# Captured Glimpses of Modernity and War in Late Qing China

Aglaia De Angeli

**Abstract**: This chapter evaluates the role of photography in witnessing the modernising process in China during the late Qing period and conflicts which stemmed from it. The camera, introduced in China during the First Opium War (1839-1842), allowed Western eyes to record the establishment of trade routes and associated facilities. The photos examined here were taken immediately before and during the Russo-Japanese War. The photographs appear to have been focusing on technological developments in trade infrastructure, but they also captured the conspicuous Japanese and Russian military presence. Consequently, the photographs reveal the Western role in the «development» of China by its incorporation into global trading networks and violent conflicts fought over control of this infrastructure.

**Keywords**: Photography, China, Russo-Japanese War, Infrastructure, Trade

*Some pictures are easily appreciated at first glance, but others – and these are often the most rewarding – require some exploration before they can be fully understood.* Susan Woodford (2018, 6)

Introduction1

The history of photography in China started as a glimpse into the East for the West. The First Opium War (1839-1842) offered a chance to bring back photographic testimony of China. Although most of the photographs taken during the war are long lost, during the span between the First and Second Opium Wars, ephemeral and lasting photographic studios, and known and unknown photographers flourished in China. At the same time, the conflict initiated by Britain against China in order to open the Middle Kingdom to foreign trade, which had been confined to the port of Canton and its factory system, led to the irreversible decline of the last ruling dynasty. The Qing paid a high toll, because this clash between China and the West started a long series of conflicts, which opened China not only to foreign trade, but also to a permanent foreign pres-

1 I would like to thank Stéphane Audrand for his help in identifying and checking the Russian Navy ships. Any mistakes, however, are entirely mine.

Aglaia De Angeli, Queen's University Belfast, United Kingdom<EMAIL_ADDRESS>0000-0003-3948-3612 Referee List (DOI 10.36253/fup\_referee\_list)

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Aglaia De Angeli, *Captured Glimpses of Modernity and War in Late Qing China*, © Author(s), CC BY 4.0, DOI 10.36253/979-12-215-0242-8.11, in Rolando Minuti, Giovanni Tarantino (edited by), *East and West Entangled (17th-21st Centuries)*, pp. 137-157, 2023, published by Firenze University Press, ISBN 979-12- 215-0242-8, DOI 10.36253/979-12-215-0242-8

ence driving an inexorable modernisation process which inevitably proceeded on foreign terms. This modernisation process included and was documented by the technology of photography.

In the late Qing period, the Qing court was struggling with internal insurrections, such as the Taiping Rebellion, while external threats were eroding the tributary system on which they had established their dominant position in East and Southeast Asia. During the last 70 years of the Qing Empire, the Chinese fought against the British and French armies in the Second Opium War (1856-1860), against France for the dominion of Tonkin and Annam in 1885, against Japan during the First Sino-Japanese War (1894-1895), and against a range of Western nations during the Boxer Rebellion of 1900-1901. Their reduced power was ultimately signified by the fact that, during the Russo-Japanese conflict of 1904-1905, they were mere spectators to a war fought by other states over their former territories in Manchuria and Korea. These conflicts were all products of Western forms of modernity and the technology of photography was part of the modernising process.

This chapter aims to analyse how photography was used to document the modernising process in China and the conflicts that accompanied it. To do so, it examines photographs belonging to the Sir Robert Hart Collection and specifically, a selection of snapshots taken by Chinese Imperial Maritime Customs officer, Richard H. Strangman, as well as a number of glass slides and photographs taken by other amateur photographers. All these photos are black and white and, despite involving different media – paper and glass – all bear witness to the period immediately preceding and during the Russo-Japanese War. These photographs offer a simple visual framing of the establishment of new trading networks in the concrete form of railways, warehouses and port facilities. They also reveal the conspicuous military presence both Russians and Japanese put in place around this infrastructure. The images taken by amateur photographers captured in material form a series of transitory moments, which reveal how wartime reality differed from that of peacetime.

The camera is a visual tool and, while photographs provide visual information, they are humanly produced artefacts that require interpretation through being contextualised and scrutinised in their medium, design, structure and composition. We should ask, then, what these photographs, in all of their dimensions, reveal about the modernising process in China and the accompanying conflict, including the role of photography in these processes. The chapter will answer these questions, first by discussing the state of the art of photography in China and the modernising process in the China of the late Qing period; then it will examine photographs as a witness of modernity and war, to finally pinpoint how photography in China can be considered a cross-cultural sight between China and the rest of the world.

#### Photography in Late Qing China

The first photographic images of China to reach the West were taken by Westerners travelling in the country, described by Clare Roberts (2013, 7) as «visiting military personnel, diplomats, traders and wanderers, who sought to document what they saw in the interests of geopolitical ambition and trade, and to satisfy the curiosity of people back home».

Photography was invented in Europe and it is widely agreed that the first successful photograph was produced by the French inventor Joseph Nicéphore Niépce in 1826. The technique was then refined over subsequent decades. In 1839, Louis Daguerre developed a new technique, known as daguerreotype, which produced a one-of-a-kind image impressed on a highly polished, silver-plated sheet of copper.2 In February 1841, William Talbot patented the «calotype», a photographic impression obtained in seconds by exposure of chemically treated paper coated with salt and brushed with a solution of silver nitrate.3 While Daguerre's invention was limited to a single positive, making each daguerreotype unique,4 Talbot's invention allowed multiple positive images to be printed. The first photographic images of China were daguerreotypes, such as those taken by the French diplomat, Jules Itier, at the signing of the first Sino-French Treaty on board *L'Archimède* on 24 October 1844 (Roberts 2013, 10-12; Bennett 2009, 3-6).

While the initial phase of photography required complex processes to take and develop pictures, Roberta Wue (1997, 28) explains that the wet collodion process, invented in England by Frederick Scott Archer, «enabled an unlimited number of paper prints to be pulled from a glass negative, and it produced clear, useable images more efficiently and reliably than the earlier calotype, or paper negative, process». Consequently, «it is only with the invention and widespread adoption of the wet collodion process after 1851 that Hong Kong photographers can begin to be linked with surviving photographs». The greater practicality of the wet collodion process not only offered glimpses of China to the West, but also led to a growing interest in photography within China. Between the two Opium Wars, the first photographic studios opened, located in the Crown Colony of Hong Kong, as well as in the major treaty ports, such as Canton and Shanghai. While most were owned by foreigners, the Chinese photographer, Lai Afang had opened his own studio in Hong Kong by 1870.5 Most of these photographic studios did not last long, yet they produced many of the historical images of China still known today. In addition to the studios, there were also itinerant photographers, who practised their art as pedlars. All of them contributed glimpses of China to Western audiences, and many, such as Pierre Rossier, who worked for the London-based Negri and Zambria com-

3 For further information about Talbot, see Daniel (2004b).

4 For more details on the two types, see Lai (2011, 20).

5 Lai Afang (sometimes spelled Afong) is the best known of the early Chinese photographers.

2 Daguerre continued the work started with his partner Niépce who died in 1833. Together, they aimed to solve the question of «how to make a permanent image using light and chemistry». Though Niépce obtained rudimental results in 1826, it was not until 1839, when Daguerre patented the equipment necessary to produce the daguerreotype, that the process of photography took off. See Daniel (2004a).

pany, played a role in making the commercialisation of Chinese photographs a successful business.6 While studio and itinerant photographers were all professionals, many more amateur photographers also enjoyed the new art, and their images also contributed to the picture of China available in the West at the time.7

Technological advancements in photography resulted in shorter exposure times and the production of multiple-prints became a common business in China. From the mid-1850s, *cartes de visite* became a popular usage of studio photos. These business cards, which included a photograph mounted on a sturdy board, were the first mass-produced portrait photographs. The success of the format rested on the fact that a single glass plate could cheaply produce up to eight different photos. Being cheap they could be sold and bought by the dozen and their dimensions were practical. It became fashion for both foreigners and Chinese alike to add an inscription, an autograph or a message to the cards, which were used as mementos or keepsakes, and they became collectible items from the start (Roberts 2013, 27).

Between the First and the Second Opium Wars, the foreign presence was limited to Hong Kong and the treaty ports of Canton, Fuzhou, Amoy (now Xiamen), Ningbo and Shanghai.

Foreigners were not allowed into inland China, and this was reflected in the pictures produced by foreign photographers during this period. The Second Opium War was a watershed for photography in China because, thanks to the Convention of Beijing signed in 1860, foreigners were subsequently allowed to travel and reside in inland China, and so new subjects appeared before the photographic lens. However, Hong Kong, Canton and Shanghai remained the most important locations for foreign photographic studios, thanks to their coastal locations. The treaty ports had a growing expatriate population who bought portrait sessions and scenic pictures enabling the studios to flourish economically (Lai 2011, 23-24). The opening up of the whole of China to foreigners created opportunities for other foreign photographers with no connections to the treaty port studios. The influx of new photographers can be divided into two groups: first, those who travelled to China to open a business and second, those who travelled to China with the intention of photographing the country and returning shortly to their countries. Well-known Western photographers who worked in China, sometimes maintaining their own studios for a period, were Louis Legrand, William Floyd and William Saunders in Shanghai. Among those who travelled and returned home, John Thomson is the most significant. All these Western photographers played a role in portraying China to the West, as well as offering their services to an expatriate clientele, and also training the first generation of native commercial photographers, including Wen Dinan of the Canton Pun

6 For further information about Rossier's contribution to the photography of China as a profitable business, see Bennett (2009, 53-66).

7 For more information about the first photographic studios, itinerant photographers and amateur photographers, see Bennett (2009).

Lun studio, Luo Yuanyou in Shanghai, Ren Jinfeng in Beijing and Liang Shitai (also known as See Tay), who first worked in Hong Kong, then Shanghai, and finally in Tianjin (Lai 2011, 26-9). The first Chinese photographers learnt the art from Western photographers based in Canton and Hong Kong in the 1840s and 1850s. From the 1860s onwards, Chinese photographers, attracted by new business opportunities arising from the increased trade between China and the West, secured training from commercial photographic studios wherever they were established (Cody and Terpak 2011, 35).

The Second Opium War saw the birth of war photography and, indeed, was the first conflict in which photographic images of war circulated widely among the Western public. Although two British army officers, Major George Malcolm and Dr Richard Woosnam, took a daguerreotype on board the British Fleet as it navigated the Yangzi River during the First Opium War in 1842, none of their images have survived (Lai 2011, 20).8 Therefore, the very first images of war in China are those taken by photographers accompanying the British and French armies as they confronted the Chinese. These photographers included soldiers, some high ranking, and also diplomats. The most famous images of the Second Opium War, however, were taken by a commercial photographer, Felice Beato.

