al. 2001;Liu et al. 2005;Worland et al. 2001a, b;Zhang et al. 2006). However, the 192-bp allele of Xgwm261 was not always linked to Rht8 (Ellis et al. 2007). A recent study showed that Rht8 is located in a genetic interval of 1.29 cM (DG279-DG371), which is 1.95 cM away from Xgwm261 (Gasperini et al. 2012). Thus, the development of a robust marker tightly linked to Rht8 is the priority for marker-assisted selection.
Over the past two decades, the successful application of quantitative-genetic methodology has facilitated identification of numerous QTL for PHT in wheat (Borner et al. 2002;Cadalen et al. 1998;Wurschum et al. 2015Wurschum et al. , 2017Yu et al. 2014;Zanke et al. 2014). In our recent study, two QTL (QPht.cau-2D.1 and QPht.cau-2D.2) for PHT linked in coupling phase were mapped on the short arm of chromosome 2D (Zhai et al. 2016). Since both QTL are located on chromosome 2DS, this investigation was undertaken to determine the genetic relationship between QPht.cau-2D.1 and QPht.cau-2D.2 using segregating populations and NILs derived from a residual heterozygous line. Moreover, the diagnostic marker for each QTL was developed for markerassisted selection in wheat breeding programmes. In addition, haplotype distribution of QPht.cau-2D.1 and QPht. cau-2D.2 in diverse panels of wheat accessions was also investigated.

Plant materials
Following our previous study of QTL mapping using the recombinant inbred lines (RILs) of Yumai 8679 (Y8679)/ Jing 411 (J411) (Zhai et al. 2016), SSR (simple sequence repeat) markers were further developed for linkage map construction and QTL analysis. In generation F 9 of the RIL population, a RHL (RIL171) that carried the heterozygous segment at the genetic region from SSR markers SSR-2212 to SSR-2429 was self-pollinated to produce the F 10 generation for further study (Fig. 1). The homozygotes (F 10 ) without recombinant were selected as NILs (NIL Y8679 and NIL J411) to validate the presence of the QTL. Two plants that carried heterozygous segments covering the intervals from SSR-2212 to Xwmc503 and from Xcfd53 to SSR-2429 were identified to produce segregate populations (F 11 , population I and II) for genetic analysis (Fig. 2b). Then, the non-recombinant homozygotes of each population were selected and self-pollinated to produce NIL-I (F 12 , NIL-I Y8679 and NIL-I J411 ) and NIL-II (F 12 , NIL-II Y8679 and NIL-II J411 ).

Field experiments
Populations I and II were tested in Hebei (37°56′N, 114°42′E). NIL Y8679 and NIL J411 were grown in two locations, Shanxi (36°08′N, 111°34′E) and Hebei, and NIL-I and NIL-II were grown in three locations, Beijing, Shanxi and Hebei (Supplementary Table S5). In each environment, two segregating populations were planted with a sowing rate of 20 seeds per row (1.5 m long and 0.3 m apart), while the NILs were planted 25 seeds per line. The irrigation and other management of all sites were in accordance with local standard practices.

Phenotypic evaluation and statistical analysis
Plant height (PHT), spike length (SL) and spikelet number (SN) of the individuals in populations I and II were measured before harvest. For the NILs, five plants from each line were used for phenotypic evaluation. PHT, SL, SN and length of 5 internodes from the main tillers were measured. For NILs, the mean values over three replications at each site were used for data analysis. Spikelet compactness (SC) was calculated by dividing the SN by the SL.
Statistical analysis was performed with IBM SPSS Statistics 20 (SPSS, Chicago, USA). Significance analysis was calculated using Student's t test.

SSR marker development
Microsatellite markers were designed to construct the genetic map of chromosome 2DS. The reference sequence 1 3 of wheat variety Chinese Spring (http://www.wheat genom e.org/) and Aegilops. tauschii genomic DNA contig sequence (http://aegil ops.wheat .ucdav is.edu/ATGSP /) were extracted to design new SSR markers by using BLASTN method with the sequences of markers DG279 and DG371 (Gasperini et al. 2012) and the flanking sequence of the polymorphic SNPs in our previous results (Zhai et al. 2016). The PCR system included 5 μl 2 × Taq PCR StarMix, 2 μl primer (mixture of left and right primer), 2 μl DNA template (50-100 ng/μl) and 1 μl H 2 O. The PCR protocol was performed using PCR amplification as follows: 94 °C for 5 min; 36 cycles of 94 °C denaturation for 30 s, 55-57 °C primer annealing for 30 s, and 72 °C extending for 30 s; and finally, 72 °C for 10 min. For polymorphism detection, PCR products were separated using the method of 8% non-denaturing polyacrylamide gel electrophoresis (PAGE) (Marklund et al. 1995). The primers of 17 codominant SSR markers are listed in Supplementary Table S14.

Linkage map construction and QTL analysis
JoinMap 4.1 was used for constructing genetic linkage map of chromosome 2DS with LOD value above 5. A regression mapping algorithm (Stam 1993) and Kosambi's mapping function (Kosambi 1943) were applied to determine the marker order and convert recombination into distances.
QTL mapping was determined with Windows QTL Cartographer 2.5 (Wang et al. 2006). Briefly, composite interval mapping (CIM) was performed using model 6 with forward and backward regression, five markers, and a 1-cM scanning window as cofactors. Empirical threshold LOD scores estimated with 1000 permutations at P ≤ 0.05 detected QTL with overlapping confidence intervals (± 2 LOD away from the peaks of likelihood ratios) were considered equivalent and named as suggested by McIntosh et al. (2017). 1 3

Histological analysis
The medial sections of peduncle at flowering stage were collected from NIL-I Y8679 , NIL-I J411, NIL-II Y8679 and NIL-II J411 plants, then fixed in FAA solution (50% (v/v) ethanol, 5% (v/v) glacial acetic acid and 4% (v/v) formaldehyde) and subjected to vacuum pumping for 30 min. Next, the internodes were dehydrated in a series of ethanol solutions (75% (v/v) ethanol and anhydrous ethanol) and destained in a series of xylene solutions (3:1 ethanol: xylene, 1:1 ethanol: xylene, 1:3 ethanol: xylene, and pure xylene). The internodes were soaked in each ethanol and xylene solution for 1 h and then embedded in paraffin . Tissue sections were cut into 4-μm-thick slices, fixed on a glass slide, and stained with 1% sarranine and 0.5% fast green (G1031, http://www.servi cebio .cn/).

Sequence analysis of candidate genes
Genomic DNA for Y8679 and J411 was used to build pairedend sequencing libraries with insert sizes of approximately 500 bp, according to vendor-provided instructions (Illumina). An average 5 × coverage of the assembled genome, with 150-bp paired-end reads for each accession, was generated with the Illumina HiSeq X Ten platform. All the sequence reads for each parent were mapped to the newly updated genome of Chinese Spring (RefSeq v1.0) in the Burrows-Wheeler Aligner program (BWA, ver. 0.7.15) with default parameters (Li and Durbin 2009). SNPs and InDels are identified by the HaplotypeCaller module. The sequence of each candidate gene including intron, exon, 3′-UTR region and 2-kb sequence upstream of translation start codon was used as query for analysis.

Saturation of genetic Linkage map and QTL mapping of QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2
To saturate the genetic linkage map for plant height (PHT) and spike length (SL) on the short arm of chromosome 2D, 632 SSR markers were developed, among which 17 markers exhibited polymorphism between parents Y8679 and J411 (Supplementary Table S14). The collinearity of these markers with Ae. tauschii markers was analysed, and the physical positions on the reference sequence of Chinese Spring are shown in Fig. 1. These 17 polymorphic SSR markers were used to genotype the 191 RIL lines for QTL analysis. The resulting genetic linkage map of chromosome 2D consisted of 75 markers spanning 70.11 cM in length (Supplementary Table S13). Consistent with our previous finding, two stable QTL in coupling phase controlling PHT (QPht.cau-2D.1 and QPht.cau-2D.2) were detected on chromosome 2DS by reanalysing the phenotypic data from 191 RIL lines in 9 individual environments and 1 combined analysis (BLUP) (Zhai et al. 2016). QPht.cau-2D.1 was located between SNP markers BS00022234_51 and BobWhite_rep_c63957_1472, which explained 4.39-11.94% of the phenotypic variation. QPht.cau-2D.2 was mapped to the genomic interval between SSR markers SSR-2062 and Xgwm484, which explained 4.12-12.96% of the phenotypic variations under different environments ( Fig. 2a, Table 1). In the same positions of QTL for PHT, two major QTL for SL (QSl.cau-2D.1 and QSl.cau-2D.2) were also detected under all environments, which explained 30.94-40.63 and 31.31-41.95% of the phenotypic variations, respectively. All increasing alleles for PHT and SL came from J411 (Table 1). For simplification, hereafter, the two genomic regions covering PHT and SL were designated as QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2, respectively.

