The Lymphatic Anatomy of the Breast and its Implications for Sentinel Lymph Node Biopsy : A Human Cadaver Study Background Current understanding of the lymphatic system of the breast is derived mainly from the work of the anatomist Sappey in the 1850s , with many observations made during the development and introduction of breast lymphatic mapping and sentinel node biopsy contributing to our knowledge . Management of the lymph nodes has been an important part of breast cancer treatment since the time of Halsted 's radical mastectomy .1 Until the 1970s it was based on the perceived need to remove cancer cells before they spread via the lymphatics beyond the reach of the surgeon . The national surgical adjuvant breast and bowel project B04 study questioned the therapeutic benefit of axillary clearance , however it remained part of the treatment of invasive breast cancer because of the prognostic significance of the axillary lymph nodes status . Sentinel node biopsy -LRB- SNB -RRB- has replaced axillary dissection in clinically node negative patients ,2 -- 4 with trials confirming a false negative rate of 5 -- 10 % . While it is agreed that SNB is appropriate , the optimal injection sites of dye and/or colloid has not been defined .5 -- 7 There are advocates of peritumoral ,8 -- 9 dermal and subdermal10 -- 12 or subareolar and periareolar injection13 -- 14 of the tracers . These recommendations regarding injection sites are derived from clinical experience rather than from anatomical studies . The anatomical basis of axillary clearance was derived from the publications of Sappey in the 1870s15 who showed the breast lymphatics as separate from those of the underlying torso , with a subareolar plexus of lymphatics , and a small number of large lymphatic vessels draining into axillary lymph nodes -LRB- Fig. 1 -RRB- . Despite the revolution in the surgical approach , there has been no advance in the underlying anatomical knowledge of the breast lymphatics . Fig. 1 . Sappey 's drawing of the superficial lymphatics of the upper torso -LRB- left -RRB- and female breast -LRB- right -RRB- in 1874.15 The lymphatics start as an avalvular lymph capillary network . In the skin they commence in the superficial dermis with diameters of 20 -- 70 μm .16 They drain to precollectors that are up to 300 μm diameter , contain valves , have some smooth muscle cells in their wall ,17 and drain this network to subcutaneous lymph collecting vessels -LRB- Fig. 2 -RRB- . The lymph collecting vessels -LRB- lymph collectors -RRB- have a smooth muscle layer18 and are the vessels commonly identified as lymphatics . The breast develops as a modified skin gland appendage and expands into the subcutaneous tissues .19 Fig. 2 . Schematic diagram of the relationship between the lymph capillaries , precollectors , and lymph collecting vessels . Progress in microsurgery has enabled new methods for investigating the lymphatic system . We developed a protocol for injecting radio-opaque contrast medium into lymph vessels of < 0.3 mm diameter in cadavers . The contrast agent fills the draining lymph vessels , which are then radiographed .20,21 We have previously published detailed investigations of the lymphatics of the upper limb .22 -- 24 This study addresses the lymphatic anatomy of the breast and anterior upper torso . Our findings may explain the clinical experience in lymphatic mapping and sentinel node biopsy , and also the persistence of a false-negative rate of 5 -- 10 % irrespective of the experience of the surgeon . Materials And Methods Bilateral anterior upper torso specimens , which included both breasts , were harvested from 10 cadavers -LRB- 4 male and 6 female -RRB- with incisions across the root of the neck , down the posterior axillary line and across the abdominal wall , just above the umbilicus . Another four unilateral studies were obtained from separate cadavers -LRB- 1 male and 3 females -RRB- by a midline incision of the sternum , thus resulting in a total of 24 sides . Protocol refinements at the beginning of the study limited early results and there were always time limits to complete the work , especially on the contralateral side of bilateral subjects , before the specimen became putrefied . The injection and dissection for each side of a specimen took 4 -- 5 weeks . The specimens were stored at − 20 °C prior to dissection and at 4 °C during the dissection . The lymphatic mapping technique has been described previously .20 -- 21 Briefly 6 % hydrogen peroxide was injected into the tissue being examined . Bubbles appear in the tissue and the lymphatic collectors and an operating microscope -LRB- Stemi 2000 , Carl Zeiss Pty. Ltd. . Germany -RRB- is used to identify and cannulate the lymphatic vessels with a 30-gauge needle -LRB- Precision glide needle : Becton Dickinson & Co. , USA . -RRB- or an extruded glass cannula for very small vessels . Radio-opaque contrast medium containing lead oxide -LRB- P3O4 Red lead , AJAX Chemicals , Australia -RRB- was injected antegrade with a micromanipulator -LRB- UM-3C , Narishige Co. , Japan -RRB- . The contrast stopped after a variable distance because of coagulation requiring further cannulation and injection . This was repeated until the first lymph node for each collector was reached -LRB- sentinel node -RRB- . The procedure was performed : around the entire periphery of the specimens in the subcutaneous plane ; around the internal thoracic artery on the inner aspect of the chest ; and in the vicinity of the perforating branches of the internal thoracic artery to identify superficial lymphatics , internal mammary lymphatics , and perforating lymphatics respectively . Indocyanine Green dye -LRB- Pulsion Co. , Germany -RRB- was injected into the internal mammary artery to demonstrate the artery and its perforating branches , which supply blood to the medial aspect of the breast , thus facilitating the identification of associated deep perforating lymphatics . A mixture of blue dye -LRB- Ultamarine , Educational Colours Pty Ltd. , Melbourne -RRB- and hydrogen peroxide was used to stain the lymph collecting vessels originating from the nipple and areolar region . In previous studies of the upper limb we have first injected this blue dye into the dermis before injecting hydrogen peroxide into the subcutaneous tissues to help identify the collecting lymphatics .22 This was helpful in identifying lymphatics in the fingertips , where they exist at high concentration . This dye was injected also into the dermis over the breast mound but was only helpful in the nipple areolar area where again the lymphatics were concentrated . The specimen was radiographed after completion of all injections to give the two-dimensional views of the lymph vessel anatomy . To provide the three-dimensional views , the female specimens were sliced parallel to the lymph vessels as they passed towards the axilla . These slices were then placed on their sides and radiographed to show the position of the lymphatic collectors with respect to the skin and the breast tissue . Radiographs were photographed -LRB- Nikon D100 , Nikon Co. , Japan -RRB- and transferred to the computer , then each lymphatic collector was traced -LRB- Adobe Photoshop CS , Adobe System Inc. -RRB- to its first-tier node and color coded . All collectors draining to the same first-tier node were assigned the same color after tracing them retrogradely from the sentinel nodes . Results Superficial Lymphatic System Lymphatic collectors were identified in the subcutaneous tissues near the peripheral cut edges of the specimens and the lateral border of the sternum . The collectors often branched in the peripheral region then combined to form larger collectors that remained approximately uniform in diameter until they reached the first lymph node -LRB- Figs. 3 and 4 -RRB- . Some collecting lymph vessels joined with others within the same sentinel node territory , rarely with others , before reaching the lymph node . All superficial lymph vessels in these dissections entered a lymph node in the axilla , which was always close to the lateral edge of the pectoralis minor muscle . The findings were similar in both sexes -LRB- Fig. 4 -RRB- . In all of the specimens we examined , many of the lymphatic collectors that passed over or through the breast ended by draining into the same first-tier lymph node . In some , almost the entire breast drained to one sentinel node . In others however , there was at least one other node that was the first-tier node for a collecting lymphatic that passed through part of the breast . Fig. 3 . Radiographs in antero-posterior views of a male -LRB- left -RRB- and female -LRB- right -RRB- specimen after completing injections with the lead oxide mixture . Note that the torso lymph vessels radiate centripetally towards the axilla.Fig . 