From 1860 onward, the whole of China was opened to Western merchants, traders, diplomats and missionaries. Although now at peace with the foreigners, Chinese military forces faced the massive and bloody Taiping Rebellion. The turbulence of the rebellion impacted photographic activity in the early 1860s. Terry Bennett (2010, xi) explains that business «was slow and the commercial viability of full-time studios in Shanghai and Hong Kong was not apparent until the end of the Taiping Rebellion in 1864». In fact, it was hard to make a living, and «when a photographer went out of business, the stock was generally sold, invariably becoming part of the buyers' inventory and traded under the name of the new studio, or just plain pirated». Roberts (2013, 27) underlines how such practices create difficulty in determining with any certainty which photographs were taken by which photographers during this period.

Despite the difficulties caused by the Taiping Rebellion, the growing interest in photography in China after the Second Opium War can be attributed, in part, to a new modernisation process known as the Self-Strengthening Movement.9 Jeffrey Cody and Frances Terpak assert that the Self-Strengthening Movement was

a means of learning from foreigners so that ultimately Chinese practitioners could surpass foreigners in the use of technologies – military and otherwise – that foreigners seemed to have mastered […]. Within this historic context, photography can be viewed as one of several technologies – along with weaponry,

8 They are «recorded to have taken a daguerreotype of Se-shan while sailing up the Yangtze River on July 16, 1842, the eve of Britain's victory in the First Opium War. […] Sadly the photographs they took have not survived; otherwise they would constitute the earliest known photographic images of China ever made». See also Bennett (2009; 2010).

9 On the Self-Strengthening Movement, see Kuo and Liu (1978).

the railroad, and the telegraph, to name a few – that became significant catalysts for reform during a period of intense upheavals in late Qing China (Cody and Terpak 2011, 34-35).

By the end of the nineteenth century, another type of photograph had gained impetus: the lantern slide. Photographic lantern slides were invented in 1849 by the Langenheim brothers of Philadelphia. They replaced the hand-painted glass plates previously projected by use of a lens with photographs (Shepard 1987). The wet collodion process, discussed above in relation to prints, «enabled details to be captured in higher quality, using cheaper materials and shorter exposure periods» and was in turn substituted by the dry plate in the 1870s (UC Arts Digital Lab, n.d.). This process was even cheaper and more efficient, as the plates could be prepared in advance and the photographer did not need to transport the plates, chemicals or a conveyable dark room for immediate development, which would have been cumbersome for travellers. The reduced size of the lantern slides (for instance 8.25 cm square) made them light to transport and easy to take. A glass plate was covered with another thin layer of glass and both were kept together by strips of gummed paper tape. Originally in black and white, colour could be hand-painted using transparent oil pants, aniline dyes or water colours. In the twentieth century, paint specifically designed for lantern slides was developed. The technical evolution of lantern slides transformed them from a tool for storytelling to an acceptable instrument for education, scientific study and travelogues and they became a favourite device for missionaries, who used them in their evangelising lectures in China, as well as in fund-raising presentations in their home countries (Kuo 2016, 29).10

By the turn of the twentieth century, photography in China had again been transformed by the snapshot revolution resulting from the appearance of the cutprice Box Brownie camera, which allowed anybody to take cheap photographs in large numbers. Supplies of photographic materials and facilities for developing film rolls became available in all large cities in China (De Angeli and Reisz 2017, 4; De Angeli 2018, 164-65).

#### Photographs and Historical Context

The sets of photographs presented in this chapter have not been selected so much for their aesthetic qualities as for the amount of information they can provide to the viewer and the historian. These black and white images were taken by amateur photographers in two different formats: paper prints and glass slides. Some printed photographs come from albums of snapshots taken by Richard H. Strangman, a customs official based in Tianjin.11 Strangman took 98 pho-

10 Cody and Terpak (2011, 53) provide the examples of the French Franciscan missionary Michel de Maynard who «used lantern-slide images to record daily life and, subsequently, to raise funds back in France for the work of his mission in China». 11 For Strangman's career in the CIMC, see De Angeli (2018, 174).

tos on the border of Zhili Province on two occasions, the first in the winter of 1903-1904 and the second shortly afterwards, although the precise date was not recorded.12 A second set of printed photographs belong to the personal collection of Sir Robert Hart. The photographer who took these pictures has yet to be identified, but it is thought that most were probably taken by an official in the Chinese Imperial Maritime Customs in order to illustrate to the inspector general the situation in Niuzhuang during the Russo-Japanese War.13

The glass slides are part of a collection which belonged to Kathleen Carrall (1883-1971) composed of three sets of slides. One set is hand-tinted and was produced by the Lantern Slide Bureau, the Methodist Episcopal Church in China and the Mission Photo Bureau, Shanghai, while the other two boxes contain black and white glass slides taken by another anonymous amateur photographer, apparently as mementos and keepsakes. These two boxes contain 118 glass slides, none of which are labelled or annotated. Nonetheless, these images were all taken in a short span of time just before or during the Russo-Japanese War, a period which foreshadowed the launch of the last reforms by the ultra-conservative Manchu court, which hoped to retain power. Although the conflict was a battle between two other powers in the Northeast of China, it was the fate of the regional trade and network infrastructure and its tax revenues that was at stake.

The Russians and Japanese battled on the plains of Manchuria and in the Yellow Sea following a surprise Japanese attack on the Russian navy docked in Port Arthur. The conflict was short but entailed heavy losses on both sides. From February 1904 to September 1905 the total number of causalities «fluctuates slightly according to the various sources, it is often cited at around 130,000-150,000, of which the Japanese lost about 88,000», of whom 70% in action or wounded, and 30% from disease (Kowner 2006, 80-81). The war resulted in a Japanese victory over Russia, whose imperial government in February of the same year had to face the First Russian Revolution. News of the attempted revolution of 1905 in Russia influenced the emerging Chinese bourgeoisie and students, who constituted the first form of modern public opinion in China. Internationally, the conflict was interpreted as the victory of a constitutional monarchy over an autocracy, and this galvanised anti-Manchu opinion in China and encouraged Chinese revolutionaries, who, in the 1900s, saw Japan as an example for their own revolution. Indeed, many Chinese radicals lived and studied in Japan during this period. In 1911, the Xinhai Revolution succeeded in bringing down the Qing court and establishing the Republic of China, the first republic in Asia. Qing China had survived the succession of internal rebellions scourging the country since the late eighteenth century, but it crumbled under the pressure of competing em-

12 For more information about Strangman's collection, see De Angeli (2018, 163-65).

13 MS15/6/4 is a collection of various formats and topics, mostly giving account of the foreign presence in China, for example, the commemoration at the arch dedicated to the German plenipotentiary Baron von Ketteler, killed in June 1900 (MS16/5/4/9-11, 13, 15-16), or the photos of the signature of the Boxer Protocol on 7 September 1901 (MS16/5/4/21-22).

pires which had «carved up the Chinese melon». The demands of war indemnity payments indebted China to European powers and Japan's bondholders, and a plethora of foreign powers established spheres of interest in China. From 1898 onward, Britain, France, Germany, Japan and Russia all established naval bases on Chinese shores, took control of railways running through most Chinese provinces, even the remotest, and obtained concessions for the exploitation of Chinese mines. Consequently, the already indebted Qing Empire was further impoverished. The Russo-Japanese War marked the end of an era, as it was the last major conflict between competing imperial powers in China before the Xinhai Revolution of 1911. Although the republic was an emerging weak power in the region, and its beginning was ravaged by a dozen years of warlordism, the established Western powers were, from that moment on, in retreat, while Japan emerged as the growing power in the region. The images of the Russo-Japanese War were an exceptional, first-hand account of the ending of an era.

#### How Photography Bore Witness to War in China

In China, war and photography were linked from the arrival of the technology in the mid-nineteenth century and, as noted above, war photography as a genre can be traced back to the work of Felice Beato. The images of the Second Opium War, which made Beato famous in the West, were taken with a technology that was primitive when compared to what was available during the Russo-Japanese War, almost half a century later. In 1858, Beato used albumen on glass negatives to produce albumen silver prints. Explaining Beato's technique, Anne Lacoste observes that the albumen technique available in the 1850s «allowed negatives to be made in advance before being exposed and was remarkable for its sharpness and overall penetration of details» (Lacoste 2010, 25), but that the process required long exposure and was more suitable for immobile subjects. From 1855, however, Beato started to use the wet collodion method, reducing the exposure times to very few seconds, «although the negative plate had to be prepared, exposed, and developed promptly while it was still damp» (Lacoste 2010, 25). Therefore, Beato's travelling photography still required him to carry «cumbersome and fragile equipment, including camera and tripod, a dark tent, potentially dangerous chemicals (collodion contained an explosive ingredient), and large, breakable glass plates of about 25 to 30 centimetres (the size of his final images). Beato had to adapt the complex processes involved to the field and weather conditions, which could interfere with the final result» (Lacoste 2010, 25). Besides the mastery of these challenging techniques, Beato is remembered for «his innovative work portraying death in war photography» and, therefore, for opening up a new pathway in the genre (Lacoste 2010, 28). He is considered the forerunner of war photojournalism. Owing to the technical limitations that existed at that time, Beato needed the image in front of the camera to be almost stationary. In time, however, the camera evolved to make photography instantaneous, thus able to cut through the heat of the battlefield and trigger the emotional reaction in the viewer that comes with the sight of death (Ritchin 2010, 119-20). By the late 1880s, commercial photography had been democratised by the invention in 1888 of the Kodak, the first ready-to-use camera, by the American George Eastman. The next generation model of the Kodak was the one used by Richard H. Strangman for his snapshots.

The images of the Russo-Japanese War from the Sir Robert Hart Collection were the snapshots of amateur photographers, who appear to have taken them as mementos for themselves or to be seen by a close circle of people. The intention with these images does not appear to have been to please an audience or for them to be commercialised.

The glass slides of the Russian Imperial Navy ships, *Askold* and *Tsesarevich*, 14 present a different kind of war image from Beato's work. These photographs are historically significant as they are the only contemporary images of ships engaged in the Battle of the Yellow Sea, the first ever naval confrontation between modern steel battleship fleets. The *Askold* was a first-rank protected cruiser built by the Krupp Company in Kiel, Germany, in 1898, to fulfil the 1897 Russian special programme of accelerated shipbuilding for the needs of the Russian Far East (MS15/6/21B/3). The *Askold*'s role was to carry out long-range reconnaissance for a squadron. The favourite ship of Tsar Nicholas II, who visited it twice while it was docked in Kronstadt, it sailed from the main base of the Russian Baltic Fleet to the Russian Far East, arriving six months later in February 1903 at Port Arthur, where it joined the Pacific Squadron.

