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Comparative Immunogenicity in Rabbits of the Polypeptides Encoded by the 5′ Terminus of Hepatitis C Virus RNA

Recent studies on the primate protection from HCV infection stressed the importance of immune response against structural viral proteins. Strong immune response against nucleocapsid (core) protein was difficult to achieve, requesting further experimentation in large animals. Here, we analyzed the immunogenicity of core aa 1–173, 1–152, and 147–191 and of its main alternative reading frame product F-protein in rabbits. Core aa 147–191 was synthesized; other polypeptides were obtained by expression in E. coli. Rabbits were immunized by polypeptide primes followed by multiple boosts and screened for specific anti-protein and anti-peptide antibodies. Antibody titers to core aa 147–191 reached 105; core aa 1–152, 5 × 105; core aa 1–173 and F-protein, 106. Strong immunogenicity of the last two proteins indicated that they may compete for the induction of immune response. The C-terminally truncated core was also weakly immunogenic on the T-cell level. To enhance core-specific cellular response, we immunized rabbits with the core aa 1–152 gene forbidding F-protein formation. Repeated DNA immunization induced a weak antibody and sustained proliferative response of broad specificity confirming a gain of cellular immunogenicity. Epitopes recognized in rabbits overlapped those in HCV infection. Our data promotes the use of rabbits for the immunogenicity tests of prototype HCV vaccines.

Introduction
Nucleocapsid (core) protein of hepatitis C virus (HCV) is the most conserved HCV antigen capable of inducing strong broadly cross-reactive responses, and therefore an attractive component of a genotype-non-restricted HCV vaccine. As such, it has been included in a number of HCV vaccine candidates including ones reaching primate trials [1]. The responses observed were described as limited. In immunizations, HCV core demonstrated features of a weak immunogen capable of inducing mainly CTL and low or no CD4+ T-cell responses with moderate IFN-gamma, weak IL-2 production, and no antibodies [2,3]. In primate trials, HCV core induced stable low-level T-cell response of CD4+ and CD8+ T-cells manifested by IFN-gamma, but no IL-2 or IL-4 responses, weak T-cell proliferation, and low titer of core-specific antibodies [4][5][6][7][8]. Attempts to achieve a more efficient anticore immune response met with difficulties [9][10][11] even when using viral vectors [12].
Interestingly, in natural infection HCV core acts as a strong humoral immunogen inducing an early potent antibody production, but limited cellular response. Furthermore, in patients developing chronic infection, antibody response to HCV core protein continues to expand, whereas the cellular responses shrink [13]. This scenario points at a limited (low to no) protective potential of core-specific humoral 2 Journal of Immunology Research responses. At the same time, in primate trials, the responses to structural HCV proteins including core were shown to significantly correlate with primate protection against HCV challenge (whereas no protection was rendered by immunizations with nonstructural proteins) [1]. This indicates a potential positive input of anticore response (moderate as it was) on the observed protection effects, emphasizing the necessity to achieve an effective core-specific cellular response. Achieving stronger core-specific responses required the addition of recombinant HCV core protein or core-derived peptides [9,14], involvement of the Th2-tilting carriers as HBcAg [15], or coadministration of cytokines such as IL-2, IL-4, or granulocyte-macrophage CSF [16], altogether pointing at the necessity of a shift towards the Th2-type T-helper cell response. Interestingly, these particular responses (of CD4+ T-cells) are involved in the spontaneous clearance of HCV infection, contrary to the CTL response reported to be stunned and ineffective [13,17].
The reasons for a deficiency of such response in natural infection are not yet fully understood. Several explanations can be named, firstly, the well-known immunomodulating properties of HCV core protein [18][19][20]. The other reason could be the abundance of HCV core as an antigen. The core antigen quantity correlates with the virus load and can reach high levels in chronic HCV infection [21,22], whereas the induction of potent cellular response appears to rely on the low immunogen doses [23]. An interference was also implied by the proteins translated from the HCV alternative reading frames (ARFs) [24,25]. Most of the core gene products appear to be contaminated with the proteins translated from the HCV alternative reading frames (ARFPs) [24,25]. The difference in anti-F response between chronic and self-limiting infection, the cross-reactivity irrespective of genotype, and the correlation of anti-F response to the response against other structural and nonstructural HCV antigens pointed at the immune response to F-protein as an integral part of the natural HCV infection [26]. As in case of HCV core, strong antibody response to F-protein correlates with the chronical course of HCV infection [27]. Kong et al. showed recently that presence of anti-F-specific antibodies negatively correlates with HCV RNA viral load suggesting that F-protein may participate in viral clearance [28]. However, other results suggest the potential involvement of F-protein (as of core antigen) in increasing the frequency of CD4+CD25+FoxP3+ T-cell-like population and IL-10-producing CD4+CD25+ T-cells [24] and biased cytokine responses (significantly decreased IFNand/or IL-2 and significantly increased IL-4 and/or IL-5 levels) [25] predisposing to persistent HCV infection. ARFPs may induce some of the negative effects ascribed to HCV core [29] and also sidetrack the immune response away from HCV core. The true role of anti-ARFP responses in resistance to viral infection or vaccine protection is yet unknown.
In this work we aimed to directly compare immunogenicity of protein products encoded by 5 end of HCV RNA in comparatively large animals, namely, in rabbits, which have numerous advantages over mice and are regularly used prior to testing vaccines in primates. Specifically, we compared immunogenicity of the main form of HCV core, core aa 1-173, its shorter form core aa 1-152, the C-terminal core aa 147-191, and F-protein as an ARFP form with the longest unique protein domain. All polypeptides generated extremely potent humoral response resembling that in chronic HCV infection. At the same time, a synthetic gene for the Cterminally truncated HCV core forbidding F-protein synthesis generated a sustained T-cell and only low antibody response indicating a clear shift towards cellular immunity deemed essential for an effective HCV vaccine.

Materials and Methods
were used for expression.

Plasmids for Expression of HCV
Core. Fragment corresponding to HCV core 1-173 aa was obtained by polymerase chain reaction (PCR) using cDNA of HCV AD78 isolate genotype 1b (GenBank accession number AJ132996 [30]) as a template and two primers: forward 5 -GATCCATGGGCA-CGAATCCTAAACCTCA contained NcoI site and reverse 5 -GTGATGAGATCTAGAGCAACCGGGCAGATTCCC-TGTTGCA contained BglII site. Second codon AGC from AJ132996 was substituted for GGC and thus gave us S to G substitution. NcoI/BglII PCR fragment was ligated into NcoI/BglII pQE-60 plasmid (Qiagen). The resulting plasmid was named pQE/core 173 (GenBank accession number KT824963).
Amplification of the DNA fragment corresponding to 1-10 aa of core and in +1 frame of the core from aa 11 to aa 143 and two additional aa (LE) was performed by PCR using 5 -GAGCATATGAGCACGAATCCTAAACCT-CAAAGAAAACCAAACGTA as forward primer and 5 -GTGGTGCTCGAGTGGTGGCGCCGACGAGCGGA as reverse primer; harboring NdeI and XhoI restriction sites, respectively, was done from plasmid bearing HCV core fragment corresponding to 1-191 aa of HCV 1b isolate 274933RU (GenBank accession #AF176573 [31]). After amplification and treatment with restriction endonucleases NdeI and XhoI fragment was ligated into NdeI/XhoI pET22b(+). pET22b(+) plasmid contains T7 promoter and 6xHis-tag coding sequence at 3 end of the cloned DNA fragment. The resulting plasmid was named pET22/ARFP.
HCV core-derived synthetic peptides used in analysis of immune response were purchased from GL Biochem (Shanghai, China) or kindly provided by Mati Sällberg (Karolinska Institutet, Sweden); and F-protein-derived peptides were purchased from Peptron (South Korea). Peptides were purified Table 1: A panel of overlapping peptides derived from HCV core and F-protein used in the tests of humoral and cellular immune response. First and last amino acid position are given according to HCV AD78 isolate genotype 1b (GenBank accession number AJ132996 [30,36]

Sources of HCV Polyproteins.
Expression of HCV core aa 1-152 (core 1-152) and core aa 1-173 (core 1-173) was carried out in the E. coli strain JM109 as was described earlier in [32] and [33], respectively. F-protein was expressed in E. coli BL21(DE3) transformed with pET22/ARFP. Transformed bacterial cells were grown at 37 ∘ C in 2x TY medium (16 g/L bacto-peptone (Difco), 10 g/L yeast extract (Difco), and 5 g/L NaCl), supplemented with 100 g/mL ampicillin, to an OD 540 of 0.8-1.0, and protein expression was induced with 0.2 mM IPTG. Induction was continued for 4 h at 37 ∘ C; after that cells were sedimented by low-speed centrifugation (10 min at 4,000 ×g) and frozen at −20 ∘ C. Frozen biomass was thawed and suspended in 10 volumes of 8 M urea containing 100 mM Tris-HCl, pH 8.0, and ultrasonicated with ten 60 s ultrasound pulses of 22 kHz. After ultrasonication incubation on ice was continued for 60 min. After clarification (30 min at 10000 ×g), supernatant was collected and dithiothreitol (DTT) was added to 100 mM and incubation was continued overnight by shaking on rotary shaker at 4 ∘ C. After repeated clarification (30 min at 10000 ×g) before loading onto immobilizedmetal affinity chromatography (IMAC) Ni-superflow agarose (Qiagen, Hilde, Germany), buffer exchange was performed with Sephadex G-25 column to replace 100 mM DTT with 5 mM -mercaptoethanol ( -ME). The recombinant protein was purified by IMAC under denaturing conditions (8 M urea, 5 mM -ME, 100 mM Tris-HCl, and pH 8.0) according to the manufacturer's instructions. F-protein containing fractions were pooled, and purified protein was diluted to final concentration of 0.5 mg/mL. The proteins were subsequently dialyzed two times (overnight and for 4 to 6 h) using refolding buffer I (2 M urea, 100 mM PB (Na 2 HPO 4 : 94.7 mM; NaH 2 PO 4 : 5.3 mM), pH 8.0, 0.5 M arginine, 5 mM glutathione reduced [GSH], and 0.5 mM glutathione oxidized [GSSG]) and refolding buffer II (100 mM PB, pH 8.0, 0.5 M arginine, 5 mM GSH, and 0.5 mM GSSG) and then PBS with 10% glycerin. Soluble proteins were concentrated using Amicon Ultra-15 10 K centrifugal filter device 10,000 MWCO (Millipore, Ireland). Its purity according to Coomassie blue staining of the SDS-PAGE gel was 95%.