4 . Tracing distally of lymphatics of both hemi upper torsos -LRB- male : A and C , female : B and D -RRB- from each first-tier lymph node colour coded ; pectoral node -LRB- green , orange , black and yellow -RRB- , subclavicular node -LRB- light blue -RRB- , and internal mammary node -LRB- red -RRB- . Note -LRB- i -RRB- that the lymph collecting vessels from the nipple and areolar region on each specimen drain into the green-colored lymph node ; -LRB- ii -RRB- the similar pattern of chest and breast drainage between the male and female studies ; -LRB- iii -RRB- that the breast lies in the pathway of collecting lymphatics that start peripherally and -LRB- iv -RRB- that , although the majority of the breast drains to one sentinel node in D , every breast area is drained by more than one first-tier node in each study . The lymphatics deep to the nipple and areola area were different from those of the other areas we examined . Microscope-assisted dissection of the areolar region revealed a dense network of lymph capillaries and precollectors in the dermis . This structure is presumably the subareolar plexus identified by Sappey . In most areas , dye injected subcutaneously over the breast mound was not taken up by the lymphatics . Dye injected in the nipple and areolar region was taken up by 2 or 3 superficial lymph collecting vessels via lymph capillaries and precollectors -LRB- Fig. 5 -RRB- each side of the areola and these drained into a first tier node in the pectoral group of axillary nodes -LRB- shown green in Fig. 4 -RRB- . Fig. 5 . Photograph of dissection in the areolar region after injecting the mixture of dye and hydrogen peroxide into the nipple . Lymph capillaries and precollectors -LRB- black arrows -RRB- , and lymph collecting vessels -LRB- white arrow -RRB- were stained with the blue dye . The radiograph of the cross-sectioned female specimen showed that the lymphatics have a `` wavy '' path through the superficial part of the subcutaneous tissue of the chest -LRB- Fig. 6 -RRB- . In the breast region most of the lymph collectors ran between the dermis and the breast tissue . However , some passed through the breast tissue itself . After traversing the breast all of the collectors passed deeply to reach axillary lymph nodes . Fig. 6 . The diagram -LRB- above -RRB- shows how the breast of Fig. 4B was sectioned to provide cross-section radiographs -LRB- middle of the breast and its lymphatic drainage -LRB- below -RRB- . Note that some lymph collectors originating from the lower torso run through the breast tissue in both A and B sections . Internal Mammary Lymphatic System The internal mammary lymphatic vessels were identified alongside the internal mammary artery and vein , deep to the parietal pleura , with lymph nodes present in the intercostals spaces . Collecting lymphatics were identified beside the perforating branches of the internal mammary arteries that were found in the deep medial aspect of the breast tissue -LRB- Fig. 7 -RRB- . A single collector passed though the intercostal fascia and muscle beside each perforating artery to join the internal mammary lymphatic system . Fig. 7 . The appearance of the perforating lymphatics -LRB- orange -RRB- along the branch from the internal mammary artery -LRB- green -RRB- in the right lateral sternal region of the female specimen . There was no apparent connection between the collecting lymphatics accompanying the branches of the internal mammary artery and the superficial collecting lymphatics . Contrast injected into the superficial collecting lymphatics never flowed into the perforating collecting lymphatics . Even in the subcutaneous tissues overlying the lateral edge of the sternum , the superficial collecting lymphatics passed laterally towards the axilla . We found no evidence of direct anastomosis between the superficial collecting lymphatics and the collectors associated with perforating arterial branches , at the size of the collecting vessels . However , this does not exclude a connection at either the precollector or lymphatic capillary network as we were unable to demonstrate them radiologically . Discussion Cruikshank was the first to report the lymphatics of the breast in his book published in 1787.25 He used the cadaver of a pregnant woman injecting mercury into the mammary duct from the nipple . He reported seeing by chance mercury-filled lymphatics but did not make any diagrams of his findings . In 1874 , Sappey applied the same method ,15 injecting mercury into the dermis of a semiputrefied cadaver to identify the superficial lymph vessels . A very thin adult cadaver was used and he described the superficial lymphatic drainage of the torso , dividing it into four territories by a sagittal midline and a horizontal line at the L2 level . The upper torso lymphatics always drained into the ipsilateral axilla -LRB- Fig. 1 -RRB- . Sappey reported that the lymphatics of the breast collected in a subareolar plexus and then drained towards the axilla via lymph collecting vessels . Sappey 's description of the breast lymphatics was adopted by anatomists and became the theoretical basis for the subareolar injection of dye and/or isotope for lymphatic mapping as part of the sentinel node biopsy for breast cancer more than one century later .13 -- 14 In 1903 , Poirer and Cuneo summarized Sappey 's results .26 They added their results from foetal studies using with Gerota 's method27 using oil painting dye to stain the lymphatics , they reviewed other people 's studies and published a comprehensive anatomy book of the lymphatic system . Poirer and Cuneo 's famous picture of the lymphatics of the breast -LRB- Fig. 8 -RRB- was redrawn and is still being used in Gray 's Anatomy .28 Fig. 8 . Poirier and Cuneo 's summary diagram of the breast lymph drainage .26 This diagram was composed based on the anatomical and clinical findings of several people , including Sappey . In 1959 , Turner-Warwick performed photographic and radiographic studies of the collecting lymphatics injecting them with iron Prussian blue or radioisotope gold -LRB- Au198 -RRB- during surgery in breast cancer patients .29 He demonstrated lymphatic pathways that passed direct from the tumor injection site in the breast to the axillary lymph nodes that bypassed the subareolar plexus . He found that all quadrants of the breast drained to the axilla and either the internal mammary or posterior intercostal nodes . He suggested that Sappey had mistaken the mammary duct for a lymphatic vessel , thereby overemphasizing the importance of the subareolar plexus . The more recent users of lymphoscintigraphy examinations of the breast are also skeptics of the centripetal lymphatic route towards the subareolar plexus . Tanis30 and Uren -LRB- Uren RF : personal communication , May 2007 -RRB- stated that there is no constant route via the subareolar plexus . Our current anatomical knowledge of the breast lymphatics still depends on Sappey and Poierer and Cuneo 's diagrams . Since Sappey used thin adult cadavers and Poirer and Cuneo used infant cadavers , the relationship between the superficial lymphatics and the adult breast tissue , and also the relationship of the lymphatic drainage of the breast tissue with that of the surrounding superficial tissues , has not been adequately described . Controversy still exists over the role of the subareolar plexus in the lymphatic drainage of the breast . There has been no concrete evidence of a centripetal anatomical lymphatic pathway that drains the breast tissue towards the subareolar plexus and then , via this plexus , towards the sentinel node . Our direct injection technique , however , gives a comprehensive image of the lymphatic system in individual specimens . Each lymph vessel that enters a lymph node can be traced retrogradely to provide an accurate map of the tissue for which that lymph node is `` sentinel . '' We have photographed , radiographed , and recorded actual lymphatic pathways . In addition , cross-sectional studies have been performed to obtain three-dimensional images of these lymph collecting vessels . We have shown that some of the torso vessels pass from the periphery through the breast tissue on their way towards the axilla . This is discordant with the conventional understanding that they run just underneath the skin . We compared the torso lymphatic vessel pathways and breast tissue lymphatic vessel pathways in adult female cadavers . Although our results do not show conclusively that the lymph drainage of the breast tissue is via the collecting lymphatics that pass through or immediately superficial to the breast substance , this is likely to be the case since -LRB- i -RRB- the lymph collecting vessel has a microporous nature and drains surrounding tissue fluid along its entire course31 and -LRB- ii -RRB- lymphoscintigraphy in breast cancer patients after peritumoral injection , coupled with similar studies with radioactive gold by Turner-Warwick , usually reveals a direct pathway from the injection site to the axillary lymph node ,29 -- 30 not via a subareolar plexus . Therefore we conclude that the lymph collecting vessels that pass through the breast contribute to breast