Figure 1 – The *Askold* in wartime black hull docked at Port Arthur harbour, summer 1904. MS15/6/21B/3.

14 For a detailed account of the blockade of Port Arthur and the Japanese blockade involving the *Askold* and the *Tsesarevich*, see Grew (1904-1906, II: 28-45, 200-40).

The *Askold* appears in the glass slide with a newly repainted black hull used in wartime. It was docked in a harbour and it can be seen that the photograph was taken during the summertime, as the Chinese boatmen in the foreground were dressed in light summer clothes and the passengers in their boats were using umbrellas to protect themselves from the sun, while two lines of sailors' clothing had been hung up to dry from the bow to the mainmast. The *Askold* appeared to be in perfect condition, prepared for battle. In fact, the *Askold* had prevented a surprise attack by Japanese destroyers on the Russian ships in Port Arthur on the evening of 26 January 1904, though the Japanese succeeded in their second attempt on the night of 8-9 February, sparking the Russo-Japanese War. Then on 28 July, the Russians exited the harbour trying to break through to Vladivostok. The *Askold* led the line ahead of the cruiser squadron. When the Russian flagship lost control and the remaining ironclads turned back towards Port Arthur, the *Askold*, followed only by the scout cruiser *Novik*, 15 broke through under a hail of fire from the Japanese ships. Thanks to her powerful engine capable of reaching 24 knots, the *Askold* disappeared beyond Japanese range, but the damage was serious. Unable to reach Vladivostok, she retreated to the neutral port of Shanghai, where she was interned until 1905. Therefore, the image could only have been shot in Port Arthur before 27 July 1904 (Grew 1904-1906, II: 207-208).

The other glass slide shows a detail of the stern of the shelled battleship *Tsesarevich* (MS15/6/21B/7); one of the twelve Canet Model 1892 6-inch/45 quick-firing guns mounted on a turret on the upper deck; and one of the eight Maxim quick-firing 37-millimetre (1.5 in) guns placed in the superstructure next to the military mast.

The *Tsesarevich* was a Borodino-class battleship built in France and characterised by a «tumblehome» hull, meaning that it narrowed above the waterline. Launched in February 1901 from the French Forges et Chantiers de la Méditerrannée shipyard in Marseille, the *Tsesarevich* entered service in August 1903 and sailed for Port Arthur to join the Pacific Fleet where, like the *Askold*, she arrived shortly before the Russo-Japanese War, on 2 December 1903. She was considered the best battleship of the Imperial Russian Navy.

The glass slide reveals details of the pounding taken by the *Tsesarevich* from the Japanese on 7 August 1904, during the attempted breakout from Port Arthur, when the temporary commander of the First Pacific Squadron, Admiral Wilhelm Withöft, was hit by shrapnel.16 The *Tsesarevich* had just returned to service following five months in dock after being torpedoed by the Japanese on the night of the surprise attack which triggered the war. The paper photo, in contrast, shows the *Tsesarevich* harboured at Qingdao after the Battle of the Yellow Sea, when, on 10 August 1904, the Russian fleet, led by the *Tsesarevich*, was engaged by the Japanese fleet as it tried to exit Port Arthur harbour and sail to safety to Vladivostok. The heavy fighting saw the *Tsesarevich* hit on the conning tower by two heavy calibre shells. The effects can be seen in the photograph. Fragments bounced across the

15 The *Novik* was the fastest cruiser of her time reaching 25 knots.

16 He is also known as Wilgelm Vitgeft (1847-1904).

unprotected bridge, which was destroyed, instantly killing Withöft, as well as the helmsman and close to a dozen crew members. After the battle, most of the ships returned to Port Arthur, but during the night, the *Tsesarevich* had to take refuge in the Germany colony of Qingdao, where it was interned until the end of war, from where it sailed in early 1906 to join the Baltic Fleet.

Figure 2 – Details of the shelled stern of the *Tsesarevich* after the pounding from the Japanese on August 7, 1904. MS15/6/21B/7.

These three images of Russian Imperial Navy ships are snapshots taken by amateur photographers standing on the pier and focusing on the Russian ships docking at Port Arthur and Qingdao harbours before or after the battles. The conflict was ongoing, but daily life continued as evidenced by the casual posture of people visible in the photographs. In the glass slide of the *Askold*, Chinese passengers can be seen boarding a boat and the sailors' laundry hangs in the sun on the stern, while in the other glass slide, Russian sailors stand in casual posture, chatting and smoking on the upper deck of the damaged *Tsesarevich*. The paper photo of the *Tsesarevich* at Qingdao harbour shows Russian crew members standing on the upper decks, while small steamboats approach the seriously damaged battleship, and a couple of rowing boats were taking on board and transporting part of the crew to shore. Meanwhile, other crew members were working on the starboard side of the upper deck towards the stern, covering shell damage to the hull. These details suggest that the photograph was taken soon after the *Tsesarevich*'s arrival at Qingdao in mid-August.

The last photograph of the Russo-Japanese War depicts another type of scene. Taken from afar, it shows a Chinese crowd assembling in front of the main gate and along the fence surrounding the Russian Civilian Administrator's residence, where the French flag was flying from the flagpole.

Figure 3 – Capture of Newchwang by Five Japanese Scouts at 4.30 P.M. on Monday, 25 July, 1904. View of the Russian Civil Administrator's Residence (French Flag Flying) with the Victors at the Gate. MS15/6/4/5.

The photo was taken on the capture of Niuzhuang (now Yingkou) by five Japanese scouts, who are standing close to the gate at 4.30 pm on 25 July 1904 (MS15/6/4/5). The image is different from the others not only in subject matter, but also in size (28.5 cm x 21.2 cm) and it appears to have been taken by a camera using plates and not film, as suggested by the fact that people are not standing still, but rather walking or running, and thus appear as shadows. This theory is also corroborated by a second picture taken from exactly the same point one hour later, from which we can assume that the camera was mounted on a tripod.

Figure 4 – Capture of Newchwang by Five Japanese Scouts on Monday, 25 July, 1904. Deputation of the Native Guilds at 5.30 P.M. to invoke the protection of the Victors. MS15/6/4/6

This image again shows a crowd in front of the gate and it is captioned as the «deputation of the native guilds at 5.30 pm to invoke the protection of the victors» (MS15/6/4/6).

Lying on the right bank of the Liao River and on the north-eastern shore of Liaodong Bay, Niuzhuang was one of the treaty ports opened in May 1861 after the Second Opium War (China 1904, 1-2). This locality, renowned as the largest port for free trade in the north of China, remained unaffected by the Russo-Japanese War until late July 1904, when these two images were taken.

On 25 July, the newly arrived Japanese scouts saw the Russians replacing their consular flag with the French flag, as shown in the photograph, but the building was neither the French nor the Russian consular premises, but rather, the administration house and the Chinese Imperial Maritime Customs (CIMC) offices.17 On July 29, the dragon flag of the CIMC was again hoisted over their property, and later on that very same day, the flag over the West Chinese Customs changed again from French to Japanese. By this time, the Russian consul and administrator, V. F. Grosse, and the manager of the Russo-Chinese Bank, Mr Blanche, had left Niuzhuang for Tianjin. By 7 August, the Japanese were in full control of the town (*North China Herald* 1904).

An insight into the event portrayed by the images is given by a Western correspondent:

Four Japanese scouts rode into the square. They were mounted on sorry, tired horses, all mud splattered, and rough. The men wore uniforms stained and torn by campaigning. The people regarded them with interest but without any display of emotion. They were not welcomed, nor was their intrusion resented. Then arrived four more, and later another four, with a non-commissioned officer. To him came the chiefs of the Chinese guilds with greetings. The foreign residents held aloof for the expected army, the officers and the generals. A short while before British ladies had provided free teas and free refreshments for the Russian troops arriving from outlying camps, and for recruits doing their drill-ground exercises. No one had anything to offer these tired, battle-worn men; they tendered not so much as a light for a cigarette, or a drink of water to the thirsting horses the men rode. The Chinese looked on with as much indifference as the foreigners showed (Grew 1904-1906, III: 60).

The account shows a rare display of bias against the Japanese according to Keith Stevens (2003: 144-45), and contrasts with the tone of the inscription in the photographs; but both agree on the fact that the Chinese guild representatives came to the gate of the seized CIMC premises to greet the Japanese scouts. This attitude of the Chinese guilds towards the Japanese army is explained by the fact that «Russian proclamations in the interior warned the people against conveying their harvest» to Niuzhuang, otherwise they would suffer loss, while the Japanese occupiers reversed the condition of trade, as they «closely invested, allowed unre-

17 A similar action had been taken previously by the Russians on 4 August 1900, during the Boxer Rebellion (China 1904, 11).

stricted importation and shipment» (China 1905, 2). The images were taken either by a customs official or someone commissioned by the local customs in order to be sent to the Inspector General, Sir Robert Hart. They are not images of battle, but rather of actions contingent to the war itself. They do not represent the photojournalism of well-known professionals like Beato, but they do contribute to displaying accessory details of the conflict, such as how the legacy of the battles affected everyday life and business, and they can provide evidence against narrative bias.

#### Infrastructure Photography: Focus on Chinese Modernisation

The modernisation process in China started with the arrival of foreigners. After the Second Opium War, the Manchu court launched the Self-Strengthening Movement, a modernising programme in which Western technology was introduced into China in service to the country, but on the premise that such technology would only be adopted as a tool and not as an element for a change of mentality. The Chinese believed that, to fight and win against the Western powers, they had to use the same weapons and equipment. And so, the Confucian doctrine remained the central doctrine of the state until the abolition of the imperial examination system in 1905. It is undoubtedly true, however, that the modernisation of the country led to the final abandonment of that doctrine.

The Self-Strengthening Movement covered a long period of the late Qing era, as it was launched in 1860 and concluded in 1895 with the First Sino-Japanese War. The war was seen as proof of the failure of the Self-Strengthening Movement. Yet, this modernising process drew China out from isolation to launch itself on a path of technological progress. China started to build its first tranche of modern infrastructure, including railways, steamships, modern harbours and docks, lighthouses, telegraph and radio networks, shipyards and arsenals, to name but a few. Photography was an extremely useful tool for bearing witness to this modernising process. Photographs could show the advancement and accomplishment of projects, such as the construction of buildings, the laying of ballasts and rails, the reinforcement of wharves, and many more.