Development of near isogenic lines harbouring QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2
To further evaluate the genetic effects of QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2 on PHT and SL, one line (RIL171) from the F 9 generation of the RIL population was selected for investigation because of its residual heterozygosity at the target QTL genomic regions. First, one pair of NILs (NIL Y8679 and NIL J411 ) from the self-pollinated progeny of RIL171 (F 10 ) harbouring QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2 were genotyped using the wheat 660 K SNP array (https ://wheat .pw.usda.gov/ggpag es/topic s/Wheat 660_SNP_array _devel oped_by_CAAS. pdf). Of 630517 SNP markers with detected signal, only 434 (0.07%) exhibited polymorphism between NIL Y8679 and NIL J411 , indicating that the genetic backgrounds of these two NILs had very high similarity (Supplementary  Table S6). Notably, based on the information of SNP markers with genomic position, 154 of 434 polymorphic SNP markers (35.48%) were located on genomic  Table S7). Second, the progenies of the above NILs (F 11 ) were used for phenotypic analysis. As shown in Table 2, significant differences were observed for SL, length of peduncle (PED) and length of 1st internode under peduncle (1IL) between NIL Y8679 and NIL J411 (P < 0.001) under different environments. By contrast, the differences in length of the second internode under peduncle (2IL), length of the third internode under peduncle (3IL) and length of the fourth internode under peduncle (4IL) were only detected under a specific environment. In addition, no difference was observed for spikelet number (SN). Consequently, PHT of NIL J411 was 12.62 and 15.08% (P < 0.001) higher than that of NIL Y8679 under two different environments. Correspondingly, spikelet compactness (SC) of NIL Y8679 was significantly (P < 0.001) higher than that of NIL J411 ( Table 2).  (Fig. 2b). The single marker analysis for PHT, SL and SN was performed using the derived segregating populations I and II (F 11 ). As expected, polymorphic SSR markers in each population were significantly (P < 0.001) associated with PHT and SL (Table 3). To further test the pleiotropic effects of QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2, two sets of NILs (F 12 , NIL-I and NIL-II) (Fig. 3) were developed from the two F 11 populations. For PHT, SL and PED, NIL-I J411 and NIL-II J411 (genotype same with J411) had significantly higher values than those of   NIL-I Y8679 and NIL-II Y8679 (genotype same with Y8679) under all environments, with average differences of 9.97% and 12.62, 16.25 and 19.56%, and 13.43% and 18.11%, respectively (Fig. 2c, Supplementary Table S9). In addition, the 1IL and 4IL of NIL-II J411 were also much higher (P < 0.001) than those of NIL-II Y8679 under all environments (Supplementary Table S9).

Effects of QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2 on culm cell length
In wheat, internode elongation is caused by cell division and/or cell elongation (Chowdhry and Allan 1966;Gasperini et al. 2012). To investigate the underlying physiological bases of two QTL, longitudinal cell length in the median section of peduncle at the flowering stage in NILs of each QTL was measured. For QPht/Sl.cau-2D.1, NIL-I Y8679 showed no difference in cells length compared to NIL-I J411 (Fig. 6a, b and e). By contrast, there was significant difference in cell length between NIL-II Y8679 and NIL-II J411 of QPht/Sl.cau-2D.2. The cell length of NIL-II J411 with increasing allele was significantly longer than that of NIL-II Y8679 with decreasing allele for PHT (Fig. 6c, d, and f, P < 0.001).
Collectively, our data exhibited that the alleles of STARP-2001-Y and SSR-2433-Y were significantly associated with the height-reduced alleles of QPht/Sl.cau-2D.1 and QPht/ Sl.cau-2D.2, respectively. To further explore implications of our findings on wheat breeding, the haplotype of 724 common wheat varieties/ lines from China was first analysed using diagnostic markers  Table S11). The frequency of SSR-2433-Y allele in different zones was in the order of II (86.67%) > X (78.05%) > IV (51.22%) > III (48.15%) > I (46.51%) > VIII (33.33%) (Fig. 5b, Supplementary Table S11). When comparing the alleles of STARP-2001-Y and SSR-2433-Y in the same zone, the frequency of SSR-2433-Y was much higher than that of STARP-2001-Y in zones I, II, III, IV and X, especially in the major wheat production zone X (Fig. 5a, b, Supplementary Table S11). Furthermore, 442 common wheat varieties/lines from other countries were tested by the two diagnostic markers. The STARP-2001-Y allele was present in 48 of 442 accessions (10.86%), spreading in five continents, including Asia, Europe, North America, South America and Oceania. The SSR-2433-Y allele was detected in 63 of 442 accessions (14.25%), which are derived from Asia, Europe, Africa and North America (Fig. 5c, d, Supplementary Table S12).
To trace the origin of dwarf genes in the QTL intervals of south-western winter wheat region; V, southern winter wheat region; VI, north-eastern spring wheat region; VII, northern spring wheat region; VIII, north-western spring wheat region; IX, Qinghai-Tibet spring-winter wheat region; X, Xinjiang winter-spring wheat region Table S2). In addition, a diverse panel of 86 accessions of Aegilops tauschii, the D-genome progenitor of T. aestivum, was also analysed. The results exhibited that the STARP-2001-Y allele was not detected in 86 accessions of Aegilops tauschii, but the SSR-2433-Y allele presented in 39 of 86 accessions (45.35%) (Supplementary Table S4). NS, no significance P = 0.05; ***, t test P < 0.001. Bars represent the standard deviation

Sequence variations in candidate genes for QPht/ Sl.cau-2D.1 and Rht8
To identify the candidate genes for QPht/Sl.cau-2D.1 and Rht8, we analysed the gene models in the mapping interval of the Chinese Spring RefSeq v.1.0 sequence. In rice, WRKY transcription factor Dlf1 and gibberellin-inactivating 2-betadioxygenase gene played important roles in the regulation of PHT (Cai et al. 2014;Lo et al. 2008). Interestingly, of 38 predicted genes in the 2.5-Mb interval of QPht/Sl.cau-2D.1 (BS00022234_51-BobWhite_rep_c63957_1472), TraesC-S2D01G051000 and TraesCS2D01G051500 encoded two WRKY transcription factors, and TraesCS2D01G049700 encoded gibberellin 2-beta-dioxygenase, which may be the candidate genes (Supplementary Table S10). DNA sequence analysis showed that there were 5 SNPs and 3 InDels between TraesCS2D01G051500 of parental lines Y8679 and J411, including 3 SNPs in exon 1, 1 InDel in 3′-UTR region, 1 InDel in intron 2 and 2 SNPs and 1 InDel in the upstream region of translation start codon (Fig. 8, Supplementary  Table S15). However, no sequence difference between Y8679 and J411 was detected for TraesCS2D01G049700 and TraesCS2D01G051000.
The well-known reduced height genes Rht-B1 and Rht-D1 in wheat and several plant height related genes in rice (DLT, SMOS1/SHB and SLR1) encoded transcriptional factors belong to the GRAS family (De Vleesschauwer et al. 2016;Qiao et al. 2017;Tong et al. 2009). In rice, OsCCC1 was involved in cell elongation by regulating ion (Cl − , K + , and Na + ) homoeostasis to maintain cellular osmotic potential, which affected PHT in turn (Chen et al. 2016). Based on the new reference sequence of Chinese Spring (RefSeq v.1.0), the mapped region of Rht8 between SSR-2062 and Xgwm484 contained 419 predicted genes, among which TraesCS2D01G055700 and TraesCS2D01G058700 encoded two GRAS transcription factors, and TraesC-S2D01G059300 encoded a cation-chloride cotransporter, which may be the candidate genes (Supplementary  Table S10). DNA sequence analysis revealed that there were 38 SNPs and 8 InDels between TraesCS2D01G055700 of Y8679 and J411, 3 SNPs and 3 InDels in the exon, and 35 SNPs and 5 InDels in the upstream region of translation start codon. For TraesCS2D01G058700, only 2 SNP variants were found, which were located in intron 4 and the upstream region of translation start codon, respectively (Fig. 8, Supplementary Table S15). By contrast, the sequence of TraesC-S2D01G095300 in Y8679 was identical to that in J411.

Two QTL for PHT/SL linked in coupling phase on chromosomes 2DS
Neighbouring QTL that are linked in coupling phase are commonly observed in primary QTL analysis (Fan et al. 2017;Wu et al. 2015). To date, many studies have tried to dissect QTL in coupling phase using NILs or residual heterozygous lines and found that coupling QTL were partially attributed to tightly linked independent QTL (Chemayek  Shen et al. 2011). For example, Wu et al. dissected two tightly linked QTL for PHT (qPH3) and yield per plant (qYD3) by using two rice near isogenic populations (Wu et al. 2015). In wheat, tight repulsion linkage between Sr36 and Sr39 was revealed by genetic, cytogenetic and molecular analyses (Chemayek et al. 2017). Recently,using 191 RILs derived from Y8679 and J411,2) controlling PHT and SL were mapped on the short arm of chromosome 2D. However, it is difficult to conclude whether QPht/Sl.cau-2D.1 is a shadow or genuine QTL because it is only 7 cM away from QPht/Sl.cau-2D.2 (Zhai et al. 2016). Here, we dissected these two QTL (QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2) using segregating populations and NILs derived from a residual heterozygous line (RIL171). They were linked in coupling phase on chromosome 2DS, with increased alleles from the same parent (J411). QPht/Sl.cau-2D.1 was located between SNP markers BS00022234_51 and Bob-White_rep_c63957_1472, and QPht/Sl.cau-2D.2 was located between SSR markers SSR-2062 and Xgwm484. The results of NILs indicated that the genetic effect of these two QTL is similar, which could reduce plant height by 4. 79-14.32 and 9.49-14.43 cm under different environments (Table 2). Our present study provides a new example of QTL dissection in wheat, but the underlying molecular basis of two QTL (QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2) is still an area for further elucidation.