lymph drainage and must be the same vessels shown in lymphoscintigraphy examinations . Embryologically , the development of the human peripheral lymphatics is poorly understood . However , the lymphatics of the foetus represent similar arrangements to those of adults .32 -- 33 We assume that some of the lymph vessels are trapped inside the mammary gland as the breast develops around preexisting superficial lymph collectors . We have color coded a diagram of our dissection to simulate various injection sites in lymphatic mapping and sentinel node biopsy for breast cancer . Figure 4 shows that , if the tracer is injected deep around the purple-colored vessel -LRB- lower outer quadrant -RRB- , it reaches both the green and orange lymph node in the pectoral group . However , if the tracer is injected into the subareolar region or into the lower outer quadrant intradermaly near the tumor , it reaches only the green node . This anatomical analysis suggests a mechanism for false negative sentinel node biopsy since more than one sentinel node drains the breast in this example . False-negative results following sentinel node biopsy are of the order of 5 -- 10 % in different series .34 -- 37 Reasons given to this have been technical ; either related to : The surgeons ' experience with the technique orThe size of the radioactive tracer which may not reach the lymph node , especially if sited in a peritumoral position . Our results now offer an anatomical explanation for these false negative results as a distinct third possibility . Perforating lymphatics that pierce the deep fascia are critical when discussing breast lymph drainage . The lymphatic system is classified conventionally into the superficial system and the deep system because of their relationship to the deep fascia . However , we subdivided the system draining the tissues above the deep fascia into the superficial system and the perforating system -LRB- Fig. 9 -RRB- . The perforating system is connected to the deep lymphatic system and these collecting vessels have the same appearance as the superficial lymphatics as they course with the internal thoracic blood vessels . Variations of the blood supply to the breast have been reported .38 -- 42 Therefore , we hypothesize that the perforating lymphatics may have the same variations . This helps explain the fact that in all quadrants of the breast , cancer has the potential to metastasize via the internal mammary lymphatics especially if the tumor is medial or deep in the breast parenchyma .43 -- 44 It is accepted that intradermal injection of tracer rarely demonstrates the internal mammary lymphatics .10 -- 12 The variable contribution of perforating lymphatics along the branches of the internal mammary artery to lymphatic drainage of the breast can not be predicted clinically and therefore the likelihood of sentinel nodes being located along the internal mammary artery is also unpredictable . Our anatomical analysis suggests that accurate lymphatic mapping requires peritumoral injection . Fig. 9 . Our concept of the breast lymph drainage , drained by both the perforating lymphatic system and the conventional horizontal superficial lymphatic system with their relationship to the lymphatic system beneath the deep fascia . Conclusions Current anatomical knowledge of the breast lymphatics is derived from the work of Sappey , Poirier , and Cuneo . Our anatomical cadaver study of the relationship of the lymphatics of the torso and the adult breast goes a step further and provides additional information which has special significance for sentinel lymph node biopsy . We have used a refined protocol to accurately record the anterior upper torso lymphatics in adult male and female cadavers . The patterns of the superficial lymphatics were no different between sexes and frequently more than one sentinel node drained the breast . The cross-sectional studies of the female breast showed some lymph vessels of the torso coursing through the breast tissue . We found also perforating lymphatics coursing with similar branches of the internal mammary artery and vein . This anatomical analysis suggests that -LRB- i -RRB- peritumoral injection is preferable for identification of the sentinel lymph node for breast cancer treatment and -LRB- ii -RRB- may help explain the incidence of negative sentinel node studies where only the subareolar plexus is injected with the radioisotope tracer .

O O O O O O O O O O B I I I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

sentinel lymph node biopsy
lymphoscintigraphy
peritumoral injection
subareolar injection
cadaver study


DoD2006 : molecular biology database update DoD2006 , an updated version of Database of Databases is an online resource maintained collectively by ProGene Biosciences and Department of Inorganic and Analytical Chemistry , Andhra University . It links to all molecular biology databases that appeared in Nucleic Acids Research 2006 database issue . DoD2006 includes 873 databases , of which , 858 are derived from Nucleic Acids Research database issue and 15 are collected from In Silico Biology , Bioinformation journals and Google Scholar search . Each database has a search option , keyword help and a brief description with direct link to the database home page . The database is freely available online at http://www.progenebio.in/DoD/index.htm Background Online availability of databases is increasing due to their utility by structural and molecular biologists . These databases are generated using a variety of programming languages and they differ in content and access methods . -LSB- 1 -RSB- The information explosion in biology resulted in database proliferation and this necessitates the importance of data integration , storage and retrieval . Many new databases tend to appear every year , with ever increasing information or knowledge from biology , driven by genome sequencing projects and easy internet access . -LSB- 2 -RSB- On the contrary , fund crunching is a limitation for data collection , constant updates , infrastructure facilities and maintenance of databases . Biological databases with infrastructural funding are hosted by academic , governmental organizations and industry as well as non-profit organizations . -LSB- 3 -RSB- Collection and analysis of data from various databases is essential for computational biologists . But it is time consuming and proportional with increasing number of databases . In this context , there is a need to integrate all databases at a single interface , usually with one search engine to query all databases . Therefore , we partially address the issue by maintaining the molecular biology databases at a single interface , with links to their respective pages . In practice , there are both legal and technical impediments to cross-database communication which include digital locks , firewalls and the inability of search engines to recognize the data . -LSB- 4 -RSB- Methodology DoD2006 Content DoD2006 is an updated version of DoD . -LSB- 5 -RSB- It is a cross-database interface with 858 databases collected from Nucleic Acids Research -LSB- 6 -RSB- , Bioinformatics -LSB- 7 -RSB- , BMC Bioinformatics -LSB- 8 -RSB- and other journals -LSB- 9 -RSB- and 15 databases collected from In Silico Biology -LSB- 10 -RSB- , Bioinformation -LSB- 11 -RSB- journals and Google Scholar -LSB- 12 -RSB- search . The current update with 873 databases against 719 reported in 2005 -LSB- 5 -RSB- represents additional databases that continue to grow despite the demise of a few . -LSB- 13 -RSB- A small number of inaccessible databases -LRB- MitoPD , ISSD -LSB- 14 -RSB- -RRB- reported in 2005 are unchanged in DoD2006 so as to enable the users to know the type of database that once existed . The complete list of databases is given in the website -LSB- 15 -RSB- and their categorization is given in Table 1 . During the search , results are displayed in a separate window as this helps in further database scan without losing the home page . The screen-shot of nucleotide database is given in Figure 1 . A hyperlink to ` Keyword help ' lists keywords used to scan respective databases and the description of different links is given in our earlier publication . -LSB- 5 -RSB- Features of DoD2006 Newly appended databases in DoD2006 are distinguished from the existing ones with a ` + ' sign and inaccessible ones are recognized by a ` * ' sign before the name . All databases in each category are given alphabetically . The appended databases are colored blue and the remaining in green color . Those who wish to have their database to be listed can contact the authors . DoD2006 provides an excellent and essential resource for molecular and computational biologists . Research through computational tools and software using databases has been recognized as a front-line research coupled with experimental design . Our database suits to deliver the wide biological content through the cross-database interface . Updates to DoD shall be made on yearly basis .