The set of photographs of modern infrastructure in China reproduced here belongs to an album compiled by a customs officer, Richard H. Strangman, from January to February 1904 and sent to the CIMC Inspector General. We know that Hart collected pictures of the customs service, because photographs of customs service compounds and related infrastructure allowed him «to keep a close eye on staff and operations in remote corners of China even after he largely gave up travelling in his later years» (De Angeli and Reisz 2017, 40). The album was compiled between December 1903 and Chinese New Year (16 February) 1904 and of its 50 snapshots, half focus on customs activities in Tanggu and Dagu, while the other half feature a train journey from Shanhaiguan to Qinghuangdao, including stops in locations along the Beijing-Mukden (Shenyang) line.18 The

18 For more information about Strangman's journey, its destination, and scope, see De Angeli (2018).

following images show the state of the railways and customs service infrastructure in the north of China just before the outbreak of the Russo-Japanese War.

Figure 5 – Four fifth of Birds Eye View of Ching Wang Tao. C.E. s M C.so Ste Hsiping out hanging at Pier. Japanese Transport «Nagata Maru» at Anchor. One fifth of the Sea left out. Jan, 1904. MS15/6/11E/33

The first is a panoramic view composed of a set of four superimposed photos19 entitled *Four Fifths of Birds Eye View of Ching Wang Tao [Qinghuangdao]* (MS15/6/11E/33). As the inscription explains, the fifth picture in the set was left out as it showed the sea view. In fact, the four photos are paired, and the part that is cut corresponds to the sea at the centre. The panorama taken from the height of the hill beside the coal yards depicts, on the right side, the town of Qinghuangdao, which, in 1904, saw the establishment of the Chinese settlement,20 and on the left the pier and the harbour located far from the town centre. At the pier, the Chinese Engineering and Mining Company steamer *Hsiping* [Xibing] is discharging mechanics and materials for building the pier and the harbour infrastructure (China 1904, 147), while close by, the Japanese transport cargo ship *Nagata Maru* is at anchor, and a steam locomotive is drawing a train back to the station. In the foreground, a few men can be seen working on the rails. The quality of the panorama is excellent, though incomplete, and provides a good sense of the harbour and the rail infrastructure. Strangman's bird's-eye view imitates a style in vogue in Europe and North America since the 1860s, which aimed «to showcase the allure of cities in the context of industrial and capitalistic expansion» (Cody and Terpak 2011, 57). If the Western professional photographers and their buyers wanted to show the capital and the potential of their godowns, customs houses, churches, and steamers in China to Western viewers, it was, according to Cody and Terpak, because they «wanted to show the strength and anchorlike extent of the Western presence» (Cody and Terpak 2011, 57).

19 The Kodak snapshots were cut to a different length, but all were 5.9 cm in height with a maximum length of 11 cm.

20 Chinese settlement began in 1904 and by 1905 the town had a population of approximately 3,000 people (China 1904, 148; 158).

The next two photographs were also taken in Qinghuangdao, but they were close-ups of the construction and maintenance of the harbour and railway infrastructure. It seems that Strangman took the panorama view close to where the following photographs were taken.

Figure 6 – Ching Wang Tao. Blasting Rock and boarding it on cars. MS15/6/11E/37.

In the first, a steam stone-breaker is blasting rocks on a barren hillside. On the left of the knapper a worker stands close to the gatehouse door, while on the right three workers are close to the cars, which are ready to load (MS15/6/11E/37).

Figure 7 – Ching Wang Tao. Dumping Rock from cars to form breakwater. MS15/6/ 11E/38.

In the second, entitled *Dumping Rock from cars to form breakwater* (MS15/6/11E/38), Strangman focuses his lens on the railway track, where the cars loaded with stones have reached the end of the pier. Three men are unloading a car, while on their left, another man works alone unloading another car. It is noticeable that the stones are being unloaded next to the rails, as a boat is docked at the bottom of the pier end, with only its mainmast visible. Built in 1901 and connected by rail to Tanghe station, the pier was partly destroyed by a storm that very same year, and so the photograph shows the ongoing repairs (China 1904, 147). Qinghuangdao harbour was built by the partnership of Sir John Wolfe Barry and Lt. Col. Arthur John Barry. Established in 1891, they had a long experience of engineering projects in China, such as the Shanghai-Nanjing and the Chinese Central railways, and the Chinese Engineering and Mining Company Limited.21

Figure 8 – Lanchow Bridge. MS15/6/11E/46.

The last photograph shows another panorama: the *Lanchow Bridge* (MS15/6/11E/46) located on the Luan River at Luanzhou. This locality, like Qinghuangdao and other sites visited by Strangman during his train journey in early 1904, was considered of strategic importance, due to its location on the still incomplete Beijing-Mukden railway line, which was built between 1881 and 1907. From the time that the line reached Mukden (now Shenyang), one of the major cities in Manchuria, Mukden became a crucial rail hub, serving those travelling from China, from Port Arthur (now Dalian) and from Harbin.

21 For information about some of their activities, see Crisp (1906).

The Mukden rail hub was built along the Japanese-controlled South Manchuria Railway and was connected to both Beijing, via the Beijing-Mukden railway, and Harbin, 350 miles further north, which served a major station on the Russian-built Trans-Manchurian Railway. The Beijing-Mukden railway and Qinghuangdao harbour were both major strategic infrastructural assets, because they were vital to the transport and commercial networks connecting the Chinese capital with the Northeast of China. The Beijing-Mukden railway constituted the backbone of the railway system in Manchuria. Its first section, connecting Tangshan to the Kaiping coal mines, built in 1879 on the orders of Governor-General Li Hongzhang, was extended westwards towards Tianjin and eastwards beyond the Great Wall at Shanghaiguan. The section between Shanghaiguan and Tianjin came under British governance after the Boxer Uprising and the British extended the line to Beijing, while the Russians extended the section east of the wall at Shanghaiguan. Following the British restitution to the Chinese authorities in 1904, construction continued as far as Xinmindun (Constant 1933, 121; Huang and Chen, 2014). Moreover, Shanghaiguan was a key station on the Beijing-Mukden railway and an infrastructural hub connecting the railway line to Qinghuangdao harbour, which served both commercial and passenger steamers. Qinghuangdao was also the locality where the Great Wall of China entered the sea. Both physically and symbolically, it marked the border between Manchuria and Zhili province, the borderland and the centre.

The infrastructures of Qinghuangdao harbour and the Beijing-Mukden railway were significant strategic objectives for both the Russian and the Japanese empires in China. While Qinghuangdao was vital in linking maritime communication and trade networks to rail networks, the Beijing-Mukden railway offered a route penetrating deep into the Chinese heartland. The Chinese authorities were well aware of the potential military, strategic and logistic uses that Russia or Japan could derive from these infrastructures. Consequently, the CIMC tried to maintain close control of them. Strangman's photographic images reveal both the infrastructure itself and the presence of the CIMC to which he belonged.

#### Conclusion: Western Photography Appraising Chinese Modernisation

The series of images from the Sir Robert Hart Collection, despite being taken by different amateur photographers and developed on different media, were all taken during a short but pivotal period for China. These photographs record war-related scenes and infrastructures just before or during the Russo-Japanese War, the very first political conflict covered by an illustrated Chinese magazine, the *Shanghai Biweekly*. The photographs published in the *Shanghai Biweekly* were used to further debate around China's resilience in the face of the imperialist drive and the extent to which the Chinese had the ability to save their country and culture from foreign domination (Wu 2011, 15).

The Russo-Japanese War photographs were conceptually different from the images taken by Felice Beato during the Second Opium War, as, by the turn of the twentieth century, the technology available to photojournalists had advanced considerably. Although not yet achieving the results attained with the 35mm cameras of the 1920s and 1930s, photographers during the Russo-Japanese War were able to capture images that were more spontaneous because they required less technical preparation (Ritchin 2010, 120). Kodak's Brownie camera provided amateur photographers with capabilities that made «snapshots» of the ongoing conflict possible. There are no images of battle, because the amateur photographers were not attached to any army involved in the conflict. Yet, the glass slides provide a significant early example of photojournalism, depicting the damage sustained by the Russian Imperial Navy on two of its Pacific Fleet ships, the *Askold* and the *Tsesarevich*. These images differ, however, from the voyeuristic cliché of war photographs, because there is no ongoing battle or depiction of death (Ritchin 2010, 120). Rather, they show some Russian mariners casually standing on the decks of the ships, laundry hanging from the superstructure and commercial boat passengers going about their regular business.

The two sets of photographs on paper offer a different perspective. Both can be attributed to people working for or connected to the Chinese Imperial Maritime Customs and offer a glimpse into the activities of the service before and during the conflict, which posed obvious dangers to Chinese sovereignty and customs infrastructure. These images differ from the stereotypical images of China that became widely available in the West following the Boxer Rebellion. Sarah Fraser (2011, 106) observes that «scenes of itinerant workers, destitute poor, and military captives at the time of the Boxer Uprising reflect racial debates about the modern Chinese subject prevalent in international power relations». The photographs taken by CIMC officials cover very different subjects, because the lens is focused on Chinese modernisation.

Chinese modernisation was one of the outcomes of the cross-cultural encounter between China and the West, which passed through the camera shutter. The impetus given to modern technology in China influenced the reforming Qing state and a new group of artisans, technicians and engineers emerged from the Self-Strengthening Movement, who recognised photography as a new tool for modernisation. Photography was not a core technology of the Self-Strengthening Movement «nor was it a technology like railroads or telegraph lines, which left an obvious footprint in the landscape». Nevertheless, it had already attracted the attention of some of the highest-ranking Qing officials by the 1860s (Cody and Terpak 2011, 44). In the mid-1870s at the Shanghai Polytechnic Institute, John Fryer and Xu Shou displayed «several dozen photographs supplied by English and Belgian firms of railway locomotives and cars, mining machinery, firearms, artillery, and iron-framed buildings» as a didactic tool (Cody and Terpak 2011, 45). Accordingly, photography was both a product and a tool of modernisation in China, and Cody and Terpak (2011, 47) assert that it eventually became an integral part of the Self-Strengthening Movement's culture and commerce. This in turn tantalised Western audiences, who were eager for images and information about China.