SDS-PAGE and Western Blot Analysis.
The purified proteins were analyzed on 15% SDS-PAGE by standard procedures (under denaturing conditions). Proteins were transferred to nitrocellulose membrane (Thermo Scientific). After blocking, the membranes were probed with rabbit antibodies specific to HCV core [34] or anti-core 1-173 or F-protein antibodies obtained here (see Section 2.6) diluted 1 : 10000, followed by a protein A horseradish peroxidase-conjugated antibody diluted 1 : 1000. Detection was performed with the DAB Substrate Kit (Thermo Scientific) according to the manufacturer's protocol.
2.6. Immunization of Rabbits. All animal experiments were performed in accordance with the Russian Federation law and were approved by the institutional ethical committee for animal experiments. Moscow strain of Chinchilla grey rabbits (female, 2-month-old, 1.5 to 1.8 kg) was obtained from the laboratory animal breeders "Manikhino" (settlement Manikhino, Ivanovskoe, Moscow region, Russia) or "Krolinfo" (Orekhovo-Zuevo, Moscow region, Russia, http://krolinfo.umi.ru). The animals were maintained at 20 to 22 ∘ C and a relative humidity of 50% ± 10% on a 12 h light/dark cycle, fed with commercial rodent chow and herbal vitamin flour ("Krosha" and "Meadow grass," both from Zoomir, Russia), and provided with tap water ad libitum. The treatment of animals was in accordance with regulations outlined in the USDA Animal Welfare Act and the conditions specified in the guide for care and use of laboratory animals [35].
In protein immunizations, groups of two Chinchilla rabbits were immunized with injections of recombinant core 147-191 (numbers 87, 88), core 1-152 (89/4, 90/5), F-protein (91, 92), and core 1-173 (93, 94) or mock-immunized with PBS (95, 96). At week 0 animals were administered 100 g of the respective polypeptides in 400 L PBS mixed (1 : 1 v/v) with the complete Freund Adjuvant (CFA) and a week later (week 1) with 100 g of the respective polypeptides in 400 L PBS mixed (1 : 1 v/v) with the incomplete Freund Adjuvant (IFA). Injections were done subcutaneously at four sites along the back. Animals were boosted three times with one-month intervals by the intravenous injections of 50 g of polypeptides in 200 L PBS mixed with IFA (1 : 1 v/v). Control animals (95, 96) received the adjuvants mixed with PBS. Rabbits were bled from the ear vein two weeks after each immunization. Sera were prepared and stored at −20 ∘ C until further analysis. A portion of blood was collected in the heparinized Vacutainer tubes, and peripheral mononuclear cells (PBMCs) were isolated by Ficoll Paque gradient centrifugation.

Antibody
Assays. Sera were assessed for the levels of antibodies against HCV core-derived polypeptides and Fprotein.
Core-derived peptides (Table 1) and core 147-191 were coated onto 96-well MaxiSorp plates and core polypeptides on the 96-well PolySorp plates (both from Nunc, Denmark). Coating was done overnight at 4 ∘ C in 50 mM carbonate buffer, pH 9.6, at antigen concentration of 10 g/mL. After blocking with PBS containing 1% BSA for 1 h at 37 ∘ C, serial dilutions of rabbit sera were applied on the plates and incubated for an additional hour at 37 ∘ C. Incubation was followed by three washings with PBS containing 0.05% Tween-20. Afterwards, plates were incubated for 1 h at 37 ∘ C with the protein A horseradish peroxidase-conjugated antibody (Sigma, USA) diluted 1 : 20000. Following three washes with PBS containing 0.05% Tween-20, the substrate OPD (Sigma, USA) was added, incubated at room temperature for 15 min in the dark, and stopped with 1 N H 2 SO 4 . Plates were read on an automatic reader (Multiscan, Sweden) at a dual length of 492 versus 630 nm. Immune serum was considered positive for anti-core antibodies whenever a specific OD value exceeded, by at least twofold, the signals generated by preimmune serum reacting with core-derived antigen and by immune serum reacting with BSA-coated wells.

PBMC Proliferation Assay.
Peripheral mononuclear cells (PBMCs) were isolated by Ficoll Paque gradient centrifugation of blood which was collected in heparinized Vacutainer tubes. PBMCs were subjected to in vitro stimulation with core-derived synthetic peptides (Table 1) using the procedure described by us earlier [38]. In brief, T-cell proliferation assay was performed in triplicate with RPMI containing HCV core-derived peptides, all at 1 mcg/well; phytohemagglutinin (PHA; 10 mcg/well) was used as positive and RPMI alone and control peptide representing aa 605-613 of gp41 of HIV-1 were used as negative controls. Data were expressed as stimulation indices (SI) defined as the ratio of a mean value of [3H]thymidine incorporation in the antigen-stimulated cultures to a mean value of radioactivity incorporation in medium containing negative control peptide from gp41 or RPMI, the highest of the values selected. SI values of 2.0 and above were considered positive. Data sets were discarded if SI by PHA was lower than 2.

Statistical Analysis. Statistical analysis was by paired
Student's -test, one-way ANOVA with pairwise comparisons, and two-way ANOVA with pairwise comparisons. < 0.05 was considered significant. Analyses were performed using STATISTICA AXA 10.0.

Design and Expression of Proteins Encoded by the 5
Terminus of HCV Genomic RNA. The full-length HCV core 1-191 is unstable and is quickly processed to a more stable shorter core aa 1-173 (core 1-173) [39]. We have chosen the latter as the immunogen and designed a recombinant core 1-173 of HCV 1b basing it on the isolate AD78P1 [30] with modifications that aimed to improve the prokaryotic expression (GenBank accession #KT824963). HCV core 1-173 is further degraded to the shorter forms, of which only core aa 1-152 (core 1-152) is readily detectable [40] motivating its choice as a second immunogen for the comparative immunogenicity studies. The expression of HCV core aa 1-152 variant was described by us earlier [32]. The panel of immunogens was complemented by the C-terminal fragment of HCV core aa 147-191 represented by a synthetic peptide (core 147-191).
The 5 terminus of HCV RNA encodes also the proteins from the alternative reading frame (ARF). ARF of HCV lacks an in-frame AUG start codon; its expression involves unusual translation-level events involving ribosomal frameshifting [41]. ARF encoded proteins (ARFPs) are synthesized through multiple events and sites such as codons (in phase +1) 26, 42, 85/87, and 144 yielding different ARFP forms including double frameshifts [42][43][44][45]. Of those, the main most stable form is F-protein, whereas the rest are comparatively short and proteolytically unstable [46]. The frameshift leading to the production of ARFP/F is remarkable: it leads to the shutdown of the main ORF for at least one round of translation and occurs so frequently that it causes the ribosome to translate +1 reading frame approximately 30% of the time [47,48]. This points at the abundance of F-protein and its significance as a target of HCV-specific immune response. We have chosen this longest and most stable ARFP form for the immunogenicity study in rabbits, to compare its immunogenic performance to that of the "classical" product of translation of the 5-terminus of HCV RNA. For this, we designed a recombinant protein containing the N-terminal 10 amino acids of HCV core and aa 11 to 143 belonging to Fprotein of HCV 1b variant [31]. Only the first ten amino acids of HCV core were retained as they were shown to stabilize Fprotein and support its correct folding [49]. One of the major antigenic sites of the core protein has been located away from the very N-terminus of HCV core (amino acids 9-16 [50]). Hence, we expected that sharing of the first ten amino acids will not interfere with the development of F-specific immune response.