O O B I B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O

database
molecular biology
biological databases


Lactic acid production from lime-treated wheat straw by Bacillus coagulans : neutralization of acid by fed-batch addition of alkaline substrate Conventional processes for lignocellulose-to-organic acid conversion requires pretreatment , enzymatic hydrolysis , and microbial fermentation . In this study , lime-treated wheat straw was hydrolyzed and fermented simultaneously to lactic acid by an enzyme preparation and Bacillus coagulans DSM 2314 . Decrease in pH because of lactic acid formation was partially adjusted by automatic addition of the alkaline substrate . After 55 h of incubation , the polymeric glucan , xylan , and arabinan present in the lime-treated straw were hydrolyzed for 55 % , 75 % , and 80 % , respectively . Lactic acid -LRB- 40.7 g/l -RRB- indicated a fermentation efficiency of 81 % and a chiral l -LRB- + -RRB- - lactic acid purity of 97.2 % . In total , 711 g lactic acid was produced out of 2,706 g lime-treated straw , representing 43 % of the overall theoretical maximum yield . Approximately half of the lactic acid produced was neutralized by fed-batch feeding of lime-treated straw , whereas the remaining half was neutralized during the batch phase with a Ca -LRB- OH -RRB- 2 suspension . Of the lime added during the pretreatment of straw , 61 % was used for the neutralization of lactic acid . This is the first demonstration of a process having a combined alkaline pretreatment of lignocellulosic biomass and pH control in fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime . Introduction Lactic acid is used throughout the world in manufacturing of food , chemicals , and pharmaceutical products . Recently , there is a lot of interest in biodegradable poly-lactic acid , which is an alternative to petrochemically derived plastic -LRB- Drumright et al. 2000 -RRB- . Chiral pure lactic acid is produced commercially by microbial fermentation of the carbohydrates glucose , sucrose , lactose , and starch/maltose derived from feedstocks such as beet sugar , molasses , whey , and barley malt -LRB- Narayanan et al. 2004 -RRB- . The choice of feedstock depends on its price , availability , and on the respective costs of lactic acid recovery and purification -LRB- Datta et al. 1995 ; Vaidya et al. 2005 -RRB- . As an alternative to these traditional feedstocks , lignocellulosic biomass is an inexpensive and widely available renewable carbon source that has no competing food value . Lignocellulose consists primarily of cellulose and hemicellulose ; polymers build up of mainly hexose sugars and pentose sugars , which are embedded in a matrix of the phenolic polymer lignin . The main pathway to derive fermentable sugars from lignocellulose is through enzymatic hydrolysis by cellulolytic and hemicellulolytic enzymes . A mechanical and chemical pretreatment of the lignocellulose is required to reduce particle size , to modify and/or to remove the lignin , and with that to enhance the accessibility of the polysaccharides for enzymatic hydrolysis -LRB- Claassen et al. 1999 -RRB- . Various chemical pretreatments of biomass have been studied in research and development of lignocellulose-to-ethanol production technology -LRB- Mosier et al. 2005 -RRB- . One is the use of lime -LRB- calcium hydroxide -RRB- at relatively mild temperature conditions -LRB- Chang et al. 1998 -RRB- . Lime as a pretreatment agent has promising potential because it is inexpensive , safe , and its use hardly results in sugar degradation products such as furfural and hydroxymethyl furfural . Nevertheless , this alkaline pretreatment features a relatively high pH value -LRB- > 10 -RRB- of the treated biomass , and at these pH levels , the activity of common cellulolytic and xylanolytic enzymes , necessary for the depolymerization of -LRB- hemi -RRB- - cellulose , is negligible low . Therefore , lowering the pH is essential to achieve an efficient enzymatic hydrolysis of the polysaccharides . One approach to remove calcium hydroxide is by washing the lime-treated biomass before enzymatic hydrolysis -LRB- Chang et al. 1998 -RRB- ; however , this leads to the use of high amounts of water . Another way to lower the pH of the pretreated material is by neutralizing calcium hydroxide with sulfuric acid . Yet , this results in the formation of the low value byproduct gypsum . As an alternative improvement to these approaches , we propose to use the calcium hydroxide present in lime-treated biomass as neutralizing agent for organic acids produced in microbial fermentation processes . To examine this proposed concept , lime-treated wheat straw -LRB- LTWS -RRB- was added fed-batch-wise during a simultaneous saccharification and fermentation -LRB- SSF -RRB- process in a 20-l controlled stirred fermenter containing hydrolytic enzymes and Bacillus coagulans DSM 2314 , a thermophilic bacterium capable to convert both hexoses and pentoses homofermentative to l -LRB- + -RRB- - lactic acid -LRB- Otto 2004 ; Patel et al. 2006 -RRB- . The objective of this research was to evaluate whether high alkaline-treated lignocellulosic biomass -LRB- without neutralization -RRB- can be used directly in a SSF process by -LRB- 1 -RRB- providing a carbon source for enzymatic hydrolysis and fermentation and -LRB- 2 -RRB- providing a source of alkali to control the pH in the fermentation process . Materials and methods Feedstock and pretreatment Wheat straw was selected as a lignocellulose model feedstock and was purchased from a farm in the Northeast of The Netherlands . The wheat straw was air dried -LRB- 89.5 % -LSB- w/w -RSB- dry matter -LSB- DM -RSB- -RRB- and ground through a 2-mm screen . The lime pretreatment was performed by filling two 15-l mixers -LRB- Terlet , The Netherlands -RRB- , both with 1,650 g ground wheat straw , 13 kg tap water , and 165 g calcium hydroxide . This wheat straw suspension was heated and kept at 85 °C for 16 h under continuously stirring at 30 rpm . The LTWS suspension was subsequently cooled to 30 °C , dehydrated by placing the LTWS in a cotton bag , and pressing the suspension using a manual piston press at pressure up to 9.7 kg/m2 . After dehydration , an amount of 11.45 kg LTWS with an average DM content of 27.0 % -LRB- w/w -RRB- and pH 11.8 was obtained and served as substrate for further experiments . The chemical composition of LTWS was determined as described by van den Oever et al. -LRB- 2003 -RRB- . Enzyme preparation The enzyme preparation GC 220 -LRB- Genencor-Danisco , Rochester , USA -RRB- containing cellulase , cellobiase , and xylanase activity of 116 , 215 , and 677 U/ml , respectively -LRB- Kabel et al. 2006 -RRB- , was used for this study . The preparation had a specific gravity of 1.2 g/ml and contained 4.5 mg/ml glucose , 2.9 mg/ml mannose , and 0.8 mg/ml galactose . Microorganism and preculture The bacterium B. coagulans strain DSM 2314 was used as the lactic acid-producing micro-organism . Bacterial cells were maintained in a 10 % -LRB- w/w -RRB- glycerol stock solution and stored at − 80 °C . Chemicals , unless indicated otherwise , were purchased from Merck -LRB- Darmstadt , Germany -RRB- . Gelrite plates were prepared with a medium containing -LRB- per liter -RRB- : glucose , 10 g ; Gelrite , 20 g -LRB- Duchefa , Haarlem , The Netherlands -RRB- ; yeast extract , 10 g -LRB- Duchefa -RRB- ; -LRB- NH4 -RRB- 2HPO4 , 2 g ; -LRB- NH4 -RRB- 2SO4 , 3.5 g ; Bis -- Tris , 10 g -LRB- USB , Ohio , USA -RRB- ; MgCl2 6H2O , 0.02 g ; and CaCl2 .2 H2O , 0.1 g. Glucose and Gelrite were dissolved in stock solution A -LRB- four times concentrated -RRB- . The pH of this stock solution was adjusted to 6.4 with 2 M hydrochloric acid and autoclaved for 15 min at 125 °C . The remaining nutrients were dissolved in stock solution B -LRB- 1.33 times concentrated -RRB- , which was also adjusted to pH 6.4 with 2 M hydrochloric acid but was filter sterilized -LRB- cellulose acetate filter with pore size of 0.2 μm , Minisart , Sartorius -RRB- . After sterilization , the medium was prepared by combining stock solutions A and B and Gelrite plates were poured . The bacteria were cultivated on Gelrite plates for 48 h at 50 °C . An isolated colony was used to inoculate a 100-ml broth with similar composition and preparation as described above but without the addition of Gelrite . The culture was incubated statically for 24 h at 50 °C and functioned as the inoculum for a 1,400-ml broth . This culture was incubated also statically for 12 h at 50 °C and served as a 10 % -LRB- v/v -RRB- preculture for the SSF experiments . Simultaneous saccharification and fermentation The SSF of LTWS was carried out in a 20-l fermenter -LRB- Applikon , Schiedam , The Netherlands -RRB- with pH and temperature control -LRB- biocontroller ADI 1020 -RRB- . At the start of SSF , the fermenter was filled with 6.0 kg tap water and 1,400 g dehydrated LTWS -LRB- DM content of 27.0 % -LSB- w/w -RSB- -RRB- . The following nutrients were then added to the LTWS suspension : yeast extract , 150 g -LRB- Duchefa -RRB- ; -LRB- NH4 -RRB- 2HPO4 , 30 g ; -LRB- NH4 -RRB- 2SO4 , 52.5 g ; MgCl2 6H2O , 0.3 g ; and CaCl2 2H2O , 1.5 g . The LTWS suspension was then heated to 50 °C , and the pH was adjusted to 6.0 with 101 g 3 M sulfuric acid -LRB- ~ 30 g H2SO4 -RRB- . The SSF process of LTWS to lactic acid consisted of three phases : -LRB- 1 -RRB- the prehydrolysis phase of preloaded LTWS , -LRB- 2 -RRB- the fed-batch phase with automatic feeding of LTWS from a screw feeder , and -LRB- 3 -RRB- the batch phase with pH control by a calcium hydroxide suspension and no LTWS feeding . A schematic representation of the experimental setup is shown in Fig. 1 . The prehydrolysis was initiated by the addition of 40 ml enzyme preparation -LRB- 88 mg enzyme/g DM substrate -RRB- to the LTWS suspension and was incubated for 2 h at 50 °C under continuously stirring at 250 rpm . The fed-batch phase was initiated by the addition of 1,500-ml preculture of B. coagulans DSM 2314 to the fermenter . The lactic acid produced by the bacteria was neutralized by the automatic addition of 8,623 g dehydrated LTWS -LRB- DM of 27.0 % -RRB- to the fermenter through a feeder -LRB- K-Tron Soder Feeders , Canada -RRB- and was regulated by the pH of the medium , which was set at 6.0 . Throughout the fed-batch phase , an amount of 280 ml of enzyme preparation -LRB- total enzyme loading of 98 mg/g DM substrate -RRB- was added proportional to the LTWS addition rate into the fermenter . During the batch phase , the pH was controlled at 6.0 by the addition of 20.0 % -LRB- w/v -RRB- calcium hydroxide suspension . Samples were withdrawn for DM , substrate , and -LRB- by -RRB- product analysis . Fig. 1Schematic representation of the simultaneous saccharification and fermentation of lime-treated wheat straw to lactic acid Analytical methods For the analysis of monomeric sugars , the fermentation broth samples were centrifuged -LRB- 3 min at 17,400 × g -RRB- , and the pH of the supernatant was adjusted to 5.0 with barium carbonate using a pH indicator -LRB- Bromophenolblue -RRB- followed by filtration of the liquid . The analysis was performed by high-performance anion-exchange chromatography using a Carbopack PA1 column -LRB- column temperature of 30 °C -RRB- and a pulsed amperometric detector -LRB- ED50 ; Dionex , Sunnyvale , CA , USA -RRB- . Before injection , the system was equilibrated with 25.5 mM NaOH for 10 min at a flow rate of 1.0 ml/min . For the separation of monomeric sugars , at injection , the mobile phase was shifted to deionized water for 30 min . Postcolumn addition of sodium hydroxide was used for detection of the neutral monomeric sugars . The determination of soluble oligomeric sugars was performed by centrifugation for 5 min at 3,000 rpm -LRB- Centaur 2 , Beun de Ronde , The Netherlands -RRB- of preweighed samples and freeze drying the supernatant overnight . Pellets were weighed and hydrolyzed with sulfuric acid , and neutral monomeric sugars were determined according to the method as described by van den Oever et al. -LRB- 2003 -RRB- . For the calculations , an average molecular weight of oligomers from glucan and xylan of 166 and 132 g/mol , respectively , were applied , resulting in a hydrolysis factor of 1.08 and 1.14 , respectively . For the analysis of insoluble polymeric sugars , samples of 25 g were centrifuged for 5 min at 3,000 rpm -LRB- Centaur 2 , Beun de Ronde -RRB- ; the supernatant was removed , and the pellet was washed by resuspension in 25 ml fresh demineralized water followed by a centrifugation step of 5 min at 3,000 rpm -LRB- Centaur 2 , Beun de Ronde -RRB- . The sequence of resuspension and centrifugation was repeated three times . After the last removal of the supernatant , the pellets were freeze dried overnight . The pellets were weighed -LRB- values used for DM calculation -RRB- , polymeric material was hydrolyzed with sulfuric acid , and neutral sugars were analyzed according to the method as described by van den Oever et al. -LRB- 2003 -RRB- . For the calculations , a molecular weight of glucan and xylan of 162 and 132 g/mol , respectively , were applied , resulting in a hydrolysis factor of polymer to monomer of 1.11 and 1.14 , respectively . The analysis of organic acids was performed by high-pressure liquid chromatography according to the procedure described by Maas et al. -LRB- 2006 -RRB- . The chiral purity -LRB- % -RRB- of lactic acid was determined by derivatization of all lactates using methanol , after which both enantiomers of methyl lactate were separated on a chiral gas chromatography column and detected using a flame ionization detector . The chiral purity was expressed as the area of the main enantiomer divided by the sum of areas of both enantiomers . Calculations The theoretical maximum lactic acid -LRB- LAtheor . max . -LSB- g -RSB- -RRB- production was calculated according the following equation -LRB- Eq . 