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*Article* **E**ff**ects of Increasing Nitrogen Content on Process Stability and Reactor Performance in Anaerobic Digestion**

### **Ievgeniia Morozova \*, Nadiia Nikulina, Hans Oechsner, Johannes Krümpel and Andreas Lemmer**

State Institute of Agricultural Engineering and Bioenergy, University of Hohenheim, 70599 Stuttgart, Germany<EMAIL_ADDRESS>(N.N<EMAIL_ADDRESS>(H.O.);

<EMAIL_ADDRESS>(J.K<EMAIL_ADDRESS>(A.L.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +49<PHONE_NUMBER>8; Fax: +49-(0)711-459-22111

Received: 1 December 2019; Accepted: 26 February 2020; Published: 3 March 2020

**Abstract:** The aim of this study was to analyse the effect of different nitrogen increase rates in feedstock on the process stability and conversion efficiency in anaerobic digestion (AD). The research was conducted in continuously stirred tank reactors (CSTR), initially filled with two different inocula: inocula #1 with low and #2 with high nitrogen (N) concentrations. Three N feeding regimes were investigated: the "0-increase" feeding regime with a constant N amount in feeding and the regimes "0.25-increase" and "0.5-increase" where the N concentrations in feedstock were raised by 0.25 and 0.5 g·kg−1 , respectively, related to fresh matter (FM) every second week. The N concentration inside the reactors increased according to the feeding regimes. The levels of inhibition (Inhibition) in specific methane yields (*SMY*), related to the conversion efficiency of the substrates, were quantified. At the N concentration in digestate of 10.82 ± 0.52 g·kg−1 FM measured in the reactors with inoculum #2 and "0.5-increase" feeding regime, the level of inhibition was equal to 38.99% ± 14.99%. The results show that high nitrogen increase rates in feeding regime are negatively related to the efficiency of the AD process, even if low volatile fatty acid (VFA) concentrations indicate a stable process.

**Keywords:** biogas; methane; ammonia; inhibition; acclimatization; trace elements

#### **1. Introduction**

Utilization of protein-rich substrates, such as kitchen waste, poultry manure, microalgae, green legumes, oilseeds, etc. may lead to high concentrations of nitrogen (N) in the reactor during anaerobic digestion (AD) [1–4]. High concentrations of N inside the reactor negatively affect process stability and efficiency due to ammonia formation. Total ammonia nitrogen (TAN), which is generally defined as the sum of free ammonia nitrogen (FAN, NH3-N) and ammonium nitrogen (NH4 +-N), is formed during the hydrolysis of proteins, urea and nucleic acids [5–8]. Ammonia freely passes through the cell membranes of methanogens and causes a proton imbalance [5,8,9]. Free ammonia changes the intracellular pH of methanogenic bacteria and inhibits specific enzymatic reactions [10]. Therefore, high concentrations of ammonia in anaerobic reactors lead to inhibition of methanogenesis and may cause complete failure of AD [6,11,12]. As reported by Chen et al. [13], temperature change has a direct impact on both microbial growth rates and free ammonia concentration: increased process temperature affects the metabolic rate of the microorganisms in a positive way; however, it also results in higher ammonia levels.

The chemical balance between NH3 (free ammonia) and NH4 + (ammonium) is shown in Equation (1) [14,15].

$$\rm NH\_4^+ + \rm OH^- \leftrightarrow NH\_3 + H\_2O \tag{1}$$

The shift of this equilibrium depends mainly on the process conditions, i.e., temperature and pH [7,9,14]. The concentration of free ammonia is positively correlated with temperature and pH [5,16].

Under high ammonia concentrations in the reactor, the acetoclastic methanogens (e.g., *Methanosarcina, Methanosaeta* spp.) are unable to degrade acetate, which results in its accumulation, depletion of buffer capacity and a subsequent drop in pH [16–19].

According to the literature [9,10,12,13,20], inhibition of the AD process by ammonia is indicated by the decrease in the specific methane yields along with the increase in volatile fatty acid (VFA) concentrations and a pH drop due to inhibition of bacterial growth. However, the limiting concentrations of TAN and FAN for maintaining AD without inhibition are subject to discussion (Table 1). In addition, there is a controversy whether TAN or FAN mainly inhibits methanogenesis [20].

Most authors in previous studies tend to agree that TAN ≥ 3.00 g·L −1 and FAN ≥ 0.20 g·L −1 have an inhibitory effect on AD (see Table 1). According to Table 1, very few studies have measured the level of inhibition in methane production when treating N-rich substrates.

For maintaining stable and efficient biogas production under high and/or increasing TAN and FAN concentrations, acclimatization strategies can be applied. A frequently used approach is to feed the reactor with a specific N- or ammonia-increase rate. However, no information on the maximum increase rates is available [1,4,12,21–24].

High nitrogen concentrations in the digestate are generally the result of a narrow carbon-to-nitrogen (C/N) ratio of the feedstock [9,12,25,26]. To reduce the concentrations of TAN and FAN in the digestate and thus to maximise biogas and methane yields, Shanmugam and Horan [26] recommend keeping the C/N ratio of the feedstock in the range of 15 to 20, while according to Kayhanian [9], this ratio should be between 27 and 32.

Currently, many operators of biogas plants suffer from AD inhibition and methane losses when utilizing N-rich substrates. The application of the acclimatization strategy with an optimal N-increase rate could stabilize AD and prevent or minimize methane losses. In this study, natural N-sources and microbial communities from full-scale biogas digesters were utilized. The research was conducted in continuously operated reactors under conditions similar to those in full-scale biogas plants. The aim of this investigation is to determine the effect of different nitrogen increase rates on anaerobic digestion in order to achieve an optimal process performance. The nitrogen increase in the feedstock was carried out every two weeks at rates of 0.25 and 0.5 g·kg−1 related to fresh matter. The N content of the digestate rose continuously in response to the two different increase rates in the feedstock. At the same time, the C/N ratio of the feedstock consistently decreased throughout the experiment. Thus, the influence of high nitrogen content on the stability of the fermentation process with regards to the C/N ratio could be investigated. By comparing the values of specific methane yield (*SMY*) obtained from the continuous experiment with those obtained from the batch experiment, the effect of increasing N content in the feedstock on the conversion efficiency of the substrates was studied.

**Table 1.** Limiting total ammonia nitrogen (TAN)- and free ammonia nitrogen (FAN)-concentrations (g·L −1 ) for maintaining stable anaerobic digestion (AD) under

*Energies* **2020**, *13*, 1139

*Energies* **2020**, *13*, 1139

if stated: (a) unacclimatised, (b) acclimatized.

### **2. Materials and Methods**

#### *2.1. Reactor Design*

The experiment was conducted in 12 horizontal, stainless steel, continuously stirred tank reactors (CSTR) of 20 L total volume (working volume 17 L) each, as described in [38], in duplicate repetition according to the Guideline 4630 issued by the Association of German Engineers (VDI) [39]. Different N-increase rates in the CSTR were achieved by different feeding regimes described in Section 2.2. During the experimental period, the organic loading rate related to volatile solids (OLRVS) was kept at 3 kg·m−3 ·d −1 with a hydraulic retention time (HRT) of 40 days. The temperature in each reactor was mesophilic at 37 ± 1 ◦C.

#### *2.2. Inocula and N-Increase in Feeding Regimes*

Each digester was filled with 17 L of inoculum at the beginning of the experiment. Inoculum #1 and inoculum #2 from two full-scale biogas plants were used in this trial. These inocula differed in total Kjeldahl nitrogen (TKN) concentrations, with inoculum #2 containing twice as much nitrogen as inoculum #1 (Table 2). Inoculum #1 was taken from a digester treating cattle manure (35–40%), maize silage (40%), grain whole plant silage (5%) and triticale (rest). Inoculum #2 was taken from a digester treating turkey manure (10%), cattle manure (8%), cereals (10%) and maize silage (62%).

**Table 2.** Characteristics of the substrates. Gas volumes are given under standard temperature and pressure conditions (0 ◦C, 101.325 kPa). Units are given in square brackets. Values are given as mean; the standard deviation is given in round brackets.

(a) Dry matter (DM) related to fresh matter (FM), (b) volatile solids (VS) related to DM, (c) total Kjeldahl nitrogen (TKN) related to FM, (d) ammonium related to FM, e) specific methane yield (*SMY*) related to VS.

The substrates were fed into the reactor daily are described in [38]. The daily feedstock consisted of fresh inoculum, maize silage (low nitrogen content) and soybean meal (N-rich substrate). Tap water was added in order to keep the HRT and OLRVS constant, thus resulting in 425 g of fresh matter daily feedstock. Characteristics of the substrates are described in Table 2. The values of the specific methane yield for the feeding substrates were determined by the Hohenheim biogas yield test [40,41].

For each inoculum, the different feeding regimes were separately analysed, as shown in Figure 1. These feeding regimes represent the rate of N increase in the feeding ratio. The investigated feeding regimes were "0-increase", "0.25-increase" and "0.5-increase". Under the "0-increase" feeding regime, the nitrogen content did not change over the whole course of the experiment. For the other two regimes, there was an increase in nitrogen content in the feedstock (see Figure 1a). The increase in nitrogen concentration was achieved by adding soybean meal and simultaneously decreasing the share of maize silage. In the feeding regimes "0.25-increase" and "0.5-increase", the share of soybean meal was increased stepwise, thus leading to N-increase rates of 0.25 and 0.5 g·kg−1 FM every two weeks, respectively. By contrast, the C/N ratio in feedstock was decreasing as shown in Figure 1b.

*Energies* **2020**, *13*, x FOR PEER REVIEW 7 of 20

**Figure 1.** Experimental procedure: (**a**) N content in feedstock; (**b**) C/N ratio in feedstock. The results are given separately for inoculum #1 and inoculum #2. Different line colours in the graphs and in the legend correspond to the N-increase rates "0-increase", "0.25-increase" and "0.5-increase" in the feeding regimes. Grey and white backgrounds in the graphs are related to the starting phase **Figure 1.** Experimental procedure: (**a**) N content in feedstock; (**b**) C/N ratio in feedstock. The results are given separately for inoculum #1 and inoculum #2. Different line colours in the graphs and in the legend correspond to the N-increase rates "0-increase", "0.25-increase" and "0.5-increase" in the feeding regimes. Grey and white backgrounds in the graphs are related to the starting phase and the experimental phase, respectively.