Robust diagnostic SSR marker for QPht/Sl.cau-2D.2
Numerous studies exhibited that there were major QTL/ genes on chromosome 2DS, such as the well-known and widely used dwarf gene Rht8 (Chebotar et al. 2013;Griffiths et al. 2012;Zanke et al. 2014). Recently, using a fineresolution mapping approach, the Rht8 genetic interval has been reduced from 20.5 to 1.29 cM, flanking by DG279 and DG371 (Gasperini et al. 2012). Comparative analysis revealed that QPht/Sl.cau-2D.2 of the present study is 1 3 located on the same genetic interval of Rht8 (Gasperini et al. 2012). Moreover, consistent with the effect of Rht8 on culm cell elongation, significant difference in cell length was also observed between NILs of QPht/Sl.cau-2D.2 (Fig. 6c, d). This, we speculated that QPht/Sl.cau-2D.2 may be the wellcharacterized Rht8 gene.
Due to the widespread use of Rht8 in wheat breeding, assessing the distribution of height-reducing alleles at the Rht8 is of interest for breeding purposes. Following the identification of Xgwm261, a closely linked marker to Rht8 with the 192-bp allele corresponding to a height-reducing phenotype, the gwm261 192bp allele has been taken as diagnostic marker for Rht8 (Ahmad and Sorrells 2002;Bai et al. 2004;Chebotar et al. 2001;Liu et al. 2005;Zhang et al. 2006). However, several instances were found in which the 192-bp allele was not associated with a height-reducing phenotype (Ellis et al. 2007). For example, a source variety of the Green Revolution semi-dwarfing genes, Norin10, carrying a 192-bp allele of Xgwm261, was independent of the height reduction effect of Rht8 (Ellis et al. 2007). Consistent with this, our data revealed that Xgwm261 was located in the genetic interval of QPht/Sl.cau-2D.1, instead of QPht/Sl.cau-2D.2. Thus, we developed a codominant SSR marker (SSR-2433) that was tightly linked to QPht/Sl.cau-2D.2/Rht8. Moreover, the SSR-2433 locus only has two allelic variants in the diversity panel of wheat accessions, which may be an ideal diagnostic marker for the QPht/Sl.cau-2D.2/Rht8 gene.

A novel QTL for PHT/SL tightly linked to dwarf gene Rht8
Previous studies exhibited that numerous QTL/genes controlling PHT were detected in wheat (Borner et al. 2002;Gao et al. 2015a, b;Peng et al. 2003;Quarrie et al. 2005;Singh et al. 2016;Tian et al. 2017). Specifically, 24 dwarfing genes (Rht1-Rht24) have been catalogued in wheat (McIntosh et al. 2017). However, quite a few genes for reduced stature have been used in wheat breeding, because most showed strongly negative effects on grain yield (Chapman et al. 2007;Zhang et al. 2013a). Thus, it is necessary to explore and utilize new QTL/genes controlling PHT. For example, the new reduced PHT gene Rht24 was important and extensively used in wheat breeding programmes (Wurschum et al. 2017). In the present and our previous studies, we identified a novel QTL for PHT neighbouring to Rht8, QPht/Sl.cau-2D.1, by the following evidences: (1) QPht/ Sl.cau-2D.1 was mapped to a position distal to Rht8 on the short arm of chromosome 2D (Fig. 2a, Table 1); (2) QPht/ Sl.cau-2D.1 had no significant effect on culm cell elongation, which is obviously different from that of Rht8 (Fig. 6).
Common wheat has an allohexaploid genome structure, which was hybrid by T. turgidum (AABB) and Aegilops tauschii (DD) 0.43 million years ago (International Wheat Genome Sequencing 2014; Petersen et al. 2006) To investigate the origin of the novel QPht/Sl.cau-2D.1 during wheat evolution, we analysed the allelic variation in the diagnostic marker STARP-2001 in a diverse panel of 86 accessions of Aegilops tauschii., but the STARP-2001-Y allele was not detected, which is significantly associated with the heightreduced allele of QPht/Sl.cau-2D.1. Thus, it seems that the novel height-reduced allele of QPht/Sl.cau-2D.1 was generated after the formation of allohexaploid wheat. Notably, haplotype analysis revealed that the height-reduced allele of QPht/Sl.cau-2D.1 has not been widely used in wheat breeding compared to Rht8. Thus, QPht/Sl.cau-2D.1 may be a favourable QTL in the genetic improvement in PHT in wheat, but the effect of different alleles of QPht/Sl.cau-2D.1 on grain yield and their potential utilization needs further investigation.

Candidate genes of Rht8 and QPht/Sl.cau-2D.1
To date, several genes controlled PHT have been cloned in wheat, such as Rht-B1, Rht-D1, TaSTE and GA2oxA9 (Rht18) (Ford et al. 2018;Gasperini et al. 2012;Peng et al. 1999;Zhang et al. 2013b). Specifically, Rht-B1 and Rht-D1 encoded transcriptional factors belong to the GRAS family (Peng et al. 1999;Sun 2010). Different alleles of Rht-B1 and Rht-D1 have been also identified, which produce dwarfs with a broad range of plant height (Pearce et al. 2011). Semi-dwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region, whereas the severe dwarfism of Rht-B1c gene is caused by an intragenic insertion of 90 bp within the highly conserved amino-terminal DELLA domain (Pearce et al. 2011). In the present study, we found that two of the three candidate genes for Rht8 (TraesCS2D01G055700 and TraesCS2D01G058700) encoded GRAS transcription factors. Notably, a 35-bp deletion in the coding region of TraesCS2D01G055700 was found in parental line J411 as compared to Y8679, which resulted in frame shift for translation. Moreover, another two SNP variations between Y8679 and J411 leaded to amino acid substitutions. In addition, 35 SNPs and 5 InDels in the upstream region of translation start codon were detected (Fig. 8, Supplementary Table S15). Collectively, we proposed that the role of TraesCS2D01G055700 in plant height merit for further investigation.
In rice, Dlf1, a WRKY transcription factor, acts as a transactivator to downregulate Ehd2/RID1/OsId1 in the signal transduction pathway of flowering and plays an important role in the regulation of PHT (Cai et al. 2014). Of the three candidate genes for QPht/Sl.cau-2D.1, TraesCS2D01G051000 and TraesCS2D01G051500 encoded two WRKY transcription factors, but only TraesCS2D01G051500 has sequence variations between Y8679 and J411, including one SNP (A/C) in exon 1 that leaded to the substitution of 1 amino acid (I/L) and 2 SNPs and 1 InDel in the upstream region of translation start codon (Fig. 8, Supplementary Table S15). Therefore, detailed studies would be necessary to analyse the effect of these variations in the function of TraesCS2D01G051500 and plant height.
Epigenetics regulate gene expression without effects on gene sequences, which was involved in diverse aspects of plant development (Bender 2002;Heer et al. 2018;Paszkowski and Mittelsten Scheid 1998). The repression of FLC expression in Arabidopsis by vernalization is one of the well-studied examples of the epigenetic regulation of a gene in plants (Bastow et al. 2004;Questa et al. 2016). Recently, Zhang et al.(2012) identify a DNA hypomethylation epi-allele in rice and demonstrate that repression of FIE1 via DNA methylation and H3K9me2 is essential for plant height. Thereby, although the sequences of TraesC-S2D01G049700, TraesCS2D01G051000 and TraesC-S2D01G095300 in Y8679 were identical to those in J411, these genes may also be the candidate genes and further experiment is needed to validate which one is the real gene for Rht8 and QPht/Sl.cau-2D.1.
In conclusion, two QTL with pleiotropic effects on plant height and spike length linked in coupling phase on chromosome 2DS in common wheat were separated, and the diagnostic marker tightly linked with each QTL was developed, which corresponded to a novel QTL QPht/ Sl.cau-2D.1 and well-known Rht8 gene, respectively. Of six candidate genes for two QTL, three have sequence variations between parental lines Y8679 and J411. Remarkably, the frequency of the height-reduced allele of QPht/ Sl.cau-2D.1 in diverse panels of wheat accessions is much lower than that of QPht/Sl.cau-2D.2. Taken together, these data provide a new example of QTL dissection, and QPht/Sl.cau-2D.1 may be an attractive target for genetic improvement in wheat breeding.
Author contribution statement ZN and AZ conceived the project; LC, ZC and RB carried out experiments; XC and HZ participated in field trials; LC, ZC and XC performed marker development of the QTL region of interest; LC and ZH developed the segregation populations and near isogenic lines; LC, ZC and RB performed phenotyping of the segregation populations and the near isogenic lines; QS, HP, YY, ZH, MX and WG assisted in revising the manuscript; LC analysed experimental results and wrote the manuscript.


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Diversity of yeast strains isolated from the “magnan’’ a traditional ferment used for the production of “attiéké” a cassava food in Côte d'Ivoire

Objective: This study aimed to identify the yeasts in the traditional fermented cassava root called ''magnan'', used for fermentation of cassava dough for “attieke: a fermented cassava food product’’ production in Cote d'Ivoire. Methodology and results: ‘magnan'' is a fermented cassava root used as a traditional starter and “attieke’’ is a fermented cassava food product. This study involved 99 samples of ''magnan'' taken from different production areas in eleven (11) communes in the District of Abidjan (Abobo, Adjame, Anyama, Attecoube, Bingerville, Cocody, Koumassi, Marcory, Port-Bouet, Songon and Yopougon). Samples of ‘’magnan’’ were collected and carried to the Biotechnology Laboratory of the Felix Houphouet-Boigny University, during sixmonth (May - November 2017) and treated separately on MYPG medium (malt extract, yeast extract, peptone, glucose, and chloramphenicol). One hundred and six (106) pure yeast cultures were identified at genus and species level by MALDI-TOF mass spectrometry at the bio-bank laboratory of the Pasteur Institute of Cote d’Ivoire. A total of 83 yeast isolates were clearly identified (78.30 %) and showed five genus: Candida, Saccharomyces, Trichosporon, Kloeckera, and Millerozyma. The major species included C. krusei (30.12 %), C. tropicalis (28.92 %), and S. cerevisiae (20.50 %) followed by a low proportion of C. kefyr (6.02 %), C. guilliermondii (3.61 %), T. asahii (3.61 %), K. apis (2.41 %), C. pararugosa (1.20 %), M. farinosa (1.20 %) and C. albicans (1.20 %). Conclusion and application of the results: This study showed the traditional cassava ferment ''magnan'' used for the fermentation of cassava dough for the production of “attieke ’’ on the sampled sites was colonized by several yeasts genera including Candida, Saccharomyces, Trichosporon, Kloeckera and Millerozyma in varying proportions. The frequently isolated species were composed by C. krusei, C. tropicalis and S. cerevisiae. The results obtained will constitute a database on the diversity of yeasts for the selection of starter strains for producing of “attieke ’’. In perspective, the techno-functional properties (enzyme production: amylase, cellulase, pectinase, phytase, tannase and β-glucosidase) of each strain will be determined for their impact on the fermenting process of the cassava dough. And the starter strains will be used by producers to optimize and standardize the quality of “attieke ’’.Keywords: yeast strains, traditional ferment, “attieke ’’, ‘’magnan’’