1 -RRB- : where DMsubstrate = the total dry matter of substrate LTWS -LRB- g -RRB- , Fpolysacch . = fraction polysaccharides per substrate -LRB- g/g -RRB- , HFmonsacch . / polysacch . = hydrolysis factor of polysaccharides , incorporation of water results in 1.11 g hexose from 1.00 g glucan and 1.14 g pentose from xylan and arabinan -LRB- g/g -RRB- , and FF = fermentation factor of 1.00 g lactic acid per gram of monomeric sugar . The efficiency of the enzymatic hydrolysis -LRB- % , w/w -RRB- was based on the amount of hydrolyzed polysaccharides -LRB- g ; calculated by the difference between initial amounts and analyzed insoluble amounts -RRB- divided by the amount of polysaccharides -LRB- g -RRB- initially present in the substrate . The fermentation efficiency -LRB- % , w/w -RRB- is expressed as the amount of lactic acid produced -LRB- g -RRB- divided by the amount of monomeric sugars consumed -LRB- g -RRB- by the bacteria . The overall efficiency of the SSF -LRB- % , w/w -RRB- was calculated by the amount of lactic acid produced -LRB- g -RRB- divided by the theoretical maximum amount of lactic acid -LRB- g -RRB- determined as described in Eq . 1 . Results Simultaneous saccharification and fermentation of LTWS to lactic acid The polysaccharide composition of the LTWS consisted mainly of glucan , xylan , and arabinan of 33.0 % , 19.0 % , and 2.0 % -LRB- w/w -RRB- , respectively , whereas the remaining mass constituted of lignin , ash , extractives , and uronic acids . Some of the soluble components in wheat straw were partially removed by the solid/liquid separation -LRB- dehydration -RRB- of the LTWS . The focus of this study was on the conversion of glucan , xylan , and arabinan , which are the predominant polysaccharides present in LTWS and accounted for 99.8 % -LRB- w/w -RRB- of the total polymeric sugars . Previous work showed that the cellulase preparation GC 220 , used for the saccharification of polysaccharides , functioned optimally at 50 °C and pH 5.0 -LRB- Maas et al. , submitted for publication -RRB- , whereas growth conditions for B. coagulans DSM 2314 were 54 °C and pH 6.5 -LRB- Otto 2004 -RRB- . In this study , both the enzymatic hydrolysis and the fermentation occurred simultaneously in the same reactor at compromising conditions , which were set at 50 °C and pH 6.0 . The SSF of LTWS to lactic acid was studied in a 20-l controlled stirred fermenter . Previous results showed that when this process was performed without a prehydrolysis of an initial amount of LTWS , the concentration of monomeric sugars was low and resulted , therefore , in relatively low lactic acid productivity . As a consequence , the fed-batch addition rate of the alkaline substrate to neutralize the produced lactic acid was low -LRB- results not shown -RRB- . To start the fermentation with a substantial initial amount of fermentable sugars -LRB- > 2 g/l -RRB- , a prehydrolysis of 378 g LTWS and enzyme preparation -LRB- 88 mg per g DM LTWS -RRB- in approximately 6 l volume at pH 6.0 for 2 h was introduced . This resulted in glucose , xylose , and arabinose concentrations of 2.0 , 0.4 , and 0.3 g/l , respectively -LRB- Fig. 3a -RRB- . The second phase -LRB- II -RRB- was initiated by introducing a 1,500-ml preculture of B. coagulans DSM 2314 . A minor amount of lactic acid produced in the preculture caused a slight pH decrease and was automatically neutralized by the addition of LTWS -LRB- Fig. 2a , b -RRB- . After a lag phase of 4 h , the dissolved oxygen concentration decreased rapidly within 1 h from 100 % to oxygen-limiting conditions of below 1 % -LRB- results not shown -RRB- , and lactic acid production started . At that moment , concentrations of glucose , xylose , and arabinose of 3.3 , 0.7 , and 0.3 g/l , respectively , were present -LRB- Fig. 3a -RRB- . These sugars were consumed simultaneously where glucose was utilized faster than xylose and arabinose . Simultaneous with the consumption of these monomeric sugars , lactic acid was produced , which was neutralized by the automatic addition of alkaline LTWS . By the addition of alkaline substrate throughout the fed-batch phase , the pH was maintained accurately at 6.0 ± 0.1 -LRB- Fig. 2a , b -RRB- . At the end of phase II , a total amount of 10,023 g dehydrated LTWS -LRB- ~ 2,706 g DM LTWS -RRB- and 320 ml of enzyme preparation was added to the fermenter . A lactic acid concentration of 20.5 g/l supernatant was detected -LRB- Fig. 3b -RRB- , corresponding to a total of 342 g lactic acid . The chiral L -LRB- + -RRB- purity of lactic acid was determined at 99.4 % , which is similar to that obtained with xylose as the sole carbon source -LRB- Otto 2004 -RRB- . Fig. 2Control of pH -LRB- a -RRB- during simultaneous saccharification and fermentation of lime-treated wheat straw by commercial enzyme preparation GC 220 and B. coagulans DSM 2314 -LRB- b -RRB- . The areas between the dotted lines represent the prehydrolysis phase -LRB- I -RRB- , the fed-batch phase -LRB- II -RRB- with pH control by addition of alkaline LTWS and enzymes , and the batch phase -LRB- III -RRB- with pH control by addition of Ca -LRB- OH -RRB- 2 suspension . Extra enzyme preparation GC220 was added at the times indicated by the arrowsFig . 3Profiles of glucose -LRB- empty square -RRB- , xylose -LRB- empty diamonds -RRB- , arabinose -LRB- empty triangles -RRB- -LRB- a -RRB- and lactic acid -LRB- filled diamonds -RRB- -LRB- b -RRB- in simultaneous saccharification and fermentation of lime-treated wheat straw by commercial enzyme preparation GC 220 and B. coagulans DSM 2314 . The areas between the dotted lines represent the prehydrolysis phase -LRB- I -RRB- , the fed-batch phase -LRB- II -RRB- , and the batch phase -LRB- III -RRB- . Extra enzyme preparation GC220 was added at the times indicated by the arrows At the end of phase I , a low acetic acid concentration was detected in the medium , which increased to 1.5 g/l throughout phase II but remained constant during phase III -LRB- results not shown -RRB- . This indicates that acetic acid was most likely not a fermentation product formed by B. coagulans . Acetic acid can be released upon solubilization and hydrolysis of hemicellulose during chemical pretreatment -LRB- Palmqvist et al. 1999 -RRB- . By the dehydration procedure of the LTWS , part of the acetic acid was easily separated from the substrate by removing the press water . Apparently , a remaining amount of acetic acid was fed together with the substrate to the fermenter . Furthermore , minor traces of other organic acids such as succinic acid and formic acid -LRB- < 0.5 g/l -RRB- were detected in the fermentation broth . Phase III