#### and the experimental phase, respectively. *2.3. Trace Elements Supplementation*

*2.3. Trace Elements Supplementation*  For AD process stability, the importance of micronutrients, i.e., iron, nickel, molybdenum, cobalt and selenium is described in literature [27,42–45]. After observing process instability for the reactors with inoculum #1 at the beginning of week 17, micronutrient levels were tested. In response to the identified deficiency in trace elements (TE) in the reactors with inoculum #1 and for keeping the TE in the range as recommended by Vintiloiu et al. [42], 1.23 g of BC.Pro Akut® was added to all the reactors (with #1 and #2) weekly, starting from the end of week 17 up to the end of the experimental trials. BC.Pro Akut® is a mixture of TE and other components comprising the For AD process stability, the importance of micronutrients, i.e., iron, nickel, molybdenum, cobalt and selenium is described in literature [27,42–45]. After observing process instability for the reactors with inoculum #1 at the beginning of week 17, micronutrient levels were tested. In response to the identified deficiency in trace elements (TE) in the reactors with inoculum #1 and for keeping the TE in the range as recommended by Vintiloiu et al. [42], 1.23 g of BC.Pro Akut® was added to all the reactors (with #1 and #2) weekly, starting from the end of week 17 up to the end of the experimental trials. BC.Pro Akut® is a mixture of TE and other components comprising the following active substances in the ionic form: aluminium, boron, calcium, iron, cobalt, copper, magnesium, manganese, molybdenum, sodium, nickel, selenium, tungsten and zinc.

#### magnesium, manganese, molybdenum, sodium, nickel, selenium, tungsten and zinc. *2.4. Analytical Methods*

*2.4. Analytical Methods*  The produced biogas was collected in gas bags as described by Haag et al. [38]. A gas measuring unit automatically analysed the gas quantity (Hoentzsch FA MS40, Waiblingen, The produced biogas was collected in gas bags as described by Haag et al. [38]. A gas measuring unit automatically analysed the gas quantity (Hoentzsch FA MS40, Waiblingen, Germany), as well as the content of CH4 and CO2 (AGM 10, Sensors Europe, Erkrath, Germany). The measurements were carried out once per day before feeding.

following active substances in the ionic form: aluminium, boron, calcium, iron, cobalt, copper,

Germany), as well as the content of CH4 and CO2 (AGM 10, Sensors Europe, Erkrath, Germany). The measurements were carried out once per day before feeding. Samples were taken from the reactors weekly. The dry matter content related to fresh matter (DMFM) and volatile solids content related to dry matter (VSDM) of the collected samples were determined by differential weighing before and after drying at 105 °C for 24 h and by subsequent ashing at 550 °C for 8 h, respectively. The pH was measured in each reactor three times per week with a WTW 323, using a SenTix 41 pH-electrode (WTW, Weilheim, Germany). Concentrations of Samples were taken from the reactors weekly. The dry matter content related to fresh matter (DMFM) and volatile solids content related to dry matter (VSDM) of the collected samples were determined by differential weighing before and after drying at 105 ◦C for 24 h and by subsequent ashing at 550 ◦C for 8 h, respectively. The pH was measured in each reactor three times per week with a WTW 323, using a SenTix 41 pH-electrode (WTW, Weilheim, Germany). Concentrations of VFA in the samples were determined by gas chromatography. The gas chromatograph Shimadzu GC-2010plus (Tokyo, Japan) was equipped with a FFAP 50 m × 0.32 mm column with a chemically bonded polyethylene glycol CP-Wax 58 FFAP CB 1.2 µm film, a flame ionization detector and helium as a carrier gas. Total ammonium concentrations in the digestate were determined by the automatic

distillation system Gerhardt Vapodest 50s (Koenigswinter, Germany). Total Kjeldahl nitrogen (TKN) is expressed as total nitrogen or N if not stated otherwise. The total nitrogen in the samples was determined by Kjeldahl analysis. The potassium determination was done by means of flame atomic absorption spectroscopy (AAS, Eppendorf, ELEX 6361, Wesseling-Berzdorf, Germany), operated with an acetylene gas. For the determination of phosphorus, a cuvette test [46] and a spectrophotometer UV–VIS 1240 (Shimadzu, Tokyo, Japan) were used. All the analyses were carried out according to standard methods [46]. The analysis on trace element content in the samples was done by an external laboratory in accordance with standard methods [47–49].

#### *2.5. Calculation of FAN and TAN*

The *NH*3 (free ammonia) concentration was calculated by using the equation described in [14].

$$\mathrm{NH\_3} = \mathrm{K\_{NH\_4}} \cdot \frac{\mathrm{NH\_4^+}}{H^+} \tag{2}$$

where *NH*+ 4 is the ammonium concentration in g·kg−1 related to FM; *KNH*4 is the ionization constant of ammonium (for 37 ◦C, *KNH*4 = 1.14·10−9 [21]); *H*+ is the hydrogen ion concentration (*H*+ = 10−*pH* [14]). *NH*3 was recalculated to *NH3*-N (FAN), and *NH*+ 4 was recalculated to *NH*+ 4 -N (ammonium nitrogen) according to their molar masses. The concentration of TAN was calculated as the sum of FAN (*NH*3-N) and *NH*+ 4 -N.

#### *2.6. Statistical Analysis*

For data processing and visualization, Microsoft EXCEL 2016, SAS 9.4, R and RStudio (version 1.1.463) were used.

#### 2.6.1. Inhibition in SMY

The inhibition in specific methane yields (Inhibition) for different *N* increase-rates in feeding regimes is defined by Equation (3):

$$\text{Inhibition} = \frac{1}{n} \sum\_{i=1}^{n} \frac{\text{SMY}\_{t} - \text{SMY}\_{m}}{\text{SMY}\_{t}} \cdot 100\% \tag{3}$$

where *n* is the number of observations over the experimental period taken for the analysis. The theoretical methane yields (*SMYt*) were calculated based on the amounts of VSDM added to the reactors and the *SMYVS* of the substrates obtained by the Hohenheim biogas yield test (Table 2). The measured SMY (*SMYm*) was based on the measured value of methane yield divided by the amount of VS added to the reactor. This inhibition can also be described as the conversion efficiency between the theoretical *SMYt* values obtained from the batch experiment and the measured *SMYm* values obtained from the continuous experiment.

The one-sided Tukey test was applied to identify whether the difference between the *SMYt* and *SMYm* was statistically significant. The analysis was done in Excel and Rstudio.

#### 2.6.2. Analysis of the effect of TAN and FAN on inhibition

Based on the experimental data for the three investigated feeding regimes along with inocula #1 and #2, the effects of TAN and FAN concentrations in the reactor on the level of inhibition were analysed. For this purpose, mixed modelling for repeated measurements was applied [50]. This model was selected for serial correlation among observations on the same experimental unit. The experimental unit, in our case, was the reactor [50]. Analyses were based on the experimental data starting from week 17 of the trials after the TE supplementation was started. The applied data were checked by using the normality test on the studentized residuals. For meeting the requirements of the mixed model, the square-root transformation of the data on inhibition in *SMY* (sqrt\_Inhibition) was used. Several types of models (independent, compound symmetry, autoregressive, unstructured) were checked; on the grounds of the normally distributed residual plots and the lowest Akaike information criterion (AIC) value, the compound symmetry type was selected as the best-fitting model.

The applied model is given in Equation (4):

$$y\_{itk} = \mu + a\_i + r\_t + b\_{tk} + e\_{itk} \tag{4}$$

where *yitk* is the dependent variable; *i* is the *i*-th observation, *t* is the weekly measurement and *k* is related to the interaction between the fixed factor and the point in time (t); µ describes the general effect of the model; α*i* is the *i*-th observation of the fixed factor; *rt* is the replicate of a weekly measurement; *btk* is the random effect of a week and the interaction between week and the fixed factor; *eitk* is the random deviation associated with *yitk*.

The sqrt\_Inhibition was used as the dependent variable; the TAN and FAN were separately analysed as the fixed factor. The influence of time and interaction between time ("WEEK", in our case) and a fixed factor was analysed on a random effect in the model. The "MIXED" procedure of SAS was used to fit the model.

#### **3. Results and Discussion**

The reactors were continuously monitored over the whole period of the trials. The measured values for N, TAN, FAN, acetic acid (HAc), pH, *SMYm* and inhibition are shown in Figure 2. The trial period included a starting phase and an experimental phase.

#### *3.1. The Starting Phase*

During the starting phase, the OLRVS was increased until the aimed values were achieved.

The starting phase was needed for the microorganisms to adapt to the operating conditions. During this phase, all reactors were fed with a constant N feeding ratio equivalent to the "0-increase" variant (Figure 2a) to establish stable conditions. The stable operation was determined by monitored VFA concentrations (Figure 2d) and specific methane production (Figure 2f). In week four to six, the TE concentrations were additionally tested, which showed sufficient nutrient levels according to Vintiloiu et al. [42] (see Table 3). For inoculum #1, the starting phase lasted for 48 days. For inoculum #2, the starting phase took 90 days.

The values provided in Figure 2 for the starting phase can be relevant for farmers and biogas operators when utilizing protein-rich substrates in biogas plants. However, these values are excluded from the statistical analysis described in Section 3.3.

**Figure 2.** Measured values of the following parameters in the continuously stirred tank reactors (CSTR) under different N-increase rates in feeding regimes: (**a**) total nitrogen (N); (**b**) total ammonia nitrogen (TAN); (**c**) free ammonia nitrogen (FAN); (**d**) acetic acid (HAc); (**e**) pH; (**f**) the measured values of specific methane yield (*SMYm*); (**g**) inhibition in specific methane yield (Inhibition). The results are given separately for inoculum #1 and inoculum #2. Different line colours along with different marks in the graphs and in the legend correspond to the N-increase rates "0-increase", "0.25-increase" and "0.5-increase" in the feeding regimes. Grey and white backgrounds in the graphs are related to the starting phase and the experimental phase, respectively. The vertical line in the graphs corresponds to the beginning of regular weekly trace elements (TE) supplementation. **Figure 2.** Measured values of the following parameters in the continuously stirred tank reactors (CSTR) under different N-increase rates in feeding regimes: (**a**) total nitrogen (N); (**b**) total ammonia nitrogen (TAN); (**c**) free ammonia nitrogen (FAN); (**d**) acetic acid (HAc); (**e**) pH; (**f**) the measured values of specific methane yield (*SMYm*); (**g**) inhibition in specific methane yield (Inhibition). The results are given separately for inoculum #1 and inoculum #2. Different line colours along with different marks in the graphs and in the legend correspond to the N-increase rates "0-increase", "0.25-increase" and "0.5-increase" in the feeding regimes. Grey and white backgrounds in the graphs are related to the starting phase and the experimental phase, respectively. The vertical line in the graphs corresponds to the beginning of regular weekly trace elements (TE) supplementation.