Polypeptides Derived from the 5 Terminus of HCV RNA
Induce Potent Antibody Response in Rabbits. Rabbits were immunized by the repeated injections of the polypeptides representing core aa 1-173 (core 173), core aa 1-152 (core 1-152), core aa 147-191 (core 147-191), and F-protein. All polypeptides were highly immunogenic on the humoral level; maximum antibody titers after completion of immunization cycle reached 10 6 and the titer of antibodies to aa 147-191 reached over 10 5 (Figure 2(a)). The strongest antibody response was achieved after immunization with HCV core 1-173 and F-protein (Figures 2(a) and 2(b)). HCV core 1-152 devoid of C-terminus generated a weaker antibody response with the maximum titer of 5 × 10 5 despite an identical immunization scheme and almost identical antigen structure of the proteins (except for the lack of C-terminus) (Figures 2(a) and 2(b)). A 44-amino-acid long core 147-191, although used in immunization without carriers (which normally ensure strong antibody response against the synthetic peptides), induced a strong specific immune response with the titers reaching 10 5 and the same kinetics of the antibody response as the longer polypeptides (Figures 3(a) and 3(b)). No anti-HCV core or anti-F-protein antibodies were detected in control rabbits 95, 96 receiving adjuvant alone (data not shown).  (Figure 2(a)).
We have used a panel of synthetic peptides (Table 1) to map the B-cell epitopes of HCV core and F-protein recognized in rabbits. In HCV core aa 1-173, nine epitopes were identified which were distributed throughout the protein with the dominant region located at N-terminus of the protein (Figure 3(a)). The sera of core 1-152 immunized rabbits recognized only the immunodominant epitope at aa 1-35 (titer 5.5 × 10 4 , Figure 3(a)). Similar analysis was performed for the epitopes of F-protein (Figure 3(b)). B-cell epitopes of F-protein recognized in rabbits were localized at aa 30-49, 45-64, 60-79, and 90-109 (Figure 3(b)). The titer of antibodies against linear epitopes of F-protein was on the average 10-fold lower than against the linear epitopes of HCV core indirectly indicating a dominance of the HCV corespecific immune response over that against F-protein, at least in the rabbit model. The analysis of B-cell reactivity against HCV core and F-protein in rabbits uncovered similarity to the B-cell responses observed in HCV infection [27,50,54,[56][57][58][59]. Most of these epitopes were also shown to be recognized in mice [60,61]. This reveals a promiscuous character of HCV core and F-specific B-cell response. Our findings also indicate that the recombinant F-protein obtained here is immunologically identical to the one formed after translation of viral RNA in infection and can be utilized in the diagnostic and possibly vaccine studies. We have further characterized the nature of crossreactivity between anti-HCV core and anti-F-protein sera seen in Western blotting (Figures 2(c) and 2(d)). The crossreactivity of anti-HCV core 1-173 and anti-F-protein sera amounted to 10% of the total reactivity of both HCV core and F-protein immunized rabbits (Supplementary Figure  S1). Immunization with HCV core 1-173 did not induce any antibodies reacting with F-protein-derived peptides. Immunization with F-protein did not induce an immune response reacting to core peptides except for the region aa 61-95 (Figure 3(a)). Analysis of the sequences of HCV core 1-173 and F-protein did not reveal any amino acid homologies, indicating that cross-reacting anti-F-protein antibodies might have recognized not a linear but a conformational epitope at aa 61-95 which could be reproduced by the synthetic peptide. Indeed, preblocking with the peptide encompassing aa 61-95 had no effect on the cross-reactivity of anti-Fprotein sera with core 1-173 in Western blotting (i.e., anti-F-protein antibodies reacting to the peptide core aa 61-95 in ELISA were unable to recognize this sequence in the context of the denatured core 1-173; data not shown). Importantly, although 10 amino acids overlap between HCV core 1-173 and F-protein at N-terminus, anti-F-protein sera did not recognize synthetic peptide representing aa 1-35 of HCV core (Figure 3(b)). Altogether, this indicated that the crossreactivity was apparently due to the immune recognition of His-tag.
Thus, all polypeptides derived from the 5 terminus of HCV genomic RNA were found to be extremely immunogenic on the antibody level. Furthermore, we have demonstrated a similarly strong immunogenicity of the HCV core and F-proteins. Albeit no function has yet been attributed to F-protein (or other products of ARFPs), it represents a target of immune response equal in potency to HCV core [26,54,62]. Supposedly nonfunctional but abundant ARFPs may induce a decoy response leading to the immune system away from addressing "the meaningful" viral proteins; its high immunogenicity in rabbits confirms a possibility of their  Figure 3: Fine epitope mapping of antibody response to linear epitopes of HCV core (a) and F-protein (b) recognized by rabbits immunized with HCV core aa 1-173 (nn 93, 94), HCV core 1-152 (89/4, 90/5), and F-protein (91, 92). Graphs demonstrate the highest antibody titers reached throughout immunization and represent the result of two to three independent ELISA runs. Unspecific antipeptide reactivity in control rabbits receiving adjuvant alone was below 5 × 10 2 .  [63].
Since F-protein has not yet been ascribed any function in the virus, while functions of HCV core are well known and essential, we concentrated our further immunogenicity studies on the cellular immune response against HCV core. Peripheral blood mononuclear cells (PBMCs) of rabbits immunized with HCV core 1-152 and core 1-173 were collected prior to and after each boost and subjected to stimulation with HCV core-derived peptides. Weak infrequent Tcell responses with stimulation indexes (SI) exceeding 2 were repeatedly observed only in rabbits immunized with HCV core 1-152, but not in the naïve or adjuvant or core 1-173immunized animals (Figure 4, data not shown). Proliferative response of rabbit PBMCs was observed after two priming HCV core 1-152 immunizations (week 4) and was not boosted except for a single response to the epitope at the HCV core N-terminus observed in the rabbit 90/5 at week 20 (Figure 4(a)). Stimulation of the hyperimmune rabbit 8 Journal of Immunology Research PBMCs with recombinant HCV core 1-152 induced a weak proliferative response independent of antigen concentration (Figure 4(b)).
Analysis of HCV core-specific humoral and cellular responses revealed that the C-terminally truncated HCV core form had somewhat weaker humoral immunogenicity than HCV core aa 1-173: antibodies were two to three times lower in titer and of restricted specificity targeting mainly the N-terminus of the protein (Figures 2 and 3). At the same time, only core 1-152 was able to induce a specific T-cell response, albeit of a very low level. Apparently, the truncation of the C-terminus led to a partial loss of B-cell immunogenicity (in terms of both breadth and potency) and at the same time the induction of the T-cell arm of immune response. We have recently shown that HCV core devoid of the N-terminus upregulated the transcription of a ROSgenerating enzyme cytochrome P450 2E1 [64]. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1 . The latter triggers the efflux of Ca 2+ ions from ER to mitochondria via mitochondrial Ca 2+ uniporter, leading to the generation of superoxide anions and possibly also H 2 O 2 [64]. ROS have a physiological role in signaling extending to every cell type involved in the induction of immune response; ROS were the first molecules found to suppress the T-cell function [65]. As with any signaling mechanism, ROS can become cytotoxic if the signal is too strong and/or too prolonged. ROS help to mediate T-cell activation; however, T-cell activation also depends on the capacity of accessory cells to maintain sufficient level of glutathione and is compromised by the oxidative stress [66]. Furthermore, excessive amounts of ROS can oxidize the protein kinases and phosphatases that regulate critical cell signals and distort the activation of signaling pathways including regulation of the lymphocyte functions [67]. Immunosuppressive effects of ROS may also be due to the fact that Tregs cells are more resistant to ROS than the effector T-cells and pertain their downregulating activity when the effector T-cells fail [66]. An excessive oxidative stress may thus be detrimental for the normal T-cell functions. This would explain the observed deleterious role of the high HCV core protein "doses" for the specific T-cell immunity in a mouse model [9]. For humoral response, on the contrary, ROS appear to contribute to Th1/Th2/Th17 cell fate decisions during T-lymphocyte activation and enhance immunoglobulin production by B-lymphocytes [68]. Our data indicates that (as in HCV infection) high levels of HCV core support the strong multiepitopic B-cell, but low or no Tcell response, and point at the role of certain core domains, specifically at the C-terminus, in tilting the response towards the humoral one. Involvement in ROS induction may explain an unexpectedly strong humoral immunogenicity in rabbits of a peptide covering aa 147-191 derived from the ROSinducing core fragment. . High levels of circulating HCV core antigen in HCV infection would induce high levels of ROS and promote strong humoral response but little immunity on the Tcell level, a scenario of immune response in the chronic HCV infection, whereas the immune success and viral clearance coincide with a weak or no antibody response [69] and potent cellular immunity manifested mainly CD4+ Tcells [13,17]. To strengthen the cellular immune response component, one would need to both decrease the immunogen dose and delete the ROS-inducing/B-cell activating signals.

Immunogenicity in Rabbits of DNA Encoding Core aa
To evade both the potential pitfalls as immune suppression induced by an excess of ROS and the immune competition from the ARF products, we exploited a synthetic gene encoding HCV core devoid of the C-terminus (DNAcore152) with a forbidden frameshift (not supporting F-protein formation [36]). Rabbits were immunized with DNAcore152 by two closely spaced priming injections (double prime, four rabbits), in two rabbits followed by a series of boosts performed first with one-month and then with four-month intervals. The latter scheme was applied in view of earlier experiments in chimpanzees which demonstrated gradual increase if there were proliferative responses to HCV core after repeated boosts performed under long period of time [4]. Contrary to the HCV core 1-152 immunized rabbits, rabbits receiving injections of DNAcore152 exhibited low but consistent proliferative response to both HCV core and core-derived peptides ( Figure 5(a)), boosted by the booster DNA injections ( Figure 5(b); Supplementary Figure S2). Rabbits immunized with repeated injections of DNAcore152 developed also a low-level humoral response to HCV core, weakly boosted after the repeated gene administrations ( Figure 5(c)). No anticore responses were registered in rabbits mock-immunized with empty vector DNA (data not shown).
Immunization with DNA encoding HCV core devoid of the 39 amino acids on the C-terminus allowed shifting the immune response to almost exclusively Th1 type as manifested by weak but consistent core-specific proliferative responses of PBMCs and low level of anti-core antibody production resembling the profiles observed in the primate trials of the multicomponent immunogens including diverse forms of HCV core (DNA, recombinant virus, protein) [4][5][6]. These results fall in line with our earlier observations made in the DNA-immunized mice; namely, the induction of cellular response to HCV core does not require high levels of HCV core protein (low amounts provided by cells in vivo transfected at immunization sites appear to be sufficient). Furthermore, we could show that truncation, at least partial, of the ROS-inducing core domain may rescue cellular response. Additional positive input was possibly made by forbidding the formation of Fprotein. The actual role of immune competition from the Fprotein is currently being assessed in a series of mouse immunizations.
Again, as in the case of protein immunizations, the specificity of B-and T-cell response to HCV core-derived peptides in DNA-immunized rabbits resembled that observed in the HCV infection [70]. Core-specific T-cell responses were persistent and boostable, resembling responses observed in the self-limiting rather than chronic HCV infection which is characterized by a gradual loss of the specific T-cell response [17,71].  Figure 5: Anti-HCV core immune response induced by single and repeated immunizations with synthetic gene encoding core aa 1-152 (DNAcore152). Rabbits were regularly bled; PBMCs were isolated and subjected to stimulation with HCV core-derived peptides and recombinant HCV core aa 1-152. Stimulation indexes (SI) observed after double priming (week 4) (a); boosting of T-cell response in rabbits receiving multiple injections of DNAcore152 illustrated by stimulation indexes exhibited by PBMCs of rabbits 101 and 102 before and after boost 5 at weeks 54 and 56, respectively (b); dynamics of antibody response to HCV core aa 1-152 in rabbits receiving repeated injections of DNAcore152 (c). All antigen stimulation tests were performed in triplicate; SI values represent an average with standard deviation. Test results were discarded if radioactivity incorporation values demonstrated by mitogen PHA were below 1000 counts per minute and if stimulation indexes in response to PHA were below 2. HCV core-specific antibodies response represent an average optical density exhibited by sera of each of the rabbits collected at given time points in two ELISA runs with standard deviations. OD of sera of rabbits immunized with empty vector DNA collected at the same time points did not exceed the optical density of 0.3 (data not shown).