INTRODUCTION "attiéké '' is a fermented cassava semolina cooked with steam from water which has a slightly acidic taste. It is a food that is much appreciated by many people in Côte d'Ivoire, in the West African sub region and elsewhere in the world (Europe, USA) where it is exported through the Africans in diaspora (Bouatenin et al., 2016;Assanvo et al., 2006). "attiéké '' has now become a profitable dish, produced on a large scale because of the ever-increasing demand and then, its commercialization generates enough gain for both producers and the Ivorian economy (Krabi, 2016). In Côte d'Ivoire, "attiéké '' is mainly produced by housewives and has several types depending on the main ethnic groups producing it. According to Assanvo et al. (2000), Djeni et al. (2015), there are in Côte d'Ivoire: "attiéké Ebrié'', "attiéké Adjoukrou'', "attiéké Alladjan'' and "attiéké Avikam''. However, despite its popularity, the preparation of "attiéké '' remains informal and is based on empirical knowledge passed down from generation to generation. Its production requires a preliminary and essential stage of fermentation of the cassava dough from a traditional cassava inoculum commonly called "magnan" which is obtained naturally after 2-3 days of spontaneous fermentation of cassava roots (about 1/10 of the dough to ferment) previously boiled or braised. Therefore, regardless of the type of "attiéké '' resulting from the fermentation of the cassava dough, there is a great variability with the final organoleptic quality in the same producer or from one producer to another even of an ethnic group to another (Djeni et al., 2008). This fluctuation in the quality, can probably be attributed to the variability of the involved fermentative germs (Oyewole and Sanni, 1995 ). Thus, it would be wise to effectively control the fermentation process which is a very decisive step in obtaining quality products. This control necessarily implies a precise identification of the fermentative germs implicated. According to previous work, it has been reported that the major microorganisms of the fermentation of '' magnan '' for the production of "attiéké '' consisted of yeasts, lactic acid bacteria, and Bacillus . However, there is insufficient data on yeast and its diversity in this fermentative process for the production of "attiéké '' for comparisons, despite the fact that yeast is a major microorganism in the production of an "attiéké '' of good organoleptic quality. In Africa, Asia and Brazil, yeast(s) have been reported to play an important role in the fermentation of cassava-based foods such as: ''tapioka, lafun and fufu...'', several yeast species have been isolated from these fermented products (Schwan et al., 2007). For the production of ''fufu'', for example, six different yeast species, Pichia saitoi, Pichia anomala, Candida krusei, Candida tropicalis, Zygosaccharomyces bailii and Saccharomyces cerevisiae have been identified (Oyewole, 2001). In the case of "attiéké '', only the species of C. tropicalis, C. krusei and S. cerevisiae have been identified . Thus, information on the yeast diversity of ''magnan'' could be an important database for the selection of the best starter-type strains for the production of "attiéké''. The aim of this work is therefore to isolate and identify the different yeast species involved in the fermentation of the ''magnan'' for the production of "attiéké '' in Côte d'Ivoire, especially in eleven municipalities of the District of Abidjan (Abobo, Adjamé, Anyama, Attécoubé, Bingerville, Cocody, Koumassi, Marcory, Port-Bouet, Songon and Yopougon). The choice of these municipalities was related to their importance in the production and marketing of attiéké in Côte d'Ivoire.
MATERIAL AND METHODS Collection of samples: Sampling was carried out in eleven (11) municipalities in the District of Abidjan (Abobo, Adjamé, Anyama, Attécoubé, Bingerville, Cocody, Koumassi, Marcory, Port-Bouet, Songon and Yopougon) (Figure 1). The criteria for choosing these municipalities was defined in the introduction (Krabi, 2016). In each of these municipalities, three major producers were randomly selected. At each of them, three pieces of boiled and fermented cassava were taken with a stainless steel spoon and placed in sterile Stomacher bags, and introduced in a cooler and taken to the laboratory for yeasts isolation. Sampling took place over a six-month period between May and November 2017. Isolation, purification and storage of yeasts: Once at the laboratory, the samples were treated separately by municipality. The surfaces of the traditional ''magnan'' ferments were scraped with a spatula and mixed, then divided into three aliquots from which the stock solutions were prepared. For the preparation of the stock solutions, 10 g of each aliquot were homogenized in 90 mL of buffered peptone water (EPT, AES Laboratory, France) and incubated at 30 °C for 18 hours to revitalize the microorganisms of the traditional ferment. After incubation, MYPG agars with 100 mg/L chloramphenicol (chloramphenicol was used to inhibit the growth of bacterial flora) were seeded by striae and incubated at 30°C for 48 hours. Colonies identified as yeasts by their macroscopic aspects on agar medium and their microscopic observations in the fresh state were purified by striae on MYPG agar. One hundred and six (106) purified isolates were obtained and stored at (-80°C) in MYPG broth with 20% (v/v) of glycerol added (Nielsen et al., 2005). Identification of yeasts by MALDI-TOF mass spectrometry: The identification of yeasts at the genus and species level by borrowing mass of ribosomal proteins was done by the MALDI-TOF (Vitek MS BioMerieux) mass spectrometer, which is a spectrometer using a matrix-assisted laser ionization source (MALDI = Matrix-Assisted Laser Desorption/Ionization) and a time-of-flight analyzer (TOF = Time-Of-Flight). The separation of molecules by this technique is softer than with other methods. It allows the ionization of large, low-volatility and heat-sensitive molecules without degrading them. MALDI-TOF identification is done in three steps: sample preparation followed by sample analysis and data processing (Karas et al., 1985).
For samples preparation, the 106 strains of yeasts isolated from the cassava ferment ''magnan'' were cultured in MYPG agar medium for 48 hours at 30 °C. Afterwards, a colony of the calibrating strain, Escherichia coli ATCC 8739, was put on the MALDI-TOF plate with 1 µL of CHCA matrix (acid α-cyano-4-hydroxycinnamic, BioMerieux SA, ref 411071). Then, using a sterile oese calibrated at 1 µL, a colony of yeast to be tested was collected and put on the target wells. The sample was tested in duplicate. On both deposits, 0.5 µL of 25% formic acid (vitek MS-FA, BioMerieux SA, ref 411072) was added. After air drying, 1 µL of CHCA matrix was put on each spot and dried again. Subsequently, the deposition plate was introduced into the Vitek MS for sample analysis after transferring the data from the Prep Station, which is a module consisting of a computer and an optical scanner used to introduce the sample data and their location on the slide to the Vitek MS. Once in the device, the target or sample to be tested was subjected to laser beams to allow the matrix to be vaporized and the molecules in the sample to be ionized. The ionized molecules thus formed migrate to the detector and were separated according to their charge and molecular weight. Then the detector sends the recorded information to the analyzer for the data processing in the form of a spectrum where each peak corresponds to a type of molecule. And the device integrates the peaks recorded in comparison with the database to search for the identification of the corresponding germ. The interpretation of the results involved two important parameters: the percentage confidence degree and the confidence level displayed by different colours. First, a confidence degree between 99.9 and 60% with a green colour indicates good identification and second, a confidence degree of less than 60% with an orange coloration corresponds to a low probability of identification. And an absence of identification is manifested by non-percentage with a red coloration.

RESULTS
Identification by MALDI-TOF mass spectrometry allowed a formal identification of 83 strains out of the 106 strains submitted for the analysis with confidence degree of 99.9 %. This represented an identification rate of 78.30% against a lack of identification of 23 strains with a rate of 21.70 %. Figure 2 shows an example of a ribosomal mass borrowing profile using MALDI-TOF mass spectrometry of a Saccharomyces cerevisiae species.  (Table 1).