was initiated by changing the pH control from the addition of alkaline LTWS to a 20 % -LRB- w/v -RRB- calcium hydroxide suspension . To maintain the pH at 6.0 , the addition of calcium hydroxide suspension occurred relatively fast but shifted , however , after a few hours to a lower addition rate indicating a decline of the volumetric lactic acid productivity -LRB- Figs. 2b , 3b -RRB- . To exclude limitation -LRB- e.g. , by inactivation -RRB- of enzymes , an extra dosage of enzyme preparation -LRB- 80 ml -RRB- was added to the fermenter after 23.5 h of incubation . This resulted immediately in a slight acceleration of the calcium hydroxide addition rate indicating an increased lactic acid productivity and limitation of enzymatic activity -LRB- Fig. 3b -RRB- . Nevertheless , after 29.7 h of incubation , a decline of the calcium hydroxide addition rate was observed again . Therefore , a second extra dosage of the enzyme preparation -LRB- 240 ml -RRB- was added and resulted this time in a slight accumulation of glucose and xylose of 1.5 and 1.0 g/l -LRB- Fig. 3a -RRB- , respectively , indicating that microbial conversion instead of enzymatic hydrolysis was rate limiting . After 32 h of incubation , a lactic acid concentration of 37.1 g/l was obtained , with a chiral l -LRB- + -RRB- - lactic acid purity of 99.4 % . Continuation of the SSF process to a total incubation period of 55 h resulted in a slightly increased lactic acid concentration of 40.7 g/l supernatant -LRB- ~ 37.8 g lactic acid/kg fermentation broth -RRB- with an overall volumetric lactic acid productivity of 0.74 g l − 1 h − 1 . At this stage , a chiral l -LRB- + -RRB- - lactic acid purity of 97.2 % was analyzed . This slight decline in lactic acid purity is possibly a result of infection with other undesired lactic acid-producing microorganisms . Because the substrate used was not sterile and also the chemical pretreatment and fermentation occurred in an open system under nonsterile conditions , microbial contamination throughout the SSF process is possible . Conversion efficiency The efficiency of the enzymatic hydrolysis of the polymeric material present in LTWS is shown in Fig. 4 . The insoluble polymeric fraction was determined at various time points throughout the SSF experiment . At the end of the prehydrolysis -LRB- 2 h -RRB- of 378 g LTWS , 36 % of the insoluble glucan -LRB- Fig. 4a -RRB- , 55 % of xylan -LRB- Fig. 4b -RRB- , and 62 % of arabinan -LRB- Fig. 4c -RRB- were converted to soluble saccharides including monomeric sugars and oligomeric sugars . After the fed-batch phase -LRB- 13 h -RRB- , 2,706 g LTWS was added and resulted in a conversion of 42 % of glucan , 57 % of xylan , and 63 % of arabinan to products including soluble saccharides and lactic acid . Between 13 and 32 h of incubation , further hydrolysis of the polymeric sugars was observed . However , during the last 23 h of the SSF , minor hydrolysis of the polysaccharides occurred , and this corresponded with the decline in lactic acid productivity during this phase . After 55 h , 398 g of glucan , 130 g of xylan , and 11 g of arabinan was still present as insoluble polymeric material . With these values , the hydrolysis efficiency of the initial glucan , xylan , and arabinan present in LTWS were calculated as 55 % , 75 % , and 80 % , respectively . Fig. 4Insoluble fraction -LRB- empty bars -RRB- and hydrolyzed soluble fraction -LRB- filled bars -RRB- -LRB- g ; calculated by the difference between initial amounts and analyzed insoluble amounts -RRB- of the polysaccharide glucan -LRB- a -RRB- , xylan -LRB- b -RRB- , and arabinan -LRB- c -RRB- at various time points during the simultaneous saccharification and fermentation of lime-treated wheat straw . The figure represents also the percentage of polysaccharide hydrolyzed into soluble products -LRB- filled triangles -RRB- . The error bars denote the deviation of duplicate analysis The monomeric sugars , derived from the LTWS , were partly converted to lactic acid -LRB- 711 g -RRB- by B. coagulans and accounted for 81 % -LRB- w/w -RRB- of the theoretical maximum , indicating the formation of other products such as microbial biomass and carbon dioxide . An overall conversion yield of 43 % -LRB- w/w -RRB- of the theoretical maximum was calculated according to Eq . 1 . The fate of polysaccharides initially present in LTWS after 55 h of incubation is shown in Table 1 . A part of the polysaccharides present in LTWS remained as insoluble polysaccharides -LRB- 37 % w/w -RRB- , whereas a minor part was converted into soluble oligomeric -LRB- 5 % w/w -RRB- and monomeric -LRB- 3 % w/w -RRB- sugars . Another part of the initial polysaccharides present in the LTWS was not recovered in the form of saccharides or lactic acid and was therefore ascribed as ` unaccounted . ' Table 1Fate of polysaccharidesa initially present in lime-treated wheat straw after 55 h of simultaneous saccharification and fermentationFractionPercentage -LRB- % w/w -RRB- Polysaccharides -LRB- insoluble -RRB- b37Oligosaccharides -LRB- soluble -RRB- 5Monosaccharides -LRB- soluble -RRB- 3Lactic acid -LRB- soluble -RRB- 43Unaccounted -LRB- insoluble/soluble -RRB- c13Presented values are averages based on duplicate analytical measurements.aTotal of glucan , xylan , and arabinanbPart of the initial polysaccharides remained present as insoluble polysaccharides.cPart of the initial polysaccharides was not recovered and therefore denoted as ` unaccounted . ' Neutralization of acid by alkaline substrate The lactic acid produced -LRB- 342 g -RRB- during the fed-batch phase -LRB- II -RRB- was neutralized with alkaline-pretreated wheat straw . During this phase , an amount of 2,328 g LTWS was added to the fermenter . Together with this substrate , an amount of 230 g calcium hydroxide was added to the fermenter and accounted for a ratio of 0.67 g calcium hydroxide per gram of lactic acid . The lactic acid -LRB- 369 g -RRB- produced during the batch phase -LRB- III -RRB- was neutralized with 163 g calcium hydroxide resulting in a ratio of 0.44 g lactic acid per gram calcium hydroxide . Discussion Lignocellulosic feedstocks are considered as potential attractive substrates for the production of bulk chemicals . Pretreatment of biomass is required to break open the lignocellulosic matrix , and an enzymatic hydrolysis is necessary for the hydrolysis of polymeric carbohydrates . The lime pretreatment has proven to enhance enzymatic digestibility of the polysaccharides present in lignocelluloses -LRB- Chang et al. 1998 ; Kaar and Holtzapple 2000 -RRB- and results , in comparison to other pretreatment routes , in minor inhibitor formation . However , before the enzymatic hydrolysis , it is essential to adjust the pH to a level optimal for enzymatic activity . In this study , the reduction in pH by washing or neutralization was omitted by using the