Conclusions
In primate trials, the responses to the structural HCV proteins including the nucleocapsid (core) were shown to significantly correlate to the protection against HCV challenge [1] implying an input of anticore response on the observed protection effects. This emphasizes the necessity of the experiments aimed at achieving an effective core-specific cellular response in larger animals than mice. Rabbits are widely used in the toxicity and safety testing of medical devices, drugs, and vaccines because of both genetic heterogeneity and possibility of the longitudinal follow-up experiments. Here, we used the rabbit model to evaluate the immunogenicity of polypeptides encoded by the 5 terminus of HCV RNA. By polypeptide immunization, we have induced in rabbits a strong humoral immune response to an abundant HCV core form aa 1-173 and the most stable ARFP form, F-protein.
Immunization with HCV core aa 1-173 led to a B-cell response of broad specificity targeting multiple linear epitopes. The Cterminally truncated core 1-152 induced a weaker antibody response directed only against the N-terminus of the protein implicating the role of the C-terminus in promoting humoral immunogenicity. Delivery of the C-terminally truncated HCV core by DNA immunization with a plasmid forbidding frameshift led to the induction of weak but sustained T-cell response to multiple epitopes within the protein. Both B-and Journal of Immunology Research T-cell responses observed in rabbits mimicked that in HCV infection which indicates the promiscuity of major epitopes localized in the polyproteins encoded by the 5 -terminus of HCV genomic RNA. These are promising findings which allow a step forward in the development of the HCV core based prototype HCV vaccines, as the previous data indicated that although HCV core is the main target of an immune response in the infected individuals [72], it is not so immunogenic in the larger species as humans [73] and could even suppress the immune response [14], also heterologous [74]. The immunogenicity of DNA representing the 5 terminus of HCV RNA and of the polyproteins encoded therein and the promiscuity of the observed responses promote the use of rabbit model for the preclinical trials of HCV vaccines, although other adjuvants would be needed to comply with the requests to vaccine formulations. These considerations may be important in further development and testing of HCV vaccines based on the structural viral proteins.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Engineering Chemistry

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Enhancing Butanol Production under the Stress Environments of Co-Culturing Clostridium acetobutylicum/Saccharomyces cerevisiae Integrated with Exogenous Butyrate Addition

In this study, an efficient acetone-butanol-ethanol (ABE) fermentation strategy integrating Clostridium acetobutylicum/Saccharomyces cerevisiae co-culturing system with exogenous butyrate addition, was proposed and experimentally conducted. In solventogenic phase, by adding 0.2 g-DCW/L-broth viable S. cerevisiae cells and 4.0 g/L-broth concentrated butyrate solution into C. acetobutylicum culture broth, final butanol concentration and butanol/acetone ratio in a 7 L anaerobic fermentor reached the highest levels of 15.74 g/L and 2.83 respectively, with the increments of 35% and 43% as compared with those of control. Theoretical and experimental analysis revealed that, the proposed strategy could, 1) extensively induce secretion of amino acids particularly lysine, which are favorable for both C. acetobutylicum survival and butanol synthesis under high butanol concentration environment; 2) enhance the utilization ability of C. acetobutylicum on glucose and over-produce intracellular NADH for butanol synthesis in C. acetobutylicum metabolism simultaneously; 3) direct most of extra consumed glucose into butanol synthesis route. The synergetic actions of effective amino acids assimilation, high rates of substrate consumption and NADH regeneration yielded highest butanol concentration and butanol ratio in C. acetobutylicum under this stress environment. The proposed method supplies an alternative way to improve ABE fermentation performance by traditional fermentation technology.

Introduction
Bio-butanol is not only an important platform chemical [1,2], but also a clean/renewable liquid fuel [2] and powerful fuel additive [3]. Butanol fermentation is also referred as acetone-butanol-ethanol (ABE) fermentation. Nowadays, C. acetobutylicum and corn starch are still the dominated strain and substrate for industrial bio-butanol production in the world. In ABE fermentation, butanol, acetone and ethanol are roughly produced at a mole ratio of 6:3:1 (B:A:E), increasing butanol concentration and ratio without sacrificing the total solvent productivity has been the two major targets pursued by many researchers. ABE fermentation is characterized with severe butanol inhibition. To alleviate the problem, a couple of methods including strain mutagenesis, genetic engineering and metabolic regulation have been implemented, but the entire fermentation performance improvement could not reach the expected level [4]. ABE fermentation with in-situ butanol separation could significantly enhance the productivity of total solvents, particularly that of butanol [5]. However, the economics and the operation complexity of the in-situ butanol separation system limit its practical application. So far, traditional batch ABE fermentation remains the dominated operation mode for industrial bio-butanol production.
Traditional solvent products recovery or purification process costs a huge amount of energy, which limits development of ABE fermentation industry in the future. Actually, the solventsmixture obtained in ABE fermentation could be recognized and directly used as an excellent diesel additive for ordinary diesel engine, as it could reduce natural luminosity intensity or soot formation [3]. In that report, it was showed that the fuel additive with higher butanol ratio could improve engine ignition performance or grade/quality of the diesel, when adding 20% (v/v) ABE solvents-mixture (ABE20) into the diesel. Thus, it is attractive to distill solvents-mixture with higher butanol ratio from ABE fermentation broth in an energy-saving way. Report [6] indicated that 82% of the total solvents could be extracted in a form of ABE solvents-mixture (recovery ratio: butanol 96%, acetone 64%, ethanol 50%) in an unit using 2-ethyl-1-hexanol (EH) as the extractant (volumetric ratio of EH/ABE fermentation broth, 1:1), when the solvents are produced in the well-recognized ratio (B:A:E = 6:3:1). When the extractant containing ABE was directed into a subsequent stripper unit, an acetone-butanol-ethanol ternary azeotropic system could be formed and the solvents-mixture could be 100% stripped on the top of the tower, while the expensive EH could be fully recovered at the tower bottom, allowing its repeated utilization. Production of ABE solvents-mixture based diesel additive by this kind of recovery method would make ABE fermentation process more economic and versatile in applications.
Selectively increasing butanol concentration and ratio are very important in ABE fermentation, which would have two advantages for the diesel additive bio-production: 1) improving quality of the diesel blended with ABE solvents [3]; 2) further increasing solvents extraction yield as the butanol recovery ratio is the highest (96%) with the purification system described by the literature [6]. Many research works have been conducted attempting to obtain higher butanol concentration or ratio (or butanol/acetone ratio), including utilization of mixed substrates [7], screening of hyper-butanol strains [4,8], control of oxidative-reductive potential [9], and addition of electron carriers/pigments such as neutral red and methyl viologen [10][11][12] to create NADH enriched environment to enhance butanol production. However, in those cases, higher butanol ratio was obtained at the expense of reducing total ABE solvents productivity, increasing purification loads (pigments removal), etc. In ABE fermentation, Clostridia have the ability to simultaneously utilize glucose and butyrate to synthesize butanol. It has been well-recognized that, utilizing butyrate as the co-substrate could increase butanol concentration [13] and conversion yield of butanol/substrates [14]. In our previous studies, we enhanced butanol concentration and butanol/acetone ratio in ABE fermentations to the extents of 5*17% and 16*23% respectively, when using reductive power enriched cassava medium to replace corn-based medium [15] or consecutively feeding a small amount of butyrate in Biochemical Pharmaceutical Co., Ltd (CSBIO). CSPC and CSBIO provided support in the form of salaries and consolation money for L. Ge, J. Zhang and Y. Zhao, respectively, but the companies did not have any additional roles in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.
Competing Interests: The authors can confirm that the co-authors L. Ge and J. Zhang are employed by CSPC, and Y. Zhao is employed by CSBIO. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
corn-based ABE fermentation [16]. However, the increments were limited and far below the expectation.
As an important fermentation technique, the method of co-culturing two or more than two microorganisms has been applied in a couple of bioprocesses including food manufactures, bio-degradations, and bio-fuel productions [17,18]. The technique was also reported in a couple of studies related with ABE fermentations. A co-culturing system of C. butylicum and amylase producing B. subtilis was proposed and the system could self-generate amylase and consume dissolved oxygen, allowing ABE fermentation to be more cost effective [19]. The other report showed that, co-culturing C. beijerinckii and C. tyrobutyricum could increase butanol concentration and substrate conversion yield, as the butyrate produced by C. tyrobutyricum could be effectively utilized by C. beijerinckii as a more efficient substrate [20]. Among the potential co-culture systems for ABE fermentation, adaptively adding viable S. cerevisiae cells into C. acetobutylicum culture broth could be considered as a special co-culturing system and promising protocol for enhancing ABE fermentation performance. The translation process of S. cerevisiae was seriously restricted under high butanol concentration environment and the amino acids pools were preserved [21]. There is a possibility to improve ABE fermentation performance by co-culturing C. acetobutylicum with S. cerevisiae: 1) the amino acids might be secreted by S. cerevisiae under its stress environment and then assimilated by C. acetobutylicum, and some of them may be favorable for C. acetobutylicum survival and butanol synthesis; 2) for cells survival, C. acetobutylicum has to compete with the yeast on substrate utilization, so that substrate consumption amount by C. acetobutylicum would be enhanced and more intracellular NADH production required for butanol synthesis could be expected.
In this study, attempting to further enhance butanol concentration and ratio in corn-based ABE fermentation, we proposed a novel fermentation strategy of co-culturing C. acetobutylicum/S. cerevisiae integrated with exogenous butyrate addition, which incorporated the advantages of the co-culturing system and butyrate addition. The effectiveness of the proposed strategy was testified in both anaerobic bottles and fermentor.

Materials and Methods Microorganisms
Clostridium acetobutylicum ATCC 824 was maintained in the form of spore suspension in 5% (w/v) corn meal medium at 4°C. The methods of inoculation and pre-culture were the same as those previously described [22]. Saccharomyces cerevisiae was obtained from Angel Yeast Co., China. It was maintained at 4°C on Yeast extract-Malt extract-Glucose (YMG) agar. Clostridium tyrobutyricum GIM1.262 (ATCC 25755) was obtained from Guangdong Microbiology Culture Center (GIMCC) and maintained at 4°C on Reinforced Clostridium Medium (RCM) agar.

Media and seeds cultures
C. acetobutylicum seed culture method and medium preparation procedures for ABE fermentation were the same as those described in our previous study [23]. Seed culture of S. cerevisiae was conducted in 500 mL Erlenmeyer flasks containing 100 mL medium. S. cerevisiae seed was incubated in a rotating shaker at 200 r/min and 30°C for 24 h. The medium for yeast seed culture and cultivation in fermentor consisted of (in g/L): glucose 20, yeast extract 8.5, (NH 4 ) 2 SO 4 1.3, MgSO 4 Á7H 2 O 0.1, and CaCl 2 Á2H 2 O 0.06. Seed culture of C. tyrobutyricum ATCC 25755 was conducted in 100 mL anaerobic bottles containing 50 mL medium. C. tyrobutyricum seed was incubated in a water bath and 37°C for 24 h. The medium for C. tyrobutyricum seed culture consisted of (in g/L): glucose 30, yeast extract 5.0, peptone 5.0, (NH 4 ) 2 SO 4 3.0, K 2 HPO 4 1.5, NaCl 6.0, MgSO 4 Á7H 2 O 0.6, FeSO 4 Á7H 2 O 0.03, and pH 6.0.

S. cerevisiae cultivation
S. cerevisiae was cultivated in a 5 L fermentor (Baoxing Co., Ltd., China) under the conditions of pH 6.0, air-aeration and 30°C, by transferring 10% (v/v) seed culture broth into the medium. The standard DO-Stat method [24] was adopted for feeding concentrated glucose solution (500 g/L) to ensure both glucose and ethanol concentrations in the broth at near zero level during feeding stage. The cultivation was carried out in parallel with ABE fermentations. When the biomass of S. cerevisiae reached about 20 g-DCW/L (DCW = 0.22×OD 600 ), a small portion of broth was harvested, and then added into the ABE fermentation broth according to requirements.