DISCUSSION
This study results showed that the traditional cassava ferment ''magnan'' used for fermentation of cassava dough for "attiéké '' production was colonized by a variety of fermentative and pathogenic yeasts, such as Candida albicans. This variability of species could be explained by the lack of hygiene in which traditional fermentations took place. The identification showed that the predominant genera are Candida (72.28 %) and Saccharomyces (20.50 %) followed by a small proportion of the species Trichosporon asahii (3%), Kloeckera apis (2.41 %) and Millerozyma farinosa (1.20 %). With Candida, the majority of species were C. krusei (30.12 %) and C. tropicalis (28.92 %). These results were in agreement with those of Coulin et al. (2006), who identified in the case of "attiéké '' the strains of C. tropicalis, C. krusei and S. cerevisiae. Also, the studies of Assanvo et al. (2006) indicated that yeasts (Candida sp.: 68%, C. valida : 9%, C. homii : 7%, C. krusei : 2%, Kloeckera japonica : 7% and Saccharomyces cerevisiae : 7%) constituted a significant part of the dominant microflora of traditional cassava starters ''Adjoukrou'' in the three sampled villages. In fact, during the submerged fermentation of cassava, only yeasts and lactic acid bacteria survive under the strong acidic conditions of the fermentation medium (Oboh and Akindahunsi, 2003). According to Padonou et al. (2009), yeasts grow with lactic acid bacteria from beginning to end in fermented cassava dough used to produce the ''lafun''. In addition, some yeasts, including Saccharomyces cerevisiae, Zygosaccharomyces, Candida, Pichia, Kloeckera, Kluyveromyces, Endomycopsis, Lipomyces, etc. are known for their ability to synthesize enzymes such as: amylase, cellulase, pectinase and even the βglucosidase (Blanco et al., 1999;Hasper et al., 2002;Tansel and Cengiz, 2013). Thus, considering the rigidity of the tissue of cassava and its toxicity, we could say that our strains isolated from the traditional ferment ''magnan'' would probably contribute to the softening of cassava and its detoxification in order to provide "attiéké '' with a smooth and soft texture and a pleasant taste sought by consumers as demonstrated by Obilie et al. (2003) in Ghana as part of the Ghanaian "attiéké ''. However, this preliminary study, which consisted in identifying the yeast species present in the ''magnan'', is more objective and will later aim to characterize the techno-functional properties of each of the isolated strains except the pathogenic ones in order to formulate yeast starters for the fermentation of cassava dough.

CONCLUSION
At the end of this study, it appears that the traditional cassava ferment ''magnan'' used for the fermentation of cassava dough used to make "attiéké '' in Côte d'Ivoire, particularly in the Abidjan district, was colonized by yeasts of the genera Candida, Saccharomyces, Trichosporon, Kloeckera and Millerozyma. C. krusei, C. tropicalis and S. cerevisiae were the major species. The results of this study will provide an important database on the diversity of ''magnan'' yeasts for the purpose of selecting better starter yeast strains for the production of an "attiéké '' of good quality. In perspective, it will be necessary to evaluate the capacity of each strain to participate or not in the fermentation process of cassava dough in order to provide producer groups in the attiéké with efficient strains for the production of an attiéké of improved quality appreciated by consumers.


extfieldsofstudy: Chemistry Medicine

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Utilization of Wheat Bran Acid Hydrolysate by Rhodotorula mucilaginosa Y-MG1 for Microbial Lipid Production as Feedstock for Biodiesel Synthesis

The lignocellulosic hydrolysate was used as the fermentation feedstock of Rhodotorula mucilaginosa Y-MG1 for the production of microbial lipids as the potential raw material for biodiesel synthesis. On synthetic media and under nitrogen-limiting condition, the Y-MG1 strain produces 2.13 g/L of lipids corresponding to 32.7% of lipid content. This strain was able to assimilate a wide range of substrates, especially C5 and C6 sugars as well as glycerol and sucrose. Fatty acid composition shows a divergence depending on the nature of used carbon source with a predominance of oleic acid or linoleic acid. An effective hydrolysis process, based on diluted acid treatment, was established for providing the maximum of fermentable sugars from different characterized lignocellulosic wastes. The highest yield of reducing sugars (56.6 g/L) could be achieved when wheat bran was used as the raw material. Hydrolysate detoxification step was not required in this study since the Y-MG1 strain was shown to grow and produce lipids in the presence of inhibitors and without the addition of external elements. Operating by controlled fed-batch fermentation yielded a dry biomass and oil yield of up to 11 g/L and 38.7% (w/w), respectively. The relative fatty acid composition showed the presence of increased levels of monounsaturated (66.8%) and saturated (23.4%) fatty acids in lipids of Y-MG1 grown on wheat bran. The predictive determination of biodiesel properties suggests that this oil may effectively be used for biodiesel production.

Introduction
e search of new alternatives for fossil fuels has become necessary, especially after the worldwide energy crisis. Biodiesel, one of the promising forms of bioenergy, has attracted attention in recent years for its nontoxic, biodegradable, renewable, and ecologic properties [1]. Biodiesel is produced mainly by the transesterification of vegetable oils, whereas vegetable oil production presents many constraints related to the dependence on climatic conditions, seasons, availability of arable land, and manpower. Microbial oil, known as single cell oil (SCO), is one of the most promising renewable oils which can replace the vegetable oils since they have a similar fatty acid composition [2]. Microbial lipids are produced by oleaginous microorganisms that synthetize and accumulate lipids more than 20% (w/w) of their total dry biomass weight and can reach up to 70% in specific conditions [3]. Under nitrogen-limiting conditions, the excess of carbon sources (such as glucose) will be used not for biomass production but rather for the synthesis of lipids, mainly triglycerides, which are used mainly for biodiesel synthesis [4]. Microbial oil is produced by several microorganisms such as yeast, microalgae, bacteria, and fungi. In fact, yeasts share several advantages for lipid production compared to other oleaginous microorganisms. ey have a short duplication times, a unicellular form, and are very easy to grow in controlled and intensive systems [2]. In addition, one of the most important characteristics of yeast resides in its ability to use a large variety of raw materials for lipid production such as industrial wastewater [5], municipal organic wastes [6], crude glycerol [7], and lignocellulosic acid hydrolysate [8,9]. e most known genera of oleaginous yeast are Candida, Cryptococcus, Lipomyces, Rhodotorula, Trichosporon, and Yarrowia [10]. However, the high cost of SCO production is the major obstacle for its larger commercialization. is limitation is mainly related to the fermentation processes and the nature of raw material used [1]. e use of inexpensive raw materials as a substrate for lipid production can help avoid this constraint. In addition, the selection of more interesting and robust yeast strains (able to grow in extreme conditions and the presence of a high level of pollutants and inhibitors) can be considered as a promising way [11]. Consequently, several studies have emerged showing the cost-effectiveness of microbial lipid production processes using low-cost fermentation substrates and more efficient yeast strains [1]. Lignocellulosic materials are the most interesting feedstock as a natural, available, and renewable resource. us, the massive amounts of lignocellulosic biomass (LB) can be converted into several high value products such as biofuels, value added fine chemicals, and bioactive molecules [12]. LB is composed of three major units, cellulose about 40-50%, hemicellulose about 20-30%, and lignin about 10-25%. Cellulose, the major component of LB, is a linear chain of D-glucose molecules while hemicellulose, the second component, is composed of repeated polymers of pentoses and hexoses [12]. Lignin has the role of glue by assembling between and around the cellulose and hemicellulose offering rigidity to the lignocellulosic structure. erefore, the pretreatment is considered a crucial step to break down the lignin linkages and to retrieve cellulose and hemicellulose which are the source of sugars that will be used by microorganisms. Actually, many biotechnological hydrolysis processes were developed to generate cost-effective fermentable sugars such as physical treatment; physicochemical treatment including steam explosion, acid-based hydrolysis, alkali-based hydrolysis [1], hydrothermal hydrolysis [13], and ozonolysis [12]; and the biological treatments based on enzymatic hydrolysis [1]. Acid hydrolysis is the most studied and used technique among various pretreatment categories. It is a fast, inexpensive, and widely used method for producing sugars from lignocellulosic biomass [1]. Among several types of acids tested until now, dilute sulfuric acid was the most commonly applied catalysts [14]. Various lignocellulose derived toxins, including furan derivatives (furfural and 5hydroxymethylfurfural (HMF)), organic acids (acetic acid, formic acid, and ferulic acid), and lignin derivatives (vanillin, 4-hydroxybenzaldehyde, guaiacol, and phenol), are generated during acid pretreatment [15]. ese toxic compounds exert an inhibitory effect on the growth of microorganisms and the cost of their removal will significantly reduce the profitability of the bioconversion process [16]. In order to increase the bioconversion rate of lignocellulosic biomass, several detoxification processes have been developed to eliminate and reduce toxic compounds derived from the hydrolysis step [17]. e overliming using the calcium hydroxide and the combination of overliming and adsorption using the activated charcoal or the amberlite resin are the most studied of chemical and physical detoxification methods [1,9]. ese techniques have many disadvantages such as the massive freshwater usage and wastewater generation, loss of the fine lignocellulose particles and fermentative sugars, and the incomplete removal of inhibitors [15]. On the other hand, the biological detoxification can be considered a promising option; however, this technique requires the addition of specific enzymes and microorganisms [15]. All of these detoxification treatments lead to increase the cost of the bioconversion process, so other alternatives must be found to enhance the lipid yield production [18]. Several previous works have studied many kinds of lignocellulosic materials with different hydrolysis processes for lipids production such as corn stalk, bagasse, corncob, and wheat straw [8,[17][18][19]. e rate of lipid production depends on the nature of LB as a supplier of sugars and the potential of microorganisms that will convert sugars into energy molecules. Using acid hydrolysis, Rhodotorula glutinis CGMCC 2.703 and R. glutinis CBS strains produce 5.5 g/L (36.4%) and 1.4 g/L (11.86%) when cultured on corncob hydrolysate and wheat straw hydrolysate [8,20].
In the present report, the newly isolated oleaginous yeast, Rhodotorula mucilaginosa Y-MG1, was evaluated for its potential to use lignocellulosic acid hydrolysate as a carbon source for microbial lipid production as potential raw material of biodiesel. Different lignocellulosic wastes were tested for fermentable sugars yields releasing after acid hydrolysis treatment. Without the need for detoxification, operating by controlled fed-batch fermentation of wheat bran hydrolysate allows increasing significantly the amount of microbial lipid produced. e current study provides an alternative way of bioconversion of lignocellulosic wastes to produce renewable energetic molecules.