alkaline character of LTWS to neutralize lactic acid produced by microbial fermentation during a SSF process . The results showed that the largest part of the polysaccharides in LTWS was converted enzymatically and the resulting sugars were fermented simultaneously to mainly lactic acid by B. coagulans DSM 2314 . Between 10 and 30 h of incubation , the bacteria utilized the monomeric sugars , as soon as they appeared in the medium , resulting in relatively low monomeric sugar concentrations -LRB- < 2 g/l -RRB- . This indicates that throughout this period , the enzymatic hydrolysis was the rate-controlling step . The highest lactic acid productivity was observed during the fed-batch phase and the initial hours of the batch phase and declined rapidly after approximately 20 h of incubation , as shown in Fig. 3b . An extra addition of enzyme preparation showed a slight improvement of the volumetric lactic acid productivity but shifted within a few hours again to a relatively low production rate . A second extra enzyme addition did not affect the lactic acid productivity significantly -LRB- Fig. 3b -RRB- . This addition of new enzymes resulted in a modest liberation of hemicellulose sugars -LRB- xylose , arabinose -RRB- , but no further hydrolysis of glucan occurred . This shows that the remaining glucan was too recalcitrant or not accessible for further hydrolysis , resulting in decreasing lactic acid productivity . Another possible explanation of the decreased lactic acid productivity is the inhibition of enzymes and/or bacteria by the increasing lactic acid concentration . A lactic acid concentration of 40.7 g/l supernatant -LRB- ~ 37.8 g lactic acid/kg fermentation broth -RRB- with a relatively high chiral purity was determined after 55 h of incubation , corresponding to an overall lactic acid yield of 43 % of the theoretical maximum . Moreover , the efficiencies of the enzymatic saccharification and the fermentation were both determined . These calculations showed that , based on residue analysis , at the end of the SSF process -LRB- 55 h -RRB- , 55 % of the glucan , 75 % of the xylan , and 80 % of the arabinan present in LTWS was enzymatically hydrolyzed , which agree well with previously obtained results from experiments aiming to convert LTWS to ethanol . To improve the yield , it is necessary to decrease the recalcitrance or improve the accessibility of polymeric sugars in the LTWS by optimization of the pretreatment procedure . The concentrations of soluble monosaccharides and oligosaccharides in the medium were relatively low , which can be expected in a SSF process . A fermentation yield of 81 % was determined -LRB- related to the amount of monosaccharides released from the LTWS -RRB- and is slightly better than the results obtained by Otto -LRB- 2004 -RRB- who reported the production of 35 g/l lactic acid from 50 g/l xylose as the sole carbon source . Because no other soluble fermentation products were detected , the remaining 19 % of the LTWS-derived monomeric sugars were most presumably converted to bacterial biomass and some carbon dioxide during the aerobic part of the fermentation . Several process parameters can be listed for enhancement of the overall lactic acid yield and productivity such as improving the accessibility of polysaccharides by a more severe lime pretreatment , enzyme dosage , type of enzymes , B. coagulans strain , size and growth phase of inoculum , pH gradient in SSF , and in situ product removal of lactic acid . These issues will be subject to further studies . During the fed-batch phase -LRB- II -RRB- , it was possible to counterbalance the pH decrease caused by lactic acid production by the addition of the alkaline feedstock . This suggests that it is possible to combine lime treatment with the production of other organic acids from lignocellulosic biomass . Throughout this phase , the ratio of calcium hydroxide in LTWS added per produced lactic acid was determined at 0.67 g/g . The theoretical stoichiometric neutralization of 1.00 g lactic acid requires 0.41 g calcium hydroxide . Therefore , only 61 % of the calcium hydroxide initially added to the wheat straw was used for lactic acid neutralization . On the other hand , throughout the batch phase -LRB- III -RRB- , an alkaline/acid ratio of 0.44 g/g was calculated corresponding to 93 % of the added calcium hydroxide suspension used for lactic acid neutralization . The low efficiency of the calcium hydroxide added with the LTWS for lactic acid neutralization during phase II has three possible explanations . First , part of the calcium hydroxide could have been used during the chemical pretreatment of the wheat straw such as the neutralization of acetic acid or other organic acids and/or irreversible binding to the lignin . Second , the calcium hydroxide might be released slowly from the insoluble wheat straw fibers and could therefore partly have been used for lactic acid neutralization in the fed-batch phase . Finally , besides lactic acid production , other acidification reactions could have contributed to the decrease in pH and therefore the demand of alkaline substrate , for instance , the decrease in pH caused by the consumption of ammonium as the nitrogen source by microorganisms -LRB- Guebel et al. 1992 -RRB- . The results in this paper show that it is possible to use lignocellulosic materials for the production of lactic acid . Lignocellulosic biomass is a relatively inexpensive substrate , and this affects feedstock costs for lactic acid production positively . Nevertheless , in comparison to the traditional relatively ` clean ' feedstocks with a well-defined composition , using heterogenic lignocellulosic substrates will require a more intensified downstream processing -LRB- DSP -RRB- to recover and purify the lactic acid from the complex fermentation broth . The costs of feedstock materials and operational costs of the DSP contribute considerably to the overall production costs of lactic acid -LRB- Åkerberg and Zacchi 2000 -RRB- . Whether the cost decrease in using lignocellulosic feedstocks outweighs the potential increasing costs of DSP was not analyzed at the moment . In summary , LTWS was converted into l -LRB- + -RRB- - lactic acid by B. coagulans throughout a SSF process at a 20-l bench scale . The pentose and hexose sugars derived from the polymeric material were utilized simultaneously by B. coagulans resulting in a final lactic acid concentration of 40.7 g/l supernatant , which accounted for 43 % -LRB- w/w -RRB- of the theoretical yield . To our knowledge , this is the first paper demonstrating a process having a combined alkaline pretreatment of lignocellulosic biomass and pH control in organic acid fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime .

B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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