Butyrate fermentation
Butyrate fermentation by C. tyrobutyricum was conducted in the 7 L anaerobic fermentor. The fermentation medium was the same as the seed medium except the initial glucose concentration (60 g/L). The loading volume of fermentation medium was 4.0 L. The temperature and pH during fermentation were maintained at 37°C and 6.0, respectively. 10% (v/v) of C. tyrobutyricum inoculum was transferred into the fermentor and the broth was aerated with N 2 for 10 min to remove the dissolved oxygen. When the residual glucose concentration in broth dropped below 10 g/L, about 80 mL of glucose solution (600 g/L) was fed into the broth to bring glucose concentration up to 25 g/L. The fermentation was ended at 60 h when butyrate concentration reached about 28.5 g/L. The butyrate fermentative supernatant was then collected and concentrated in a rotary vacuum evaporator at 80°C and 0.03 MPa, to a concentrated level of 50.0 g/L. The supernatant was filtrated (0.22 μm) for sterilization and then kept in a refrigerator at 4°C. The supernatant was used in ABE fermentation to replace the chemically synthesized butyrate according to the requirement.

ABE fermentation
The corn flour (starch content about 70%, w/w) was obtained at local market. The medium was pretreated by adding a tiny amount of α-amylase (8 U/g-corn flour, heated in boiling water bath for 45 min) and then glucoamylase (120 U/g-corn flour, heated at 62°C for 60 min). Subsequently, the viscosity reduced medium was autoclaved at 121°C for 15 min. ABE fermentations were carried out at 37°C in 100 mL anaerobic bottles containing 50 mL fermentation medium (corn flour contents 15%, w/v). 10% (v/v) of C. acetobutylicum seed broth was transferred into the bottles. When C. acetobutylicum had been cultured for 24 h, S. cerevisiae broth harvested in the 5 L fermentor was added into the anaerobic bottles with the adding amount of 0.04-0.40 g-DCW/L-broth. According to the requirements, concentrated butyrate solution (pH adjusted to 6.0) amounted 4.0 g/L-broth was also added into the bottles at the same time. ABE fermentations were conducted in a 7 L anaerobic fermentor (Baoxing Co., Ltd., China) as well. A temperature controllable water bath was used to circulate warm water into the coil pipes settled inside the fermentor to maintain broth temperature at 37°C. The loading volume of fermentation medium was 3.5 L with the initial corn flour contents of 15% (w/v). 10% (v/v) of C. acetobutylicum inoculum was transferred into the fermentor and then the broth was sparged with N 2 for 10 min. The pressure inside the fermentor was controlled in a range of 0.030-0.055 MPa by a manually adjustable pressure valve after self-generated gas began to evolve. According to the requirements, specified amounts of S. cerevisiae broth and concentrated butyrate solution (4.0 g/L-broth) were supplemented at 24 h when ABE fermentation entered solventogenic phase. Here, pH was regulated to 5.0 by adding 3 mol/L NaOH solution.
Determination of the cell viability of S. cerevisiae Viable S. cerevisiae cells were inoculated into the ABE fermentation broth at about 24 h. Then, the ABE broths were diluted in triplicate in a range of 10 7 *10 8 fold at each sampling time, plated on Yeast extract-Peptone-Dextrose (YPD) agar plates and incubated for 24 h at 30°C. C. acetobutylicum could not grow on YPD plate, the viable cell amounts of S. cerevisiae were measured by counting the colony forming units (CFU) on each plate.

Analytical methods
The concentrations of solvents (butanol, acetone and ethanol) and organic acids (butyrate and acetate) were measured by a gas chromatograph (GC112A, Shanghai Precision Science Instrument Co., China) with iso-butanol as the internal standard [23]. The starch content of the broth was determined by the acid (HCl) hydrolysis method and the glucose concentration was calculated as 1.1-fold of starch concentration (g/L) [23]. The amino acids concentrations in the supernatant were determined by an HPLC (Agilent 1100, USA) [25]. The cellular morphological shapes of C. acetobutylicum and S. cerevisiae in pure cultivations and co-culturing system with/without exogenous butyrate addition were pictured by an Olympus BX53 camera (Olympus Corporation, Japan). In fermentor based fermentations, the evolved gas amount was measured by collecting the gas in a graduated tube filled with water every hour. The gas was firstly directed into two absorption bottles connected in-series filled with 6 mol/L NaOH solution, to determine H 2 release amount within a period of 15 min. Then total evolved gas amount was measured in the remaining 45 min without passing through the absorption bottles. H 2 and CO 2 evolution rates (r H2 , r CO2 ) were calculated by Eq 1.
where A H2 and A GAS were H 2 gas formation volume measured in the first 15 min and total gas evolved volume in the rest 45 min, respectively. L was the volume of ABE fermentation broth. A H2 and A GAS were then converted into the unit of mol/L-broth/h (r H2 and r CO2 ) using the ideal gas equation (25°C, 1 atm). Concentrations of residual glucose, ethanol and acetone were smoothed by polynomial fitting with fermentation time as the independent variable, and their consumption/synthesis rates (r GLC , r EtOH and r ACE , mol/L/h) at instant T were then determined by differentiating the fitted concentrations curves with regards to T. Those rate parameters (together with r H2 and r CO2 ) were then used for determining the theoretical NADH regeneration rate for butanol synthesis in C. acetobutylicum that will be described in the following section.
Estimation of formation/consumption of NADH, glucose and ethanol in C. acetobutylicum in the co-culturing system In ABE fermentation by solventogenic Clostridia, butanol synthesis and ratio are controlled by NADH (reductive power) availability or its regeneration rate [26,27]. Butanol synthesis in ABE fermentation could be enhanced if the substrates (glycerol and mannitol) with more reductive power were used [28]. In our case, experimental analysis of intracellular NADH in C. acetobutylicum is very difficult because the cells concentration was very low and the cells mixed tightly with the solid starch particles or/and S. cerevisiae cells. As a result, we had to estimate glucose consumption rate (r GLC CA ) and NADH production rate (r NADH BtOH ) required for butanol synthesis in C. acetobutylicum for the co-culturing system, based on the measurable fermentation parameters and the reasonable assumptions. As both C. acetobutylicum and S. cerevisiae produce ethanol, a parameter γ was defined as the contribution ratio of ethanol synthesis by S. cerevisiae over the total ethanol produced. Assumptions: 1) In standard ABE fermentation by C. acetobutylicum, the well-recognized stoichiometric mass balance equation of Eq 2 is generally applied [29], while ethanol synthesis by S. cerevisiae yields mass balance equation of Eq 3 (both in mole base).
2) The stoichiometric ratio of r EtOH /r CO2 (mole base) in C. acetobutylicum and S. cerevisiae metabolisms does not vary under the co-culturing operation mode, that is, Here, r EtOH , r EtOH CA , r EtOH SC , r CO2 , r CO2 CA and r CO2 SC represented total (apparent) ethanol production rate, ethanol synthesis rates by C. acetobutylicum and S. cerevisiae, total CO 2 production rate, CO 2 evolution rates by C. acetobutylicum and S. cerevisiae, respectively. The reasonability of the two assumptions will be discussed in the section of "Results and Discussions". Combining Eq 4 and Eq 5, γ could be then calculated by Eq 6.
Based on the mathematical formula reported by our previous study [15], the glucose utilization rate (r GLC CA ) and butanol synthesis oriented NADH utilization rate (r NADH BtOH ) in C.
acetobutylicum could be estimated by Eq 8 and Eq 9, using the calculated γ(T), as well as the measured parameters of r GLC , r EtOH , r CO2 , r H2 and r ACE . In addition, the total glucose amount consumed by S. cerevisiae (GLC SC ) could also be calculated by Eq 10, where MW GLC represented the molecular weight of glucose.