Chemicals and Oleaginous Yeast
Strain. Glucose, xylose, arabinose, sucrose, lactose, and glycerol were supplied from Bio Basic Canada Inc. Furfural, 5-hydroxymethyl furfural (HMF), and 3,5-dinitrosalycilic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Agroindustrial by-products (wheat bran and sugarcane bagasse) were kindly provided by a local food processing industry (White-rose group, Sfax, Tunisia). Almond shell, date palm leaves, and Posidonia oceanica balls were collected locally. All lignocellulosic residues are conserved at 4°C until use.
e new oleaginous yeast strain Y-MG1, isolated from rotten fruit, was identified in our previous work based on its internal transcribed spacer (ITS) sequence [21]. Y-MG1 strain was identified as being Rhodotorula mucilaginosa and was submitted in the National Strains Collection of Centre of Biotechnology of Sfax, CBS, Tunisia, under the accession number CTM-30138. e ITS sequence of Y-MG1 was also submitted to GenBank under the accession number ID: KX347596.1.

Acid Hydrolysis of Lignocellulosic Residues.
Various lignocellulosic by-products were used as the raw material for fermentable sugars production. Wastes were firstly mechanically milled using electric chopper (rotary shear shredder) to obtain a particle size smaller than 2 mm and then washed with sodium hydroxide solution at low concentration (100 mM), followed by intensive washing with deionized water before drying for 48 h at 50°C [22]. Prepared lignocellulosic residues were then suspended and stirred at room temperature in 2% (v/v) dilute sulfuric acid solution at a solids loading of 10% (w/v). ereafter, mixtures were autoclaved under different programs: P1: temperature 115°C, time 60 min, and pressure 0.6 bar; P2: temperature 121°C, time 60 min, and pressure 1.1 bar; P3: temperature 133°C, time 20 min, and pressure 2.1 bar; and P4: temperature 121°C, time 20 min, and pressure 1.1 bar. Hydrolysates were then filtered on Whatman No. 1 filter paper, neutralized with a 10N NaOH solution until pH 6.0, and used for the determination of reducing sugars.

Preculture Media. Y-MG1 strain was grown in 250 mL
Erlenmeyer flasks containing 50 mL of preculture medium with the following composition: glucose, 20 g/L; bactopeptone, 10 g/L; and yeast extract, 10 g/L. e pH of the media was adjusted to 6.0, and prior to inoculation, the preculture broth was sterilized at 121°C for 20 min. e incubation of the preculture was conducted at 30°C and under 180 rpm of agitation for 18 h. Cultures for lipid production were inoculated with 5% (v/v) of the preculture media corresponding to 0.2 unit of OD at 600 nm.

Single Cell Oil Production on Synthetic Media.
Cultivation of the yeast using synthetic media for lipid production was in 0.5 L Erlenmeyer flasks containing 100 mL of culture. e synthetic medium composition was the same as described in our previous work [23] containing (in g/L) Na 2 HPO 4 2.5, KH 2 PO 4  0.0001. e initial pH of yeast cultures was adjusted to 6.0, and sugar concentration in all the experiments was set at 40 g/L providing a C/N ratio equal to 100. e carbon sources were commercial sugars such as glucose, xylose (alone or in mixtures), arabinose, sucrose, lactose, and polyol (glycerol). Culture samples were withdrawn regularly, and at the end of fermentation, the yeast cells were centrifuged at 3000 ×g for 5 min and harvested for the biomass determination and lipid extraction. All experiments were done at least thrice to ensure reproducibility.

Cultivation in Bioreactor.
Microbial lipids were produced by the oleaginous yeast Y-MG1 using fed-batch fermentation in 7.0 L stirred tank bioreactor (Infors, AG GH-4103 Bottmingen, Switzerland) with a 4.0 L of initial working volume. e bioreactor (dished bottom glassjacketed reactor) was equipped with an instrument for measurement and/or control of agitation, temperature, pH, and dissolved oxygen concentration. e cultivation temperature was fixed at 30°C and culture pH was kept constant at pH 6.0 by automatic addition of KOH (1 M) and H 3 PO 4 (1 M). Agitation and aeration rates were maintained at 400 rpm and 1.5 vvm, respectively. Silicone (426 R) antifoam was added to control foam production. e fed-batch fermentation was conducted on wheat bran acid hydrolysate containing 60 g/L of reducing sugars and 2.4 g/L of nitrogen and supplemented with a mineral solution containing MgSO 4 ·7H 2 O, 1.5 g/L; CaCl 2 ·2H 2 O, 0.2 g/L; FeSO 4 ·7H 2 O, 10 mg/L; MnSO 4 ·H 2 O, 0.07 mg/L; ZnSO 4 ·7H 2 O, 10 mg/L; and CuSO 4 , 0.1 mg/L. A volume of 160 mL of concentrated hydrolysate (using a rotary evaporator) with an initial reducing sugars concentration of 280 g/L was used to feed the culture when the residual sugars level in the medium became equal to or less than 10 g/L. During fermentation, samples were withdrawn regularly for biomass determination, sugars consumption, and lipid quantification.

Lipids Extraction.
Microbial lipids were extracted according to Dey and Maiti [10] with some modifications. Briefly, a 100 mL culture sample was centrifuged at 3000 ×g for 10 min; the cell pellet was washed twice with 50 mL of distilled water and dried for 24 h at 105°C. 300 mg of dry matter was mixed with 10 mL of HCl (4 M) and incubated at 70°C for 2 h. e acid-hydrolyzed cells were stirred in 20 mL of chloroform: methanol mixture (1 : 1) at room temperature for 3 h, followed by centrifugation at 1500 ×g for 10 min to separate the aqueous upper phase and organic lower phase. e organic phase (containing lipids) was recovered and transferred to another glass tube. Finally, 20% (v/v) of 9 g/L NaCl was added to remove any residual moisture, solvent was removed by evaporation, and the total lipid was weighed. Lipid content was expressed as the percentage (w/w) of the extract on the dry biomass.

Biochemical Composition of Lignocellulosic Residues.
e dry weight of lignocellulosic residues was determined by oven drying 1.0 g of each sample at 105°C until a constant weight. Mineral components were determined as ash after incineration of an aliquot of the material at 550°C according to the NREL protocol, LAP-001 [24]. Acid-insoluble lignin content (Klason Lignin) was determined by a modified version of the method described in TAPPI T222, for acidinsoluble lignin in wood and pulp. Acid-insoluble lignin content (Klason Lignin) was determined by a modified version of the method described in TAPPI T222, for acidinsoluble lignin in wood and pulp. As lignin is insoluble in sulfuric acid, so fibers were stirred firstly in 75% H 2 SO 4 for 2 hours and then the acid solution was diluted to reach 3% of concentration. Subsequently, the solution was heated to boiling with refluxing for 4 h. Finally, the residue was filtered and washed with 500 ml of water and dried at 100°C, representing lignin fraction. Acid soluble lignin (%) was quantified from sulfuric acid hydrolysates by measuring the absorbance at 205 nm.
BioMed Research International e cellulose content of various lignocellulosic residues was determined using a Fibertec machine (Tecator, 1010 Heat extractor). Briefly, 1.0 g of fiber was firstly treated with 100 mL of a boiling sulfuric acid (1.25%) for 30 min followed by 100 mL of a boiling sodium hydroxide (1.25%). After 30 min, the residue was filtered and washed 3 times with hot water before washing with acetone (for 2 min). In the end, the residue was dried at 105°C to a constant weight and then incinerated at 550°C for 2 hours. e cellulose fraction represents the difference of residue mass before and after incineration. e holocellulose content was determined according to the standard of American Society for Testing and Materials (ASTM) D 1104-56 (Reapproved 1978): 2 g of fibers was treated with 0.2 mL of glacial acetic acid and 1.0 g of sodium chlorite (NaClO 2 ) at 75°C for 5 hours (adding the same mixture each hour). After cooling the mixture to 10°C, the residue was filtered and washed with 500 mL of water and then dried at 100°C. Hemicellulose content is the difference between holocellulose and cellulose.

Reducing Sugars, Nitrogen, and Cell Mass
Determination. Reducing sugars were quantified by the 3,5dinitrosalicylic acid (DNS) method [25]. Nitrogen concentrations in lignocellulosic acid hydrolysate and in culture samples were analyzed by the Kjeldahl method [26]. To determine the amount of cell biomass, 10 mL of cell suspension sample was centrifuged at 3000 ×g for 5 min. e cell pellet was then washed twice with distilled water and dried in a preweighed glass tube at 105°C for 24 h, and the final mass was expressed as dry cell weight.

Lignocellulosic Hydrolysate Compositions.
Wheat bran hydrolysate composition including sugars (glucose and xylose) and inhibitors (5-hydroxymethylfurfural (HMF), furfural, and acetic acids) was determined using HPLC (Agilent Technology 1260 Infinity, refractive index detector RID, Agilent, USA) equipped with a Bio-Rad Aminex HPX-87H column. Sulfuric acid at 5 mM was used as a mobile phase with a flow rate of 0.6 mL/min, and at the column, temperature was at 65°C [9]. All compounds analyzed are used as standard at a concentration of 1 g/L.