2 0.5-increase 1.36 (0.07) 1.38 (0.07) 1.35 (0.07) 0.99 (0.01) 1.79 (0.06)

*Energies* **2020**, *13*, 1139

#### *3.2. The Experimental Period*

After the starting phase, the reactors were continuously operated and monitored for 26 and 20 weeks for inoculum #1 and #2, respectively.

The lack of TE in the reactors with inoculum #1, which resulted in the accumulation of acetic acid up to 8.53 g·kg−1 FM, along with a drop in pH up to 6.40 (as described in [12,42]), was identified at the beginning of week 17 of the trials (see Figure 2d,e and Table 3). The weekly supplementation of the CSTR with TE was established thereafter in order to compensate for the deficiency in TE in the reactors with inoculum #1 and to ensure a sufficient TE supply for the remainder of the experiment. The vertical line shown at week 17 in Figure 2 marks the beginning of weekly TE supplementation. The positive effect of TE to AD process stability can be seen in Figure 2d,e in the stabilization of pH and HAc in the weeks following supplementation. The analysis of TE measured in week 24 showed that the amounts of these nutrients in the reactors were well-balanced (see Table 3).

Additionally, the total phosphorus and potassium concentrations inside the reactors were analysed. The availability of these nutrients may be of great interest when using digestate as a fertilizer. The concentrations of these macro elements within the research period were the following: for inoculum #1, P = 0.62 ± 0.13 g·kg−1 FM, K = 3.20 ± 0.35 g·kg−1 FM; for inoculum #2, P = 0.99 ± 0.09 g·kg−1 FM, K = 3.73 ± 1.11 g·kg−1 FM.

Over the experimental period, the concentration of N in the digestate was accumulating, as shown in Figure 2a. The accumulation of N in the reactors was related to the analysed N-increase rates. The average N-increase rate in the daily feedstock under the "0.5-increase" feeding regime was 35.7 mg·kg−1 ·d −1 related to the fresh matter of the input substrates. At the end of the experiment, the highest values of total nitrogen in the digestate were 10.09 ± 0.08 g·kg−1 FM and 11.49 ± 0.01 g·kg−1 FM for the reactors with inoculum #1 and #2, respectively. Accordingly, a maximum "nitrogen loading rate" (NLR) can be given; the NLR was equal to 0.25 g·L −1 ·d −1 for the reactors with inoculum #1 and 0.30 g·L −1 ·d −1 for those with inoculum #2.

Concurrently, the TAN and FAN concentrations in the digestate increased, as shown in Figure 2b,c. At the end of the experiment, the highest values of TAN were 7.72 ± 0.33 g·kg−1 FM (for the reactors with inoculum #1) and 7.95 ± 1.08 g·kg−1 FM (for the reactors with inoculum #2). The highest FAN concentration in the final samples was 0.72 ± 0.03 g·kg−1 FM and 0.74 ± 0.12 g·kg−1 FM for the reactors with inoculum #1 and #2, respectively.

The concentration of HAc in the reactors over the period of the trials is shown in Figure 2d. The average concentrations of acetic and propionic acids in the CSTR during the experimental period were 0.88 ± 0.46 g·kg−1 FM and 0.17 ± 0.32 g·kg−1 FM, respectively, independent of the inoculum. In the reactors with #1, acetate accumulation caused by TE deficiency decreased to a minimum after the start of TE supplementation, with no acetate found in weeks 31–33. In the reactors with inoculum #2, acetate remained at a stable low concentration over the entire experimental phase with zero-values at the end of the trials. The concentrations of other VFA, i.e., iso-butyric, n-butyric, iso-valeric, n-valeric and caproic acids were low over the research period; the concentration of these acids was 0.04 ± 0.16 g·kg−1 FM for both inocula.

The pH-values during the experimental phase were slightly higher than those in the starting phase. Over the entire experimental period, the pH levels in the CSTR were stable, except for the reactors with inoculum #1 under the TE deficiency with a drop in pH up to 6.40 (see Figure 2e). The average pH was 7.45 ± 0.21 for the experiments based on inoculum #1 and 7.77 ± 0.11 for those based on inoculum #2.

The values of *SMYm* during the experimental phase are given in Figure 2f. The mean *SMYm* was 289.93 ± 35.13 L·kg−1 VS and 267.20 ± 19.86 L·kg−1 VS for the reactors with #1 and #2, respectively.

The values of inhibition during the experimental phase are given in Figure 2g. At the end of the experiment, the values of inhibition for inoculum #1 were 0.57% ± 1.22% (in weeks 32–33), 18.02% ± 22.64% (in weeks 32–33) and 26.96% ± 22.88% (in weeks 29–33) for the "0-increase", "0.25-increase" and "0.5-increase" variants, respectively. At the final phase of the experiment (in weeks 29–33) the

values of inhibition for inoculum #2 were 10.91% ± 4.58%, 19.38% ± 8.93%, 38.99% ± 14.99% for the "0-increase", "0.25-increase" and "0.5-increase" variants, respectively. The 38.99% ± 14.99% inhibition determined in the reactors with #2 and "0.5-increase" feeding regime was related to N, TAN and FAN concentrations of 10.82 ± 0.52 g·kg−1 FM, 7.92 ± 0.27 g·kg−1 FM and 0.69 ± 0.10 g·kg−1 FM, respectively. As seen in Figure 2g, inhibition levels in the reactors with both inocula appear to have reached higher levels at the higher N increase rate. *Energies* **2020**, *13*, x FOR PEER REVIEW 15 of 20 "0-increase", "0.25-increase" and "0.5-increase" variants, respectively. The 38.99% ± 14.99% inhibition determined in the reactors with #2 and "0.5-increase" feeding regime was related to N, TAN and FAN concentrations of 10.82 ± 0.52 g∙kg−1 FM, 7.92 ± 0.27 g∙kg−1 FM and 0.69 ± 0.10 g∙kg−1 FM, respectively. As seen in Figure 2g, inhibition levels in the reactors with both inocula appear to have reached higher levels at the higher N increase rate.

#### *3.3. Results of Statistical Analysis 3.3. Results of Statistical Analysis*

#### 3.3.1. Results of analysis on inhibition in *SMY* 3.3.1. Results of analysis on inhibition in *SMY*

The results of the analysis on inhibition in *SMY* over the experimental phase are given in Table 4 and are shown in Figure 3. According to the results of the Tukey test, for all the analysed feeding regimes the difference between the *SMYt* and *SMYm* was statistically significant. The large variation in the *SMYm* for the reactors with inoculum #1 and the "0-increase" variant can be explained by the instability of the AD process under the TE deficiency. The highest inhibition was determined in the reactors with inoculum #2 and the "0.5-increase" variant. The results of the analysis on inhibition in *SMY* over the experimental phase are given in Table 4 and are shown in Figure 3. According to the results of the Tukey test, for all the analysed feeding regimes the difference between the *SMYt* and *SMYm* was statistically significant. The large variation in the *SMYm* for the reactors with inoculum #1 and the "0-increase" variant can be explained by the instability of the AD process under the TE deficiency. The highest inhibition was determined in the reactors with inoculum #2 and the "0.5-increase" variant. According to the results of the analysis, the N-increase rate in feeding regime had a negative

**Table 4.** The results of analysis on inhibition in specific methane yield (*SMY*). Feeding regime expresses the N-increase rate in a feeding ratio. Degrees of freedom (DF). *SMYt* and *SMYm* are the theoretical and measured values of specific methane yield, respectively. Gas volumes are given under standard temperature and pressure conditions (0 ◦C, 101.325 kPa). Units are given in square brackets. Values of *SMYt* and *SMYm* are given as mean; the standard deviation is given in round brackets. effect on the AD process efficiency. **Table 4.** The results of analysis on inhibition in specific methane yield (*SMY*). Feeding regime expresses the N-increase rate in a feeding ratio. Degrees of freedom (DF). *SMYt* and *SMYm* are the theoretical and measured values of specific methane yield, respectively. Gas volumes are given under standard temperature and pressure conditions (0 °C, 101.325 kPa). Units are given in square brackets.

\* Significant at *p*-value = 0.0001. \* Significant at *p*-value = 0.0001.

**Figure 3.** The results of analysis on inhibition in specific methane yield (*SMY*). The results are given separately for inoculum #1 and inoculum #2. Tick marks "0", "0.25" and "0.5" on the x-axis correspond to the N-increase variants of "0-increase", "0.25-increase" and "0.5-increase" in feeding regimes. The "measured" value is the measured *SMY* (*SMYm*); the "theoretical" value is the theoretical **Figure 3.** The results of analysis on inhibition in specific methane yield (*SMY*). The results are given separately for inoculum #1 and inoculum #2. Tick marks "0", "0.25" and "0.5" on the x-axis correspond to the N-increase variants of "0-increase", "0.25-increase" and "0.5-increase" in feeding regimes. The "measured" value is the measured *SMY* (*SMYm*); the "theoretical" value is the theoretical *SMY* (*SMYt*). Letters "a" and "b" denote the significant differences between the *SMYm* and *SMYt* for the same variant of N-increase according to the results of the one-sided Tukey test.

According to the results of the analysis, the N-increase rate in feeding regime had a negative effect on the AD process efficiency. 3.3.2. Results of analysis of the effect of TAN and FAN on inhibition The results of the fitted model were the following: The increase in TAN levels resulted in an

the same variant of N-increase according to the results of the one-sided Tukey test.

*Energies* **2020**, *13*, x FOR PEER REVIEW 16 of 20

3.3.2. Results of analysis of the effect of TAN and FAN on inhibition increase of inhibition in *SMY*, *p*-value = 0.0001 (Table 5 and Figure 4). The increase in FAN

The results of the fitted model were the following: The increase in TAN levels resulted in an increase of inhibition in *SMY*, *p*-value = 0.0001 (Table 5 and Figure 4). The increase in FAN concentration in the AD reactor resulted in an increase of the inhibition level, *p*-value = 0.0012 (Table 5 and Figure 5). The observed noise in Figures 4 and 5 can be associated with the fact that the inhibition does not derive only from TAN or FAN concentrations inside the reactors; this inhibition can be also affected by other parameters. concentration in the AD reactor resulted in an increase of the inhibition level, *p*-value = 0.0012 (Table 5 and Figure 5). The observed noise in Figures 4 and 5 can be associated with the fact that the inhibition does not derive only from TAN or FAN concentrations inside the reactors; this inhibition can be also affected by other parameters. The results of the data analysis show that the analysed N-increase rates can be recommended for a stable AD process. However, the level of inhibition in *SMY* depends on the concentration of TAN and FAN inside the reactors and the N-increase rate in the feeding regimes (see Figures 4 and 5).