Results and Discussion
Exogenous butyrate addition improving performance of pure ABE fermentation by promoting amino acids secretion and enhancing NADH regeneration rate Butyrate is an important intermediate metabolite in ABE fermentation. In acidogenic phase, butyrate could accumulate to a level of up to 1.0*2.0 g/L; and then it is gradually re-assimilated by C. acetobutylicum cells as a co-substrate during the solventogenic phase, the butyrate formation/re-assimilation closed-loop is thus formed. Butyrate is considered as an alternative substrate for ABE fermentation, which could regulate carbon fluxes in C. acetobutylicum metabolism and effectively increase butanol/carbon-sources conversion ratio [13,30]. Our previous report also indicated that exogenous addition of a small amount butyrate resulted a higher butanol/acetone ratio [16]. Furthermore, exogenous butyrate addition could be considered as a kind of environmental stress to C. acetobutylicum survival, so that it may affect ABE fermentation by C. acetobutylicum in other aspects. Under the stress environment, some substances, such as amino acids, etc. which are beneficial for the cells survival, might be forced to be intracellular accumulated and extracellular secreted. We conducted ABE fermentation by C. acetobutylicum with exogenous butyrate addition in anaerobic bottles and fermentor, and compared the results with those of ABE fermentation without exogenous butyrate addition (control, case #a). The fermentation results were presented in Fig 1. In fermentor case, butyrate was consecutively fed in 7 times, with a total adding amount of 4.0 g/L-broth (case #b). Compared with control, butanol concentration increased from 11.63 g/L to 13.50 g/L, and butanol ratio rose from 0.62 to 0.66 ( Fig 1A and 1B, Table 1). Actually, many literatures have reported the effectiveness of exogenously adding butyrate in improving ABE fermentation performance. The reports indicated that exogenous butyrate additions could alter metabolic flux distribution to direct more glucose into butanol synthesis route [13,16] and the added butyrate could be used as a more efficient carbon source in presence of glucose because of no NADH consumption requirement in converting butyrate into butanol [14,30,31].
On the other hand, many reports showed that, some amino acids, either self-generated or exogenously added, are favorable for butanol synthesis or cells survival in ABE fermentation by C. acetobutylicum, which would lead to the performance improvement. A study using a chemically defined medium, showed that exogenously adding aromatic family amino acids (phenylalanine and tyrosine) has positive effects on butanol synthesis (butanol concentration rose from 12.0 g/L to 14.2 g/L) and cells growth (maximum biomass increased from 3.44 g/L to 4.57 g/L) [32]. Another report indicated that aspartic family amino acids (lysine and methionine) could up-regulate butanol synthesis [33]. We also added aromatic and aspartic family amino acids, as well as their combinations, into the anaerobic bottles based ABE fermentation broth when it entered into solventogenic phase at 24 h, attempting to improve ABE fermentation performance. In our case, the largest improvement in ABE fermentation occurred when adding aspartic family amino acids (0.80 g/L-broth lysine and 0.20 g/L-broth methionine), butanol concentration and butanol/acetone ratio increased from 11.59 g/L to 12.98 g/L and 1.91 to 2.27, respectively. However, improvement in ABE fermentation performance by exogenously adding those amino acids was very limited.
By exogenously adding 4.0 g/L-broth butyrate into ABE fermentation broth in 7 L anaerobic fermentor, first of all, we found that the "favorable" amino acids of phenylalanine, tyrosine and methionine (expect lysine) could be secreted into the broth at certain amounts, while the secretions actually reflected their accumulations in C. acetobutylicum. Fig 2A and 2B depicted the secretion patterns of the four amino acids in the ABE fermentations with/without butyrate addition. With butyrate addition, those amino acids except lysine gradually secreted and their concentrations increased continuously, while the amino acids concentrations remained unchanged in control case. Based on the results, we speculated that, exogenous butyrate addition stimulated intracellular accumulation of the three amino acids favorable for butanol synthesis and cells survival. Alsaker et al. studied the metabolic mechanism of the anaerobe C. acetobutylicum based on gene-expression-based analysis, when implementing butanol and butyrate stress [34]. Their results revealed that some genes, which encoded phenylalanine and tyrosine biosynthesis, were differentially upregulated by butanol and butyrate stress. This conclusion was consistent with our findings of the amino acids secretion patterns in case of exogenously adding butyrate (case #b).
A report indicated that the exogenously added butyrate (molecule form) could penetrate into the C. acetobutylicum cells, and then the dissociation equilibrium of CH 3 CH 2 CH 2 COOH $ CH 3 CH 2 CH 2 COO − + H + was formed intracellular inducing the formation of excessive proton H + [35]. As a result, exogenous butyrate addition might not only stimulate amino acids secretions but also enhance the regeneration rate of NADH (r NADH BtOH , calculated by the prediction model Eq 9, γ = 0) required for butanol synthesis in C. acetobutylicum. As shown in higher than that of control (case #a) during the solventogenesis phase, the enhanced r NADH BtOH was also responsible for the enhanced butanol concentration and ratio. Therefore, the synergetic effect of amino acids intracellular accumulation, enhanced intracellular r NADH BtOH , as well as metabolic regulation by exogenous butyrate addition, contributed to ABE fermentation performance improvement co-operatively. However, the increments in butanol concentration and ratio were limited with this butyrate addition strategy. Therefore, further advanced fermentation strategies were explored.
The possibility of improving ABE fermentation by co-culturing C. acetobutylicum/S. cerevisiae by investigating interactions and dynamics of S. cerevisiae and C. acetobutylicum under each stress environments First of all, we speculated that S. cerevisiae has the ability to survive under its in-favorable conditions (high temperature, high butanol/butyrate concentrations, strict anaerobic, etc.) by secreting some substances, such as amino acids, etc. to resist the stress environment. If the amino acids secreted by S. cerevisiae could be assimilated or utilized by C. acetobutylicum to protect these cells against the high butanol concentration in ABE fermentation, then ABE fermentation performance improvement could be expected. The environments of ABE fermentation by C. acetobutylicum is considered to be a stress to S. cerevisiae. The doubts may be raised with regards to the yeast survival ability and the lose of investigated the butanol tolerance in a selection of microorganisms, which indicated that S. cerevisiae could tolerate 1-2% (w/v) butanol environment [36]. Another report also revealed that S. cerevisiae could still maintain about 10% relative growth under 2% butanol environment [37]. Furthermore, butyrate, either self-generated or exogenously added may further generate the stress environment to S. cerevisiae. Therefore, we conducted the following preliminary S. cerevisiae cultivation experiments in 100 mL anaerobic bottles, under the environments of strictly anaerobic, 37°C and glucose sufficiency, by intently adding butanol (B, 10 g/L), acetone (5 g/L), butyrate (4 g/L) and butanol/butyrate (B&B, 10 g/L butanol + 4 g/L butyrate) to simulate the interactions and dynamic behaviors of S. cerevisiae under ABE fermentation environments. Fig 4A and 4B showed the cultivation results, including S. cerevisiae growth, ethanol synthesis, and the secretions of the four amino acids favorable for C. acetobutylicum cells survival of and butanol synthesis in C. acetobutylicum. S. cerevisiae growth were severely inhibited but ethanol synthesis was not much repressed, under the conditions of exogenously adding butanol, butyrate and butanol/butyrate. In these cases, cells growth still continued and did not stop completely as shown in Fig 4A. The secretion amounts of the four "favorable" amino acids were effectively enhanced (Fig 4B). On the other hand, the environments of high ethanol concentration (> 10 g/L) created by S. cerevisiae cultivation could be considered to be a stress to ABE fermentation by C. acetobutylicum in turn. Fig 4C showed the impacts of exogenous ethanol addition on ABE fermentation performance including the synthesis amounts of butanol/ acetone/ethanol and total gas production amount. The results revealed that ABE fermentation performance of EtOH5 (5 g/L ethanol addition) and EtOH10 (10 g/L ethanol addition) was comparable to that of control. Therefore, it could be concluded that ethanol accumulation up to 10 g/L could not deteriorate the metabolic viability of C. acetobutylicum and its abilities of synthesizing butanol, acetone and ethanol.