Neutral Lipid Composition and Biodiesel Characterization.
Fatty acid composition of extracted microbial lipid was determined using GC/MS analysis. Firstly, the crude lipid was converted to fatty acids methyl esters (FAME) as follows: 100 mg of lipids was solubilized in 2 mL of hexane and supplemented with 0.2 mL of 2 M methanolic KOH solution. Subsequently, the mixture was incubated for 15 min at room temperature. Finally, the upper phase was recovered [27] and then analyzed by gas chromatographic system coupled to a series 5975 B Inert MSD Mass-Selective Detector (Agilent Technologies, France). 2 μL of FAME aliquot was injected employing an HP-5MS Phenyl Methyl Siloxane capillary column (30 m × 250 μm × 0.25 μm nominal). Helium was used as a carrier gas with a constant flow (1 mL/ min). e temperatures of the injector and detector were 250 and 240°C, respectively. e temperature program is as follows: 120°C for 5 min, an increase of 3°C/min to 180°C, an increase of 10°C/min to 220°C, and 220°C for 31 min. Data were evaluated using the NIST Mass Spectral Search Program.
e biodiesel is produced with a transesterification reaction where the TAGs are converted to fatty acid methyl esters with acidic or alkaline catalysts in the presence of alcohol and generating of glycerol as a by-product. e biodiesel characterization was conducted using the Biodiesel-Analyser software ( Ver. 1.1, 2013).
is analytical software was designed to predict biodiesel fuel properties (bioprospecting of FAME profile) of any oil feedstock profile determined by gas-chromatography [28].
e biodiesel characterization prediction is based on the fatty acid methyl ester profile of the oil feedstock. e estimation of the various properties from the fatty acid methyl ester profile (FAME) and the structure of the relevant fatty acids are based on 12 equations available in the study of Talebi et al. [28].

Oleaginous Yeast Selection.
e main limitation of the marketing of microbial lipids is largely related to the high cost of raw materials converted to biodiesel. Lignocellulosic residues used as feedstock for lipids production can partially resolve this constraint. However, this type of bioconversion process requires more efficient microorganisms able to assimilate and convert efficiently the C6 and mainly C5 sugars into lipids [29]. In our previous work, using the qualitative fluorometric technique (based on Nile red staining), twelve new yeast strains with an interesting lipogenic character were selected [21]. One of them, the strain Y-MG1, characterized by simultaneous use of glucose and xylose as carbon sources, was also able to accumulate intracellular lipids when it was cultivated on nondetoxified lignocellulosic acid hydrolysate showing a good resistance against the inhibitors. e Y-MG1 strain was identified, using molecular technique, as being Rhodotorula mucilaginosa [21]. e yeast R. mucilaginosa was previously described for its ability to produce interesting metabolites such as microbial lipids, carotenoids, and enzymes [30,31].

Lipid Accumulation on a Synthetic Medium by R. mucilaginosa Y-MG1.
It is necessary to evaluate the capacity of lipids production by oleaginous yeast on synthetic media before using it for lignocellulosic hydrolysate as a substrate. To this end, batch culture in shake flasks was carried out on a nitrogen-limited medium with 40 g/L of glucose as a carbon source (C/N ratio about 100).
e time courses of cell growth, lipid production, and sugar consumption are illustrated in Figure 1. During the first 24 hours of culture, exponential growth was observed and accompanied by consumption of about 15 g/L of glucose. e biomass gradually increased up to a maximum of 6.5 g/L after 144 h of cultivation. For lipid accumulation, very slight production appeared in the first 24 hours and increased during the stationary phase to reach 2.13 g/L corresponding to 32.76% of lipid content. In such nitrogen-limited conditions, the quickly depletion of nitrogen element in the growth medium causes deceleration of cell growth, and consequently, the assimilated carbon will be directed to synthesizing secondary metabolites such as fatty acids [4]. Cultivated under nitrogen-limited condition and in the presence of a high concentration of the carbon source (60 g/L of glucose corresponding to C/N ratio of 150), TJY15a, another yeast strain of R. mucilaginosa, produced 30.4% of lipid content [32]. Likewise, when it was cultivated on glycerol (50 g/L) as a unique carbon source, R. mucilaginosa produced around 3.1 g/L after 72 h of culture [33].

Carbon Source Effect on Lipids Accumulation and Fatty
Acid Composition of R. mucilaginosa Y-MG1. Besides glucose, other substances like disaccharides, polyols, and hydrophobic substrates could also serve as substrates for single cell oil production by oleaginous yeasts [34]. In addition, the fatty acids profile of accumulated lipids has been shown to be largely dependent on the nature of the carbon source used [23] which will determine the potential application of these lipids. To this end, the effect of carbon sources on cell growth, lipids accumulation, and relative fatty acids composition of R. mucilaginosa Y-MG1 was investigated and the results are shown in Table 1.
All substrates were added at an equal amount of carbon and supplemented with 0.16 g/L of nitrogen source (yeast extract/(NH 4 ) 2 SO 4 ) to reach a C/N ratio of 100. is strain is able to use a large panel of substrates, especially, C6 and C5 sugars as well as glycerol and sucrose. However, lactose and arabinose are not suitable substrates for lipids accumulation; only 15.7 and 11.7% of lipid content were observed, respectively. e highest amount of lipid was detected when Y-MG1 was cultivated on glucose (1.84 g/L corresponding to 27.65% of lipid content) followed by sucrose and xylose (1.74 g/L and 1.53 g/L, respectively). ere is no significant improvement in lipid production when glucose and xylose were mixed. is result was similar to that obtained by Sha [35] where the production of biomass and lipid was similar when glucose and the mixture of glucose/xylose were used as a carbon source by four oleaginous yeasts (L. lipofer, L. starkeyi, R. glutinis, and Y. lipolytica) [35]. Similarly, in the study of Hu et al. [36], they have tested many combination ratios of glucose and xylose for Trichosporon cutaneum AS 2.571 cultivation and lipids production. No enhancement in lipid rate was detected compared with cultivation on glucose. Lipid production was improved only in 3-liter bioreactor batch fermentation using glucose/xylose 2 : 1 combination [36]. On the other hand, the use of the glucose/ xylose combination as a carbon source significantly improves the lipids production by other oleaginous yeast strains such as C. viswanathii Y-E4 and T. cutaneum CTM-30125 (4.28 g/L and 4.58 g/L, respectively) [9,23]. Also, we noticed that Y-MG1 was able to assimilate and synthesize lipids when grown on glycerol (lipid content of 27.08%). We shall notice that glycerol is a potential waste of agri-food and biorefinery industries and its conversion into fatty acids would be an economically profitable alternative [1]. In addition, the ability of the strain Y-MG1 to assimilate sucrose is an interesting feature as many oleaginous strains cannot metabolize it efficiently. For example, the wild strain of Y. lipolytica is unable to grow on sucrose [37]. However, the expression of the gene encoding S. cerevisiae invertase in this yeast made it possible to circumvent this difficulty [38]. At the industrial scale, the Y-MG1 strain could grow efficiently on inexpensive residues which are considered as sucroserich substrates such as molasses. Lipids extracted from R. mucilaginosa Y-MG1 were transmethylated and analyzed by GC/MS (Table 1). e fatty acid (FA) composition shows a divergence according to the nature of the carbon source used. e lipids produced on glucose, xylose, and sucrose were essentially composed of oleic acid, a major compound, followed by palmitic acid and a relatively low amount of linoleic acid (maximum of 14.5%).
is FA composition profile is similar to that of vegetable oil, suggesting that the lipid produced by this yeast could be a potential candidate for biodiesel production. However, linoleic acid becomes the predominant compound in lipids extracted from glycerol, arabinose, and glycose/xylose cultures (>33%). Ahmad et al. [39] demonstrated that FA profile was slightly affected by the nature of the carbon sources used in the culture media of R. mucilaginosa with a predominance of C18 : 1 followed by the C16 : 0 and C18 : 2 [39].

Biochemical Characterization of Lignocellulosic Wastes.
In order to produce microbial lipids via an efficient bioprocess, five types of lignocellulosic materials were used (wheat bran, sugarcane bagasse, date palm leaf, Posidonia balls, and almond shell) as feedstock. Generally, lignocellulosic biomass is composed of three main elements: cellulose, hemicellulose, and lignin. e relative proportion of these components depends mainly on the origin of the material [40]. For this reason, a biochemical characterization step was carried out for the different residues based on the normalized process (Table 2). Cellulose fraction is the major compound in all lignocellulosic wastes (39.72% to 55.76%) except in wheat bran where hemicellulose is the major compound (52.7%). e high content of cellulose and hemicelluloses (sources of fermentable sugars) is of great interest in the exploitation of waste lignocellulosic material. Likewise, the study of Merali et al. [13] carried out on wheat bran shows that the major compound is hemicellulose with about 51% [13]. e compositions of sugarcane bagasse, date palm leaves, and Posidonia pellets are in agreement with those published in the literature [41][42][43] except for almond shell where hemicellulose is the major compound, with 35% [44]. For all the studied wastes, the amount of lignin was relatively low and varies between 7% and 19.94%. Generally, the low amount of lignin facilitates the hydrolysis of lignocellulosic materials and enhances the recovery yield of sugars [45]. In addition, during the hydrolysis process, the high amount of lignin increases the formation of phenolic derivatives that affect microbial proliferation [18].