**Table 5.** The effect of total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) on the inhibition in specific methane yield: the results of the fitted model. Degrees of freedom (DF). The square-root transformed values of inhibition in specific methane yield (sqrt\_Inhibition); the transformation was done for meeting the requirements of the model. **Table 5.** The effect of total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) on the inhibition in specific methane yield: the results of the fitted model. Degrees of freedom (DF). The square-root transformed values of inhibition in specific methane yield (sqrt\_Inhibition); the transformation was done for meeting the requirements of the model.

**Figure 4.** The correlation between the total ammonia nitrogen (TAN) and the inhibition in specific methane yield (Inhibition). The "0", "0.25" and "0.5" marks in the legend correspond to the N-increase variants of "0-increase", "0.25-increase" and "0.5-increase" in the feeding regimes. The regression line was built based on the results obtained from the model. **Figure 4.** The correlation between the total ammonia nitrogen (TAN) and the inhibition in specific methane yield (Inhibition). The "0", "0.25" and "0.5" marks in the legend correspond to the N-increase variants of "0-increase", "0.25-increase" and "0.5-increase" in the feeding regimes. The regression line was built based on the results obtained from the model.

*Energies* **2020**, *13*, x FOR PEER REVIEW 17 of 20

**Figure 5.** The correlation between the free ammonia nitrogen (FAN) and the inhibition in specific methane yield (Inhibition). The "0", "0.25" and "0.5" marks in the legend correspond to the N-increase variants of "0-increase", "0.25-increase" and "0.5-increase" in the feeding regimes. The regression line was built based on the results obtained from the model. **Figure 5.** The correlation between the free ammonia nitrogen (FAN) and the inhibition in specific methane yield (Inhibition). The "0", "0.25" and "0.5" marks in the legend correspond to the N-increase variants of "0-increase", "0.25-increase" and "0.5-increase" in the feeding regimes. The regression line was built based on the results obtained from the model.

*3.4. Discussion*  The inhibitory effect of urea, NH4Cl, TAN, FAN and high N concentration in feeding, as well as The results of the data analysis show that the analysed N-increase rates can be recommended for a stable AD process. However, the level of inhibition in *SMY* depends on the concentration of TAN and FAN inside the reactors and the N-increase rate in the feeding regimes (see Figures 4 and 5).

the effect of elevated ammonium (NH4+), elevated ammonium nitrogen (NH4+-N) and elevated TAN

#### on biogas and methane yields, have been previously studied [1,4,15,17,22,32,51–53]. However, no *3.4. Discussion*

results on the effects of N concentration in inoculum and N-increase rate in feedstock on the level of inhibition in specific methane yield were found. Contrary to the research results reported by Siegrist et al. [18], Chen et al. [17], Meng et al. [19] and Theuerl et al. [16], the stable AD-process was found in this study as indicated by stable pH values and a minimal accumulation of acetate (except for the reactors with #1 under the TE deficiency) (see The inhibitory effect of urea, NH4Cl, TAN, FAN and high N concentration in feeding, as well as the effect of elevated ammonium (NH4 +), elevated ammonium nitrogen (NH4 +-N) and elevated TAN on biogas and methane yields, have been previously studied [1,4,15,17,22,32,51–53]. However, no results on the effects of N concentration in inoculum and N-increase rate in feedstock on the level of inhibition in specific methane yield were found.

Figure 2e,d). During the experimental phase, the specific methane yields were kept stable in all the reactors, and their values were in a normal range (see Figure 2f). Based on the results obtained it can be assumed that the analysed feeding regimes enabled the microorganisms to adapt to changing Nconditions, which is indicated by a stable AD process. The regular supplementation of reactors with TE positively contributed to the process stability. The proposed increase rates did not have any negative effect on the process stability. Hereby the N-increase variants of "0.25-increase", "0.5 increase" and the NLR up to 0.30 g∙L−1∙d−1 can be recommended for maintaining a biogas plant in a stable way. In contrast, the efficiency of AD, which in this study corresponded to the inhibition in specific methane yield (Inhibition), was affected by N-increase rate and the level of TAN and FAN inside the Contrary to the research results reported by Siegrist et al. [18], Chen et al. [17], Meng et al. [19] and Theuerl et al. [16], the stable AD-process was found in this study as indicated by stable pH values and a minimal accumulation of acetate (except for the reactors with #1 under the TE deficiency) (see Figure 2e,d). During the experimental phase, the specific methane yields were kept stable in all the reactors, and their values were in a normal range (see Figure 2f). Based on the results obtained it can be assumed that the analysed feeding regimes enabled the microorganisms to adapt to changing N-conditions, which is indicated by a stable AD process. The regular supplementation of reactors with TE positively contributed to the process stability. The proposed increase rates did not have any negative effect on the process stability. Hereby the N-increase variants of "0.25-increase", "0.5-increase" and the NLR up to 0.30 g·L −1 ·d −1 can be recommended for maintaining a biogas plant in a stable way.

reactor. The conversion process in the reactors, which in this study is described as inhibition, became more and more inefficient due to the closer C/N ratio in feedstock (see Figure 1b). Chen et al. [17] has stated that the methane production was intensely inhibited when TAN increased to 5 g∙L−1 and they recommended to maintain the ammonium concentration below 2 g∙L−1 in the reactors for preventing the ammonium shock to the AD process. According to the review made by Chen et al. [15], in different studies there is controversial information on the level of inhibition in methane production depending on the TAN and FAN concentrations in the AD reactor: 50% of methane inhibition was observed at TAN of 1.44 g⋅L−1, 2.48 g⋅L−1 and 5.60 g⋅L−1 and FAN of 0.03 g⋅L−1 and 0.64 g⋅L−1; 100% of methane In contrast, the efficiency of AD, which in this study corresponded to the inhibition in specific methane yield (Inhibition), was affected by N-increase rate and the level of TAN and FAN inside the reactor. The conversion process in the reactors, which in this study is described as inhibition, became more and more inefficient due to the closer C/N ratio in feedstock (see Figure 1b). Chen et al. [17] has stated that the methane production was intensely inhibited when TAN increased to 5 g·L −1 and they recommended to maintain the ammonium concentration below 2 g·L −1 in the reactors for preventing the ammonium shock to the AD process. According to the review made by Chen et al. [15], in different

studies there is controversial information on the level of inhibition in methane production depending on the TAN and FAN concentrations in the AD reactor: 50% of methane inhibition was observed at TAN of 1.44 g·L −1 , 2.48 g·L −1 and 5.60 g·L −1 and FAN of 0.03 g·L −1 and 0.64 g·L −1 ; 100% of methane inhibition was identified at TAN values above 5.20 g·L −1 and FAN of 0.20 g·L −1 and 0.62 g·L −1 . Fotidis et al. [32,53] specify that at the NH4 +-N in the range of 3–5 g·L −1 , an ammonia induced inhibited-steady state in the AD reactors was observed with inhibition in methane production of 30–40%, and the authors recommend a bioaugmentation strategy for overcoming an ammonia inhibiting effect. However, in our research, under NH4 +-N of 5.03 ± 0.06 g·kg−1 FM, our TAN and FAN concentrations in the reactors were 5.34 ± 0.15 g·kg−1 FM and 0.31 ± 0.14 g·kg−1 FM, respectively, and the value of inhibition was equal to 9.46% ± 5.60%. According to the results of the data analysis, both TAN and FAN had a significant effect on the level of inhibition. As FAN levels are mostly affected by temperature and pH fluctuations [7,9,14,21], the effect of FAN was less significant than TAN in our research, since the reactors were operated under mesophilic conditions at stable temperature and pH (except for the pH values in the reactors with inoculum #1 under the TE deficiency). As the OLRVS, HRT, temperature and pH in the reactors were kept stable, the results show that the N-increase rate in the feeding regime was negatively related to the efficiency of the AD process even if low VFA concentrations indicated a stable process. In further studies, the influence of the increasing N concentrations in the digestate on the microbial population should be investigated.

The results of this study can be applied by biogas operators running their systems at high nitrogen concentrations up to 11.5 g·kg−1 FM or utilizing substrates with varying nitrogen contents.

#### **4. Conclusions**

In this study, we analysed the effect of different inocula and different N-increase rates in feeding regimes on AD process stability and efficiency. The stepwise acclimatisation strategy used for microorganisms to adapt to a new nitrogen concentration according to the feeding regime prevented failure of the AD process under high and elevated ammonia levels. The research approach applied in this study enabled us to run the CSTR in a stable way under the elevated nitrogen loading rates up to 0.30 g·L −1 ·d −1 . The highest N, TAN and FAN in the digestate at the end of the experiment were equal to 11.50 g·kg−1 FM, 9.07 g·kg−1 FM and 0.85 g·kg−1 FM. However, the study indicates that the N-increase rate was negatively related to the AD process efficiency. The level of inhibition in specific methane yield was positively correlated to the TAN and FAN concentrations in the digestate.

**Author Contributions:** Data curation: I.M., N.N. and A.L.; formal analysis: I.M.; funding acquisition: H.O. and A.L.; project administration: A.L. and I.M.; resources: H.O. and A.L.; supervision: A.L.; writing—original draft: I.M; writing—review and editing: I.M., A.L. and J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Agency for Renewable Resources (Fachagentur Nachwachsende Rohstoffe e. V. or FNR) under the framework of the project "Optimized substrate management and influence of digestate composition on the soil nitrogen and soil humus balance" (grant no. 22402412). All experiments were conducted at the State Institute of Agricultural Engineering and Bioenergy, University of Hohenheim. The APC was funded by the State Institute of Agricultural Engineering and Bioenergy, University of Hohenheim.

**Acknowledgments:** We want to thank Prof. Dr. Hans-Peter Piepho for his valuable advice and help during the statistical analysis. Ievgeniia Morozova would like to thank the German Academic Exchange Service (Deutscher Akademischer Austauschdienst, DAAD) for the financial support.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*


**2.3 Recreational drugs**

opiates (narcotics), methamphetamines, anabolic–androgenic steroids, Marijuana, and cocaine are examples of illicit drugs thatNegatively affects male fertility. Destructive effects of these drugs is on the hypothalamic–pituitary-gonadal axis, testicular structure and sperm function [13].