Effect of C. acetobutylicum/S. cerevisiae co-culturing system on ABE fermentation
Based on the results of the above mentioned preliminary experiments, a C. acetobutylicum/S. cerevisiae co-culturing system was proposed, which might potentially enhance ABE fermentation performance in the following three aspects: 1) the amino acids secreted by S. cerevisiae might be assimilated by C. acetobutylicum for more efficient butanol synthesis; 2) when C. acetobutylicum co-exists with S. cerevisiae, C. acetobutylicum has to compete with the yeast on substrate utilization to increase its substrate consumption rate, leading to a higher production rate (r NADH BtOH ) of NADH required for butanol synthesis in C. acetobutylicum metabolism; 3) Due to the facts that the yeast and Clostridia strains were competing on substrate utilizations and consuming substrate simultaneously, glucose consumed amounts might be largely increased. If some of the extra consumed glucose could be utilized by C. acetobutylicum and butanol conversion yield from glucose did not vary, then extra butanol production (concentration) would be expected. Theoretically, S. cerevisiae growth and the associated ethanol synthesis could be suitably repressed by certain manner to ensure C. acetobutylicum metabolism to be dominated in the co-culturing system. The viable S. cerevisiae adding amount and time, as well as temperature of the co-culturing system were optimized in anaerobic bottles by orthogonal design experiments. The preliminarily optimal conditions of temperature, the yeast adding amount and instant were determined as 37°C, 0.2 g-DCW/L-broth and 24 h after initiating the ABE fermentations. The major results of fermentor based ABE fermentation by co-culturing C. acetobutylicum/S. cerevisiae (case #c) and the comparisons with control (case #a) were shown in Figs 1-3 and Table 1. In this case (case #c), butanol concentration only slightly increased from 11.63 g/L to 11.91 g/L and butanol ratio dropped from 0.62 to 0.46 (though butanol/acetone ratio rose from 1.98 to 2.22), this was because final ethanol concentration accumulated to a very high level of 8.42 g/L and ethanol even became the secondly ranked end-product in ABE fermentation. In this case, the metabolic activity of S. cerevisiae was too high, and the metabolic competition between C. acetobutylicum and S. cerevisiae was not properly controlled. This actually deteriorated the ABE fermentation performance.
Similar as the case of pure ABE fermentation by C. acetobutylicum with exogenous butyrate addition (case #b), the three "desirable" amino acids (methionine, phenylalanine and tyrosine) also gradually secreted and their concentrations increased continuously, but almost no lysine secretion was observed as well ( Fig 2C). In case #c, concentrations of the four amino acids were lower than those of case #b. We speculated that the secreted amino acids in case #c mainly originated from S. cerevisiae, as C. acetobutylicum did not suffer any other extra environmental stress except the higher ethanol accumulation. As shown in Fig 4C, C. acetobutylicum metabolic activities and ABE synthesis ability could not be deteriorated under ethanol stress environment if its concentration does not exceeded 10 g/L, thus 8.42 g/L ethanol accumulation in case #c would not stimulate the extracellular amino acids secretions. In case #b, the higher amino acids secretion concentrations (Fig 2B) implied a higher intracellular amino acids accumulation in C. acetobutylicum, which led the higher butanol concentration and ratio in turn. The secreted amino acids concentrations in case #c were higher than those of control (case #a) but lower than those of case #b. There was a possibility that, some of the secreted amino acids could enter into C. acetobutylicum cells due to the existence of extracellular-intracellular amino acids concentration gradient, but this was not enough to create equivalently enriched intracellular amino acids environment as that in case #b. As a result, we came to a conclusion that, the amino acids secreted by the yeast in the co-culturing system could not improve ABE fermentation performance.
As mentioned above, in the co-culturing system, the Clostridia and yeast strains would compete on substrate utilizations and thus increase substrate consumption amounts. Certain part of the extra consumed substrate would be utilized by C. acetobutylicum for possible butanol and ABE productions enhancement. At the same time, intracellular NADH required for butanol synthesis could also be over-produced, as all of NADH is generated in the glycolysis route in C. acetobutylicum metabolism. Therefore, we attempted to use the previously described mathematical model (Eq 2-Eq 10) to estimate ratios of the extra substrate consumed by C. acetobutylicum and by S. cerevisiae, as well as the NADH regeneration rate r NADH BtOH in C.
acetobutylicum. This will definitely help us to find out the problems in the co-culturing system and to explore the ways to solve the problems. Here, a simplified metabolic network map covering both C. acetobutylicum and S. cerevisiae metabolisms was summarized in Fig 5. As shown in Fig 1 and Table 1, total glucose consumption amount largely increased from 51.3 g/L to 71.3 g/L by co-culturing the two strains. Using the proposed mathematical model, the primary secondary fermentation parameters in the co-culturing system (case #c) could be calculated, and the parameters included glucose comsumption rate r GLC CA , NADH regeneration rate r NADH BtOH and ethanol synthesis amount EtOH CA in/by C. acetobutylicum, as well as the extra glucose amount consumed by S. cerevisiae GLC SC . As shown in Fig 3, r GLC CA was effectively increased as compared with those of pure ABE fermentations by C. acetobutylicum with/without exogenous butyrate addition (case #a, control; case #b). However, the enhancement in r NADH BtOH was limited and r NADH BtOH was even lower than that of case #b. As for ethanol formation, S. cerevisiae contributed 80-90% (average γ%0.8*0.9) of the total ethanol synthesized amounts. Furthermore, the model estimated that, 72% of the extra consumed glucose was used by S. cerevisiae to over-produce ethanol while only 28% of the extra consumed glucose was utilized for ABE solvents production by C. acetobutylicum. Therefore, the ABE fermentation strategy of co-culturing C. acetobutylicum/S. cerevisiae could largely increase total glucose consumption amount and effectively increase r GLC CA , however, only 28% of the extra consumed glucose was used by acetobutylicum leading to a very limited r NADH BtOH enhancement in turn. This strategy failed to improve the entire ABE fermentation performance. It must be addressed that two assumptions were made when developing the prediction model (Eqs 2-11) to estimate the primary secondary parameters in evaluating the co-culturing based ABE fermentations. The two assumptions (Eq 5 and Eq 6) were the stoichiometric coefficients of ethanol and CO 2 formation rates in S. cerevisiae and C. acetobutylicum, and they should be reasonable under the following technical backgrounds or aspects. First of all, under the strict anaerobic ABE fermentation environment, almost 100% of glucose could only be consumed via the metabolic route of GLC ! 2 EtOH + 2 CO 2 (Eq 3) in S. cerevisiae, which was supported by the facts that the yeast growth ceased (Fig 4A and Fig 6) and only a tiny amount of glucose was used for cell maintenance. This tiny amount of glucose could be completely ignored in developing the prediction model. Secondly, in the standard ABE fermentation by C. acetobutylicum, the well-recognized stoichiometric mass balance equation (Eq 2) is generally applied, with the butanol/acetone/ethanol mole ratio of 6:3:1 [29]. There would be less doubt regarding the first assumption (Eq 5), but argument might be raised on the second assumption (Eq 6), which is if the r EtOH CA /r CO2 CA ratio of 0.0474 could still be held in the cases of co-culturing based ABE fermentations. We used ethanol and CO 2 formation rates to evaluate the coculturing system based on the following reasons: 1) ethanol and CO 2 are the only two measurable metabolites produced by both C. acetobutylicum and S. cerevisiae, therefore it is reasonable to utilize the two parameters to model the co-culturing system; 2) glycolysis route (GLC ! acetyl-CoA) in C. acetobutylicum metabolism is quite robust against environmental disturbances as no branched nodes exist in the route, and a couple of literatures [11,29] reported that the ethanol mole ratio over total ABE solvents could be solidly maintained at 0.1 (0.11/(0.63+0.315 +0.11)%0.1, Eq 2) by different but traditional operation modes without gene manipulation involvements; 3) in pure and co-culturing based ABE fermentations, 100% H 2 is from the Environment Stress Improving ABE Fermentation electron transport shuttle system in C. acetobutylicum metabolism (Fig 5). Although the higher intracellular butyrate concentration would induce excessive production of proton H + , the excessive H + could be consumed in the following two routes: NAD + H + ! NADH and 2H + ! H 2 under the catalysis of hydrogenase [35]. If hydrogenase activity is not affected by the coculturing system or/and butyrate addition (no reports were found yet regarding this), in both cases H 2 would still be produced with the fixed stoichiometric coefficient of 1.26 (Eq 2), and the excessive H + could be consumed in the route of NAD + H + ! NADH to over-produce NADH which is actually beneficial for butanol synthesis. In the case of existing large variations in butanol/acetone ratio (mole B/A, for example B/A largely varied from 2.0 to 3.0) which may associate with using the co-culturing system or/and adding butyrate, the stoichiometric coefficients variations in Eq 2 could be re-calculated by re-balancing the elements of C, H and O. Under this condition, r EtOH CA /r CO2 CA ratio and ethanol ratio over total ABE in Eq 2 only vary in the very narrow ranges (r EtOH CA /r CO2 CA : 0.0469*0.0474; ethanol ratio: 0.104*0.107). In addition, similar as the yeast metabolism, when ABE fermentation entered the solventogenesis phase, the cell growth also stopped and only very little amount of glucose is consumed on cells maintenance as well. As a result, we believed that the two assumptions (Eq 5a & Eq 5b) are reasonable and robust enough in handling and predicting the behaviors of ABE fermentations with possible/large variations in butanol/acetone ratio.
Novel fermentation strategy of co-culturing C. acetobutylicum/S. cerevisiae in coupling with exogenous butyrate addition significantly improving ABE fermentation performance In C. acetobutylicum metabolism, a portion of the exogenously added butyrate could be directly converted into butanol via the metabolic route of butyrate ! butyryl-CoA ! butanol (intracellular) ! BtOH ex (butanol in supernatant) as shown in Fig 5a. However, this metabolic route would not work without the existence of glucose, because 1 mol butyrate would consume 2 mol NADH to produce 1 mol butanol [13]. The required NADH has to be supplied by the glycolysis route in C. acetobutylicum metabolism. C. acetobutylicum has a complicated α-amylase and glucoamylase secretion structure to gradually hydrolyze the corn starch into glucose first, and then ABE synthesis is carried out by the hydrolyzed glucose. On the other hand, the pure coculturing system could significantly increase total glucose consumption amount. S. cerevisiae has stronger glucose utilization ability than that of C. acetobutylicum, the hydrolyzed glucose was utilized by S. cerevisiae in priority leading to a very low ratio of extra substrate consumed by C. acetobutylicum (28%). In principle, the exogenously added butyrate has to be quickly used out to relieve the stress pressure of C. acetobutylicum cells against the high butyrate concentration environment, thus the added butyrate could be considered as an "inducer" for accelerating glucose consumption rate in C. acetobutylicum to satisfy the requirements of the stress relieve and butyrate bio-conversion. In this case, most of the extra consumed glucose might be directed into ABE production by C. acetobutylicum, significant enhancement in ratio of extra substrate consumed by C. acetobutylicum and intracellular NADH regeneration rate r NADH B-tOH could be expected. Based on the above speculation, a novel ABE fermentation strategy integrating C. acetobutylicum/S. cerevisiae co-culturing system with exogenous butyrate stress was thus proposed. Table 1 and Fig 1D showed the ABE fermentation performance when implementing the newly proposed strategy. In anaerobic bottles case, both butanol concentration and butanol/ acetone ratio reached the highest levels of 16.34 g/L and 2.97, while the total solvents concentration reached the highest level of 25.34 g/L as well. In 7 L fermentor, final butanol concentration and butanol/acetone ratio also reached the highest levels of 15.74 g/L and 2.83, which were 35% and 43% higher than those of control respectively. Furthermore, the total solvents (ABE) concentration and productivity also increased from 18.77 g/L to 25.15 g/L and 0.29 g/L/h to 0.52 g/L/h, respectively (Table 1). Final butyrate concentration declined to a low level of 0.39 g/ L, indicating the added butyrate was mostly assimilated and utilized by C. acetobutylicum cells as a supplementary carbon source. With the novel fermentation strategy, ABE fermentation performance was significantly improved.