Selection of Lignocellulosic By-Product.
e exploitation process of LB requires three steps: the preparation of plant material called pretreatment step, the hydrolysis step that serves to release the monosaccharides, and the third step is the bioconversion that uses microorganisms [46]. In the first step, acid treatment is the chemical processes mostly used for LB degradation [47][48][49]. To this end, the different LB has undergone a heat treatment coupled with sulfuric acid hydrolysis. e sulfuric acid concentration was set at 2% while temperature, pressure, and time of treatment were varied. e result of LB hydrolysis is illustrated in Figure 2(a).
Wheat bran shows the maximum of reducing sugars under all autoclaving programs and allows 44.54 g/L of released sugar with the program 2 (121°C, 60 min, 1.1 bar).
is is due to its richness in hemicellulose which is mainly affected by acid treatment and also due to its low lignin content [17]. However, sugarcane bagasse, date palm leaf, Posidonia balls, and almond shell do not exceed 28 g/L of liberated reducing sugars. In previous works, rice straw and corncob LB treated with 1.5% sulfuric acid at 121°C for 90 min and 2.5% sulfuric acid at 135°C for 60 min release 35.2 g/L and 45.7 g/L of sugars, respectively [47,49]. e sugarcane bagasse produces 21.38 g/L of sugars when it was hydrolyzed with 2.5% HCl, at 121°C for 45 min [48]. Based on the amount of released sugars, wheat bran was selected as the feedstock for microbial lipid production by the Y-MG1 strain. Moreover, wheat bran is a cheap and abundant source of dietary fiber which is produced as a by-product in several processes of wheat treatment in milling industries [50]. Worldwide consumption of wheat, according to the World Agricultural Supply and Demand Estimates (WASDE), has been estimated to 652.18 million tons for the year 2010 (WASDE, 2010). One million tons of wheat can produce up to 0.25 million tons of wheat bran. is agriculture residue has relatively diverse applications in food, feed, health, and fermentation industries due to its richness in carbohydrates (mostly fibers), protein, and fats [51].
On the other hand, the acid concentration is also one of the important factors in the chemical hydrolysis of the lignocellulosic materials. For this purpose, different concentrations of sulfuric acid (0.5-3%) were applied for wheat bran hydrolysis using the best program (P2) (Figure 2(b)). At 1% of sulfuric acid, wheat bran releases the maximum amount of sugars reaching a concentration of 56.61 g/L. e increase in the concentration of sulfuric acids is not accompanied by an increase in the amount of sugars released. In general, the use of high acid concentrations can generate a significant amount of by-products such as HMF, furfural, and acetic acid that are unfavorable for the growth of microorganisms [48]. In addition, using low concentrations of acid reduce the environmental impact of the bioconversion process.

Lipid Production in Bioreactor by R. mucilaginosa Y-MG1
Using Wheat Bran Hydrolysate. Carbohydrate, nitrogen, and inhibitors composition of acid wheat bran hydrolysate was determined before using this raw material as a carbon source for microbial lipid production by Y-MG1 strain. e composition based on HPLC analysis was as follows: glucose 23.7 g/L, xylose 19.1 g/L, HMF 0.69 g/L, furfural 0.07 g/L, and acetic acid 0.73 g/L. Total reducing sugars and nitrogen were about 56 g/L and 2.4 g/L, respectively. During wheat bran hydrolysate fermentation, the addition of external nitrogen source was not necessary to avoid the decrease of the C/N ratio and its consequent negative effect on lipid synthesis. In addition, the presence of inhibitors such as acetic acid (the most abundant aliphatic acid formed by the hydrolysis of acetyl groups of hemicellulose), furfural, and HMF (the degradation by-products of pentoses and hexoses, respectively) can limit microbial growth. Several studies operate through detoxification techniques to increase the efficiency of the bioconversion of carbohydrates [16]. Our study was conducted to set up a cost-effective bioconversion process. To do this, we chose not to include detoxification treatments. e yeast strain R. mucilaginosa Y-MG1 was previously tested for its capacity of growing and producing lipid on acid-WBH without detoxification and in the presence of inhibitors [21]. Under batch fermentation, Y-MG1 produces about 2.3 g/L of lipid corresponding to 20.4% of lipid content [21]. Productivity and lipid yields can be enhanced using the fed-batch fermentation. is culture mode allows also avoiding problems of mixing and heat transfer due to the high concentration of sugars in the culture medium [52]. To this end, R. mucilaginosa Y-MG1 was cultivated on nondetoxified WBH (initial reducing sugars concentration was about 60.5 g/L) supplemented only by an elements solution (ZnSO 4 , MnSO 4 , FeSO 4 , and CuSO 4 ) and at fixed pH 6.0. e evolution of biomass and lipid production, as well as the consumption of sugars and nitrogen, was determined every 24 hours (Figure 3). We observed two fermentation phases. e first phase extends from the beginning of the culture until 74 hours. is phase was characterized by exponential growth and allows reaching 8.0 g/L of biomass and 1.5 g/L of lipid accompanied by a consumption of 47.6 g/L of sugars and 1.0 g/L of nitrogen. e second phase (feeding phase) begins when the concentration of residual sugars exceeds the minimum threshold of 10 g/L.
ree successive feedings were performed with each feed containing 11.2 g/L of sugars and 0.33 g/L of nitrogen.
is allowed the biomass to grow gradually to reach a maximum of 13.3 g/L after 168 h before declining at the end of the culture. e progressive addition of WBH allows producing 4.5 g/L of lipid. e conversion efficiency of sugars into lipid (Y L/S ) has evolved from 0.03 g/g (during the first phase) to 0.12 g/g (after feeding phase). However, biomass productivity (Y X/S ) was clearly superior during the first phase (0.16 g/g). is is explained by the consumption of sugars in favor of cell growth in the first phase, whereas, in the second phase, a metabolized sugar was directed toward lipid synthesis. e same result was observed when the oleaginous yeast, Cryptococcus sp. SM5S05, was cultivated on corncob acid hydrolysate under fed-batch fermentation [52] giving a final production rate of lipids of 4.5 g/L.
Exploiting new abundant LB and reducing the use of costly nutritional additives for microbial growth are a challenge to set up a cost-effective process for microbial lipid production. Different reports have exploited lignocellulosic material as raw material for the production of lipids by oleaginous yeasts (Table 3). e process of microbial lipid production was related to several factors affecting its productivity such as culture conditions, yeast species, the nature of raw material, and nitrogen source. Compared with other yeast of Rhodotorula genus, the lipid content of Y-MG1   [8,19]. Cultivation of C. starkeyi on bagasse hydrolysate under different culture modes had resulted in lipid yields not exceeding 28% [17]. In addition, C. tropicalis and T. cutaneum CX1 cultivated on palm empty fruit and corn stover hydrolysate, respectively, produce only 1.6 and 3.1 g/L of lipids using batch bioreactor culture [53,54]. However, the lipid yield of Y-MG1 was lower than that of R. mucilaginosa (6.64 g/L) using the nondetoxified wheat straw hydrolysate [19]. All of this shows that the nondetoxified acid wheat bran hydrolysate is another potential lignocellulosic source that could be used as feedstock for microbial lipid production.

Fatty Acid Composition and Biodiesel Characterization.
Fatty acid (FA) profile of lipids extracted from R. mucilaginosa Y-MG1 after growth on nondetoxified WBH was determined by GC-MS analysis. Table 4 shows that oleic acid C18 : 1 was the major compound of lipids (66.12%) followed by palmitic acid C16 : 0 (11.94%) and stearic acid C18 : 0 (11.06%). Nevertheless, the polyunsaturated fatty acids did not exceed 8%. is lipid composition is highly similar to L. starkeyi and T. cutaneum when grown on a corncob and barley hulls acid hydrolysates, respectively [9,55]. is composition, largely identical to that of many vegetable oils, was considered as ideal for biodiesel synthesis [56]. Indeed, the quality of biodiesel depends on the fatty acid composition of lipids, which must be rich in saturated and monounsaturated FAs and parallel poor in polyunsaturated FA [56]. Methyl esters from monounsaturated FAs guarantee a good quality of biodiesel because they are in liquid form at room temperature and characterized by good flow properties, unlike methyl esters from polyunsaturated FAs which induce oxidation problems during storage [56]. e theoretical characterization of R. mucilaginosa biodiesel was performed using BiodieselAnalyser software [28]. Some biodiesel properties are shown in Table 3 with US standards (ASTM D-6751). e cetane number is an indicator of the ignition quality of a diesel fuel. It measures the readiness of the fuel to autoignite when injected into the engine [57]. e biodiesel of R. mucilaginosa shows a cetane number equal to 56.68, which conforms to the norm (≥47). e iodine value influences several important properties of biodiesel, such as the clogging point of the cold filter and the oxidation stability [58]. e maximum limit of the iodine value is 120 and the biodiesel of R. mucilaginosa complies with an index of 57.9. e kinematic viscosity of biodiesel is also an important Table 3: Lipid production from lignocellulosic biomass using oleaginous yeasts.

Strains
Lignocellulosic residues Nitrogen source Culture mode X a (g/ L)   parameter because it indicates the ability of the fuel to form deposits in the engine [58]. e biodiesel of R. mucilaginosa has a kinematic viscosity of 1.39 (mm 2 /s) which is close to the lower limit 1.6 (mm 2 /s). e biodiesel density of R. mucilaginosa is slightly below the minimum limit. In the applied domain, this limitation can be circumvented by mixing the R. mucilaginosa biodiesel with other denser products. We conclude that the lipids extracted from the yeast R. mucilaginosa can be considered as very suitable for the production of biodiesel.

Conclusion
In our current study, we have shown the feasibility of producing microbial oil from a renewable raw material as lignocellulosic wastes. e newly isolated oleaginous yeast, R. mucilaginosa Y-MG1, was able to utilize acid wheat bran hydrolysate for oil production without prior detoxification. Moreover, the addition of an external nitrogen source was not needed during wheat bran hydrolysate conversion, which is beneficial for the economics process. Fed-batch fermentation allowed a significant increase in the amount of microbial lipids produced by Y-MG1 strain. e resulting Y-MG1 lipids were finally considered as a promising feedstock for biodiesel production.

Data Availability
(i) Oleaginous yeast selection and microbial lipids production and quantification, (ii) lignocellulosic residues characterization and degradation using diluted acid treatment, and (iii) the corresponding results of bioreactor fedbatch fermentation and biodiesel synthesis and characterization data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.