This novel ABE fermentation strategy (case #d) incorporated the advantages of C. acetobutylicum/S. cerevisiae co-culturing and exogenous butyrate addition simultaneously. With this strategy, intracellular accumulations of the amino acids favorable for C. acetobutylicum cells survival and butanol synthesis, as well as NADH required for butanol synthesis in C. acetobutylicum were largely enhanced. As shown in Figs 2D and 3D, secretion amounts of the four "favorable" amino acids and r NADH BtOH were at the highest levels simultaneously among the four fermentations. For the amino acids, methionine, phenylalanine and tyrosine could be secreted from both C. acetobutylicum and S. cerevisiae (Fig 2B-2D), the total secretions were roughly the sum of those obtained in cases #b and #c. Interestingly, by adopting the novel fermentation strategy, lysine secretion was extremely enhanced for 27.5-fold (from 1.67 mg/L to 45.9 mg/L, Fig 2D). A report indicated that up-regulating the synthesis of lysine-specific permease was extremely important in protecting C. acetobutylicum cells under high butanol concentration environment [33]. We could not identify lysine secretion was originated from C. acetobutylicum, or from S. cerevisiae, or from both of them. Even for the most indirect case, that is, lysine was secreted from S. cerevisiae, such a huge amount of secreted lysine is quite possible to be assimilated into and utilized by C. acetobutylicum. The similar phenomenon was also observed in ABE fermentation by co-culturing C. acetobutylicum/S. cerevisiae integrated with exogenous acetate addition (4.0 g/L-broth). In this case, lysine was extensively secreted as well, and up to 25.3 mg/L lysine was released which was about 15.0-fold higher than those of cases #a, #b and #c (S1C Fig). As a result, ABE fermentation performance was also improved correspondingly (butanol: 11.63 g/L!13.91 g/L; acetone: 5.86 g/L!8.27 g/L) [38]. As a result, the novel fermentation strategy greatly stimulated lysine secretion by exogenous additions of either butyrate or acetate. The proposed fermentation strategy also simultaneously enhanced r GLC CA ( Fig 3C) and r NADH BtOH (Fig 3D). The large amount of lysine secretion and enhancement in r NADH BtOH contributed to ABE fermentation performance enhancement co-operatively. More importantly, glucose consumption amount also largely increased from 51.3 g/L (case #a, control) to 69.3 g/L (case #d) with the proposed strategy. In this case, about 72% of the extra consumed glucose was utilized for ABE solvents production by C. acetobutylicum, as the metabolic activity of S. cerevisiae was severely inhibited, average contribution ratio of ethanol synthesis by S. cerevisiae reduced to 40-80% (γ%0.4-0.8), and final ethanol concentration only accumulated at a lower level of 3.84 g/L. We must address that the severe ethanol synthesis (by S. cerevisiae) repression in this case (case #d) was somewhat different from that of "simulated" experiments implemented in the anaerobic bottles, which indicated that ethanol synthesis by S. cerevisiae were not further repressed so much under high butanol+butyrate (B&B) concentrations environments (10 g/L + 4 g/L, Fig 4A). This was because that, glucose rather than corn starch was used, co-existence of C. acetobutylicum and competition of the two strains on substrate utilizations in that case actually did not happen. There would be a concern whether S. cerevisiae can really survive and fulfill the expected metabolic functions under the co-culturing environment. Therefore, we investigated S. cerevisiae cells viability and the theoretical glucose consumption by S. cerevisiae in the cases of coculturing based ABE fermentations with/without exogenous butyrate addition, using CFU analysis method and the mathematical prediction model (Eq 2-Eq 11). As shown in Fig 6, the yeast cells could survive and glucose could be consumed by S. cerevisiae in both cases. However, viable yeast numbers were much less, S. cerevisiae metabolic activity and the glucose amount consumed by S. cerevisiae were much lower in the case of exogenously adding butyrate. As mentioned above, the co-culturing based ABE fermentations with/without exogenous butyrate addition could largely increase the total glucose consumption amount. Compared with the control, the extra glucose consumed amounts were 20 g/L (without butyrate addition) and 18 g/L (with butyrate addition), and the relevant extra glucose amounts consumed by the yeast were 14.3 g/L and 5.3 g/L respectively. In the case of co-culturing the two strains with butyrate addition, ratio of extra substrate consumed by S. cerevisiae decreased from 72% to 29%, glucose utilization ability/efficiency of C. acetobutylicum was enhanced significantly in an indirect manner. Fig 7 showed the morphological shapes of C. acetobutylicum and S. cerevisiae (30 h and 50 h), in the pure cultivations of C. acetobutylicum and S. cerevisiae, as well as those in the C. acetobutylicum/S. cerevisiae co-culturing system with/without exogenous butyrate additions. The pictures clearly indicated that the two strains could co-exist without autolysis under the environments of 37°C and high concentrations of butanol or/and butyrate until the fermentation end (cases #c and #d). From Fig 7E-7H, it could be concluded that the amino acids secretions were originated from the viable yeast or/and Clostridia cells in the co-culturing system, rather than the release due to the yeast autolysis.
Finally, in 7 L fermentor, the proposed ABE fermentation strategy of co-culturing system integrated with exogenous butyrate addition increased butanol concentration from 11.63 g/L (control) to 15.74 g/L with an increment of 4.11 g/L. Therefore, there is a concern with regards to whether the titer increment was contributed by the added butyrate solely or by consuming glucose/butyrate co-operatively, since this is very important and closely related with the economics of the entire fermentation process. It was believed that, the enhanced butanol titer (4.11 g/L) originated from two parts: about 58% of the increment was from the extra consumed glucose (18.0 g/L), and another 42% was from the exogenously added butyrate (4.0 g/L-broth) directly. In our previous study [16], we investigated the theoretical conversion yield from exogenously added butyrate to butanol (Y BtOH/BU ) based on graph theory and experimental data fitting, and this yield was determined as 48% (weight base, 0.48 g-butanol/g-butyrate). By considering this yield (0.48) and the residual butyrate concentration (0.39 g/L, Table 1), contribution of added butyrate to the butanol concentration increment would be about 42% ((4.0-0.39)×0.48/4.11 = 0.42). As for the rest 52% of consumed butyrate, we speculated that, they were assimilated in the C. acetobutylicum cells to maintain the fermentation under enhanced intercellular butyrate concentration environments to contribute another 58% butanol titer increment in an indirect way: 1) the higher intracellular butyrate concentration would induce excessive production of proton [H + ], which is beneficial for butanol synthesis oriented NADH over-production. The exogenously added butyrate (molecule form) could penetrate into the cells, and then the dissociation equilibrium of CH 3 CH 2 CH 2 COOH $ CH 3 CH 2 CH 2 COO − + H + was formed intracellular [35]; 2) the higher intracellular butyrate concentration apparently stimulated the amino acid intracellular accumulation and secretion in either C. acetobutylicum or S. cerevisiae, some of the amino acids are favorable for C. acetobutylicum cells survival and butanol production, though we still do not understand the real mechanism of amino acids secretion under butyrate stress environment as experimental analysis of intracellular butyrate in C. acetobutylicum was very difficult in this study. On the other hand, based on the theoretical calculation of glucose consumed by S. cerevisiae (GLC SC ) using Eq 10, it was estimated that 64.0 g/L glucose was utilized by C. acetobutylicum by deleting GLC SC (5.3 g/L) from the total glucose consumed (69.3 g/L). In this case (case #d, Table 1) and in comparison with control, 12.7 g/L extra glucose was consumed by C. acetobutylicum. If the 12.7 g/L glucose was converted into butanol at the ABE yield on glucose of 39% and butanol ratio over total ABE of 60% [29], then 2.97 g/L butanol (12.7×39%×60% = 2.97 g/L) could be over-produced, which is roughly consistent with the estimation of 58% butanol titer enhancement originated from glucose (4.11×0.58 = 2.38 g/L, 2.38 g/L versus 2.97 g/L). As a result, though the apparent Y BtOH and Y ABE almost stayed at same levels as those of control, butanol yield on glucose by C. acetobutylicum (Y' BtOH ) was increased from 0.227 to 0.246 (Table 1).
Besides the amino acids intracellular accumulation and extracellular secretions, the highest r NADH BtOH and the extra glucose consumed by C. acetobutylicum achieved by the proposed fermentation strategy, the strategy might also affect the activities of the major intracellular enzymes regulating C. acetobutylicum metabolism. Among the enzymes, CoA-transferase (CoAT) encoded by ctfAB gene is most important, which could re-assimilate the organic acids (acetate and butyrate) formed, convert acetate and butyrate into acetyl-CoA and butyryl-CoA. As addressed many times, experimental analysis of the key enzymatic activities in C. acetobutylicum was very difficult in our case. However, we still measured the transcriptional levels of ctfAB in different batches by real-time fluorescence quantitative PCR analysis method. In solventogenesis phase, the normalized transcriptional level of ctfAB did not vary in different runs. The result indicated that, variations in butanol concentration/ratio were not due to variations in the transcriptional level. Table 1 summarized the comprehensive performance comparisons of ABE fermentations with different operation strategies.
The application potential of the novel ABE fermentation strategy integrating C. acetobutylicum/S. cerevisiae co-culturing system with exogenous butyrate addition Many investigators have reported the effect of exogenous butyrate addition and solventogenic Clostridia co-culturing mode on ABE fermentation performance. As shown in Table 2, exogenous butyrate addition (2.78-4.95 g/L) could enhance butanol concentration and butanol/ acetone ratio by pure Clostridia cultivation. In the case of co-culturing C. beijerinckii/C. tyrobutyricum with glucose as the substrate, butanol concentration and ABE productivity enhanced by 33.3% and 173.3%, but butanol/acetone ratio decreased by 34.5%. With the proposed ABE fermentation strategy integrating C. acetobutylicum/S. cerevisiae co-culturing system with exogenous butyrate addition, butanol concentration, butanol/acetone ratio and total ABE productivity were enhanced 35%, 43% and 34%, respectively. There would be some arguments with regards to whether total ABE production and butanol ratio over total ABE solvents are really improved when using the proposed ABE fermentation strategy, as S. cerevisiae also produces ethanol which varies butanol ratio in turn. It was true that the apparent butanol ratio over total ABE solvents only slightly increased from 62% to 63% (compared with control, Table 1), which was even less than that of pure ABE fermentation with exogenous butyrate addition (from 62% to 66%, case #b). However, we must address that, with the novel fermentation strategy, butanol ratio actually increased from 62% to 70% by C. acetobutylicum metabolism, if ignoring the extra ethanol produced amount by S. cerevisiae (contribution ratio of 71%, Fig 3B). Nevertheless, the large increase in butanol concentration would improve the quality of the ABE solvents-mixture based diesel additives, as the butanol recovery ratio is the highest (96%) in the ABE purification system described by the literature [6], while the recovery ratio for ethanol is the lowest (50%). With the ABE mixtures achieved by the proposed strategy (butanol 15.74 g/L, acetone 5.57 g/L, ethanol: 3.84 g/L, Table 1), ABE ratio in the purified diesel additive could reach a desirable level of 74:17:9 (weight base). Many reports showed that, butanol ratio and total ABE concentration could be altered by the methods of metabolic engineering or gene knockout. Butanol ratio could increase from 70% to 80.05% by disrupting the acetoacetate decarboxylase gene (adc) in C. acetobutylicum EA 2018, but total ABE production decreased from 19.1 g/L to 9.3 g/L [8]. In another report, when the alcohol/aldehyde dehydrogenase gene (aad) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol synthesis and CoA-transferase (CoAT) was down-regulated to minimize acetone synthesis, high alcohol concentrations (butanol and ethanol: 13.19 g/L and 13.82 g/L), total ABE production (30.55 g/L) as well as butanol/acetone ratio (3.72) could be achieved, but butanol ratio reduced from 64.2% to 43.2% [39]. In addition, the unstable hereditary features of the engineered mutants limit their practical applications in fermentation industrial sectors. In this study, we attempted to increase overall ABE fermentation performance by traditional fermentation technique, using wild-type C. acetobutylicum and dominated raw material-corn starch. We believed that the novel fermentation strategy is industrially practical.
Butyrate is recognized as an excellent substrate for butanol production under the existence of glucose [13,14]. However, the price of butyrate is equivalent to that of butanol, utilizing chemically synthesized butyrate as the co-substrate is not industrially feasible. In this study, we also conducted butyrate fermentation by C. tyrobutyricum in a 7 L anaerobic fermentor with glucose as the substrate, and then adding the butyrate containing supernatant into ABE fermentation broth, attempting to implement the proposed ABE fermentation process in a more economic way. The butyrate fermentation by C. tyrobutyricum ended at 60 h, and final butyrate concentration reached 28.5 g/L by consuming 75.2 g/L glucose. The yield of butyrate on glucose (Y Bu/GLC ) was 0.38, which is much higher than butanol yield on glucose (Y BtOH , 0.23*0.27). This provided the possibility for improving ABE fermentation economics. We used a vacuum flash-evaporation unit to distill water out at the top of the unit, and concentrated the fermentative butyrate supernatant from 28.5 g/L to 50.0 g/L at the bottom, then the concentrated butyrate supernatant subject to the pre-treatment was directly added into the coculturing based ABE fermentation broth to replace the chemically synthesized butyrate. Concentrating the fermentative butyrate supernatant using the vacuum flash-evaporation unit and adding the concentrated supernatant into ABE fermentation broth were because that, we wished to avoid the big volume variations in ABE fermentation due to the dilution effect. As shown in Table 1, in this case (#e), butanol concentration also increased to 14.91 g/L in ABE fermentation by adding 300 mL of the concentrated supernatant which was equivalent to 4.0 gbutyrate/L-broth, with the ABE fermentation broth increment from 3.5 L to 3.8 L. Table 3 summarized a rough economical evaluation of proposed fermentation strategy by exogenously adding butyrate synthetic butyrate and the butyrate fermentative supernatant. From the table, it could be concluded that, the co-culturing system based ABE fermentations by adding either pure chemically synthesized butyrate or butyrate fermentative supernatant would have net gross profits, but the profits would reduce at certain extent in butanol and total ABE productions when considering extra starch consumptions induced by the butyrate additions as compared with those of the standard ABE fermentation (control). However, we believed that the gross profits decrease in those cases could be compensated by the high ABE productivity, high butanol ratio, and the reduced purification cost for distilling fermentation broth with higher butanol concentrations. Finally, we must address that the proposed ABE fermentation strategy by co-culturing C. acetobutylicum/S. cerevisiae integrating with exogenous butyrate stress is only effective for corn-based ABE fermentations by C. acetobutylicum, which on the other hand, still dominate the current industrial bio-butanol production in the world. Table 3. Economical evaluation of the proposed fermentation strategy using synthetic butyrate and butyrate fermentative supernatant.